A fluorescent probe and its preparation method and application

Abstract

The present invention discloses a fluorescent probe and its preparation method and application. The fluorescent probe comprises two parts, namely 5-iodo-2'-deoxy-2'-fluorouridine and 2-(4-ethynylphenyl)-1,2,2-triphenylethylene. Due to the 5-iodo-2'-deoxy-2'-fluorouridine unit, the biocompatibility of the fluorescent probe is improved; due to the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene group, the fluorescent probe has high fluorescence intensity. The design of the fluorescent probe in the present invention well solves the problems of biocompatibility and fluorescence yield, and has multiple functions such as mitochondrial targeting, pH detection, and mercury ion recognition. The preparation method of the fluorescent probe of the present invention is simple and efficient. The fluorescent probe of the present invention has good selectivity, high biocompatibility, and convenient use of related instruments, and is a detection means with relatively high sensitivity in current chemiluminescence detection methods, and has important potential application value.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe, a preparation method thereof and an application thereof. Background Art

[0002] Fluorescent probes are an important discovery in the field of chemical sensing technology. Currently, for the research on fluorescence imaging technology, the main focus is to develop highly efficient fluorescence imaging agents. Fluorescence imaging agents based on the aggregation-induced emission (AIE) mechanism have excellent capabilities in imaging and monitoring various reactions of organisms and their biological systems, and have received great attention in recent years.

[0003] However, for typical AIE molecules, their hydrophobic chromophores constructed by multiple aromatic rings and branched large-volume spatial structures determine their low biocompatibility, which limits their applications in the biomedical field. How to design and improve the biocompatibility and sensitivity of such AIE fluorescent probes and expand their functions in environmental monitoring is also a current research hotspot.

[0004] Therefore, designing and synthesizing a fluorescent probe with good biocompatibility and high fluorescence efficiency is of great significance for developing its subsequent application research in fields such as life science and biomedical engineering. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a fluorescent probe with good biocompatibility and high fluorescence efficiency, a preparation method thereof, and also provide its applications in cell imaging, pH indication and ion response, aiming to solve the problem of low biocompatibility of existing AIE fluorescent probes.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a fluorescent probe is provided, wherein the structural formula of the fluorescent probe is as follows:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the fluorescent probe (TPE-FdU) of the present invention is provided, which includes the steps of:

[0010] Providing 5-iodo-2'-deoxy-2'-fluorouridine;

[0011] Providing 2-(4-ethynylphenyl)-1,2,2-triphenylethylene;

[0012] The 5-iodo-2'-deoxy-2'-fluorouridine is subjected to a Sonogashira coupling reaction with the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene to obtain the fluorescent probe (TPE-FdU).

[0013] Optionally, the preparation method of the 5-iodo-2'-deoxy-2'-fluorouridine includes the steps of:

[0014] Providing 2'-deoxy-2'-fluorouridine and iodine;

[0015] Subjecting the 2'-deoxy-2'-fluorouridine to a substitution reaction with the iodine to obtain the 5-iodo-2'-deoxy-2'-fluorouridine (abbreviated as FdU).

[0016] Optionally, the preparation method of the 5-iodo-2'-deoxy-2'-fluorouridine specifically includes the steps of:

[0017] Providing 2'-deoxy-2'-fluorouridine, ammonium cerium(IV) nitrate, iodine and a first solvent;

[0018] Dissolving the 2'-deoxy-2'-fluorouridine, ammonium cerium(IV) nitrate and iodine in the first solvent, heating under reflux for 1 - 4 hours, and after purification treatment, obtaining the 5-iodo-2'-deoxy-2'-fluorouridine.

[0019] Optionally, the preparation method of the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene includes the steps of:

[0020] Providing diphenylmethane, 4-bromobenzophenone, p-toluenesulfonic acid, trimethylsilylacetylene, tetrabutylammonium fluoride;

[0021] Reacting the diphenylmethane with the 4-bromobenzophenone, followed by catalytic dehydration with p-toluenesulfonic acid, and after separation and purification, obtaining 1-(4-bromophenyl)-1,2,2-triphenylethylene;

[0022] Subjecting the 1-(4-bromophenyl)-1,2,2-triphenylethylene to a Sonogashira coupling reaction with the trimethylsilylacetylene to obtain 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene;

[0023] Reacting the 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene with the tetrabutylammonium fluoride to obtain the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene (abbreviated as TPE).

[0024] Optionally, the step of obtaining the fluorescent probe by performing a Sonogashira coupling reaction between the 5-iodo-2'-deoxy-2'-fluorouridine and the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene specifically includes:

[0025] Dissolve 5-iodo-2'-deoxy-2'-fluorouridine and CuI in DMF or THF, then add Pd(PPh3)4, 2-(4-ethynylphenyl)-1,2,2-triphenylethylene, anhydrous Et3N or THF, and perform the Sonogashira coupling reaction at room temperature. After purification, the fluorescent probe (TPE-FdU) is obtained.

[0026] Optionally, in the step of performing a Sonogashira coupling reaction between the 5-iodo-2'-deoxy-2'-fluorouridine and the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene, the time of the Sonogashira coupling reaction is 8 - 12 hours.

[0027] In the third aspect of the present invention, there is provided an application of the fluorescent probe of the present invention in cell imaging.

[0028] In the fourth aspect of the present invention, there is provided an application of the fluorescent probe of the present invention in pH detection.

[0029] In the fifth aspect of the present invention, there is provided an application of the fluorescent probe of the present invention in mercury ion detection.

[0030] Beneficial effects: The present invention provides a pH-sensitive mitochondrial-targeted fluorescent probe (TPE-FdU) and its preparation method and application. The fluorescent probe includes two parts: 5-iodo-2'-deoxy-2'-fluorouridine and 2-(4-ethynylphenyl)-1,2,2-triphenylethylene. Due to the 5-iodo-2'-deoxy-2'-fluorouridine unit, the biocompatibility of the fluorescent probe is improved; due to the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene group, the fluorescent probe has high fluorescence intensity. The design of the fluorescent probe in the present invention well solves the problems of biocompatibility and fluorescence yield. The preparation method of the fluorescent probe in the present invention is simple and efficient. The fluorescent probe of the present invention has good selectivity, high biocompatibility, and is convenient to use with related instruments. It is a detection means with relatively high sensitivity in current chemiluminescence detection methods and has important potential application value. Description of the Drawings

[0031] Figure 1 It is the ultraviolet-visible light absorption spectrum diagram of the fluorescent probe in the embodiment of the present invention.

[0032] Figure 2This is the fluorescence spectrum diagram of the fluorescent probe in the embodiment of the present invention.

[0033] Figure 3 This is the result diagram of detecting the cytotoxicity of the fluorescent probe by the MTT method in the embodiment of the present invention.

[0034] Figure 4 、 5 、6 This is the cell imaging diagram of the fluorescent probe in the embodiment of the present invention.

[0035] Figure 7 This is the mitochondrial co-localization diagram of the fluorescent probe in the embodiment of the present invention.

[0036] Figure 8 This is the lysosome co-localization diagram of the fluorescent probe in the embodiment of the present invention.

[0037] Figure 9 This is the pH spectral titration diagram of the fluorescent probe in the embodiment of the present invention.

[0038] Figure 10 This is the metal ion selectivity diagram of the fluorescent probe in the embodiment of the present invention.

[0039] Figure 11 This is the mercury ion sensitivity diagram of the fluorescent probe in the embodiment of the present invention.

[0040] Figure 12 This is the mercury ion selectivity diagram of the fluorescent probe in the embodiment of the present invention. Detailed implementation manners

[0041] The present invention provides a fluorescent probe and its preparation method and application. To make the purpose, technical solution and effect of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Aggregation-induced emission is a characteristic of certain compounds. The molecular structures with this characteristic are very unique, containing multiple benzene rings or aromatic ring structures. They hardly emit fluorescence when in a dispersed state in solution, but can emit strong fluorescence when aggregated in a poor solvent or in a solid state. Utilizing this unique property, it can be applied to the detection field of biomolecules.

[0043] Based on this, an embodiment of the present invention provides a fluorescent probe, wherein the structural formula of the fluorescent probe is shown as follows:

[0044]

[0045] The fluorescent probe includes two parts, 5-iodo-2'-deoxy-2'-fluorouridine and 2-(4-ethynylphenyl)-1,2,2-triphenylethylene. Due to the 5-iodo-2'-deoxy-2'-fluorouridine unit, the biocompatibility of the fluorescent probe is improved; due to the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene group, the fluorescent probe has high fluorescence intensity. In the embodiments of the present invention, the design of the fluorescent probe well solves the problems of biocompatibility and fluorescence yield, and has multiple functions such as mitochondrial targeting, pH detection, and mercury ion recognition. The fluorescent probe in the embodiments of the present invention has good selectivity, high biocompatibility, and convenient use of related instruments. It is a detection means with relatively high sensitivity in current chemiluminescence detection methods and has important potential application value.

[0046] The embodiments of the present invention also provide a preparation method of the fluorescent probe as described above, which includes the steps:

[0047] S1. Provide 5-iodo-2'-deoxy-2'-fluorouridine;

[0048] S2. Provide 2-(4-ethynylphenyl)-1,2,2-triphenylethylene;

[0049] S3. Perform a Sonogashira coupling reaction on the 5-iodo-2'-deoxy-2'-fluorouridine and the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene to obtain the fluorescent probe.

[0050] Using the 5-iodo-2'-deoxy-2'-fluorouridine and 2-(4-ethynylphenyl)-1,2,2-triphenylethylene, the fluorescent probe (TPE-FdU) is obtained through a Sonogashira coupling reaction. The preparation method of the fluorescent probe in the embodiments of the present invention is simple and efficient.

[0051] In step S1, in one embodiment, the 5-iodo-2'-deoxy-2'-fluorouridine is prepared from 2'-deoxy-2'-fluorouridine and iodine.

[0052] In one embodiment, the preparation method of the 5-iodo-2'-deoxy-2'-fluorouridine includes the steps:

[0053] Provide 2'-deoxy-2'-fluorouridine and iodine;

[0054] Perform a substitution reaction on the 2'-deoxy-2'-fluorouridine and the iodine to obtain the 5-iodo-2'-deoxy-2'-fluorouridine.

[0055] The preparation method of 5-iodo-2'-deoxy-2'-fluorouridine will be described in detail below in conjunction with the following synthesis reaction formula of 5-iodo-2'-deoxy-2'-fluorouridine:

[0056]

[0057] Provide 2'-deoxy-2'-fluorouridine, ammonium cerium(IV) nitrate (abbreviated as CAN), iodine and a first solvent; the first solvent can be anhydrous acetonitrile (abbreviated as MeCN) or anhydrous ethanol, etc.;

[0058] Dissolve the 2'-deoxy-2'-fluorouridine, ammonium cerium(IV) nitrate and iodine in the first solvent, heat under reflux for 1 - 4 hours (such as 1 hour, 2 hours or 4 hours), and after purification treatment, obtain the 5-iodo-2'-deoxy-2'-fluorouridine.

[0059] Among them, the steps of the purification treatment can be: after the heating under reflux ends, quench with a saturated Na2S2O3 solution, extract with ethyl acetate and then concentrate the product, and perform column chromatography (the volume ratio of CH2Cl2:MeOH is 10:1) to separate and remove the liquid.

[0060] In one embodiment of step S2, the preparation method of 2-(4-ethynylphenyl)-1,2,2-triphenylethylene includes the steps:

[0061] S21. React the diphenylmethane with the 4-bromobenzophenone, and then catalyze dehydration with p-toluenesulfonic acid, and after separation and purification, obtain 1-(4-bromophenyl)-1,2,2-triphenylethylene;

[0062] S22. Perform a Sonogashira coupling reaction on the 1-(4-bromophenyl)-1,2,2-triphenylethylene and the trimethylsilylacetylene to obtain 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene;

[0063] S23. React the 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene with the tetrabutylammonium fluoride to obtain the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene.

[0064] In one embodiment, the preparation method of 2-(4-ethynylphenyl)-1,2,2-triphenylethylene is specifically as follows:

[0065]

[0066] First step: Dissolve diphenylmethane in THF, slowly add n-butyllithium (n-BuLi) dropwise under an ice bath and magnetic stirring. After 1 hour, add 4-bromobenzophenone and react overnight. Quench the reaction with saturated ammonium chloride solution. After rotary evaporation under reduced pressure, the obtained crude product is a white solid. Redissolve the crude product in toluene, add p-toluenesulfonic acid, and then reflux for 6 hours. After concentration of the product, perform column chromatography (PE) and separate to obtain a white solid 1-(4-bromophenyl)-1,2,2-triphenylethylene.

[0067] Second step: Dissolve 1-(4-bromophenyl)-1,2,2-triphenylethylene, PdCl2(PPh3)2, CuI (38.1 mg, 0.2 mmol), and triphenylphosphine in 100 mL of THF / Et3N / piperidine (volume ratio 5:4:1). After the catalyst is completely dissolved, add trimethylsilylacetylene and stir overnight at 50 °C. After extraction with ethyl acetate and rotary evaporation of the solvent, purify by column chromatography (petroleum ether) to obtain a pale yellow solid 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene.

[0068] Third step: Dissolve 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene in THF, add tetrabutylammonium fluoride (TABF), and react at room temperature for 2 hours. Purify the product by column chromatography (PE) to obtain a white solid 2-(4-ethynylphenyl)-1,2,2-triphenylethylene.

[0069] In step S3, use the 5-iodo-2'-deoxy-2'-fluorouridine and 2-(4-ethynylphenyl)-1,2,2-triphenylethylene to obtain the fluorescent probe through Sonogashira coupling reaction.

[0070] In one embodiment, the preparation method of the fluorescent probe is specifically as follows:

[0071]

[0072] Dissolve 5-iodo-2'-deoxy-2'-fluorouridine and CuI (as a co-catalyst) in DMF or THF, successively add Pd(PPh3)4, 2-(4-ethynylphenyl)-1,2,2-triphenylethylene, anhydrous Et3N or THF (providing an alkaline environment and serving as a solvent), and perform Sonogashira coupling reaction at room temperature. After concentration of the reaction product, perform column chromatography (volume ratio of PE:EA is 1:1) and separate to obtain a light yellow solid.

[0073] In one embodiment, in the step of performing Sonogashira coupling reaction on the 5-iodo-2'-deoxy-2'-fluorouridine and the 2-(4-ethynylphenyl)-1,2,2-triphenylethylene, the time of the Sonogashira coupling reaction is 8 - 12 hours.

[0074] The embodiment of the present invention also provides an application of the above-mentioned fluorescent probe in cell imaging.

[0075] The AIE characteristic group possessed by the fluorescent probe enables the molecule to emit strong fluorescence in the aggregated state. And when using a low concentration of the fluorescent probe to co-incubate with NIH-3T3 cells, cell imaging can be carried out within only 30 minutes, and it can specifically bind to mitochondria. The design of the fluorescent probe in the embodiment of the present invention well solves the interference of poor biocompatibility. The preparation method of the fluorescent probe in the embodiment of the present invention is simple and efficient. The fluorescent probe in the embodiment of the present invention has good selectivity, high biocompatibility, and convenient use of related instruments. It is a detection means with relatively high sensitivity in the current chemiluminescence detection method and has important potential application value.

[0076] The embodiment of the present invention also provides an application of the above-mentioned fluorescent probe in pH detection.

[0077] The AIE characteristic group possessed by the fluorescent probe enables the molecule to emit strong fluorescence in the aggregated state, and the fluorescence intensity of the probe is easily affected by the solution pH and changes greatly. At pH = 4, the fluorescence intensity of the probe reaches the highest value; at pH = 9.5, the fluorescence quenches. The design of the fluorescent probe in the embodiment of the present invention has the characteristics of a wide application range and high sensitivity in pH detection, and at the same time has the potential to monitor the change of environmental pH. The preparation method of the fluorescent probe in the embodiment of the present invention is simple and efficient. When the fluorescent probe is applied in pH detection, the related instruments are convenient to use. It is a detection means with relatively high sensitivity in the current chemiluminescence detection method and has important potential application value.

[0078] The embodiment of the present invention also provides an application of the above-mentioned fluorescent probe in mercury ion detection.

[0079] The fluorescent probe has a specific response to mercury ions and can cause significant fluorescence quenching. The application of the fluorescent probe in the embodiment of the present invention in mercury ion detection has the advantages of convenience, high speed, high sensitivity, and strong specificity, and overcomes the disadvantages of poor sensitivity, large interference, complex pretreatment, and expensive instruments of conventional detection methods. It is a simple and practical mercury ion qualitative analysis technology.

[0080] The following further illustrates the present invention through several specific embodiments.

[0081] Example 1

[0082] Synthesis of TPE-FdU Fluorescent Probe

[0083] 1. Synthesis of 5-Iodo-2'-deoxy-2'-fluorouridine

[0084] Dissolve 2'-deoxy-2'-fluorouridine (2.00 g, 8.12 mmol, 1 equiv.), iodine (1.24 g, 4.87 mmol, 0.6 equiv.) and ammonium cerium(IV) nitrate (2.23 g, 4.06 mmol) in anhydrous acetonitrile (50 mL), and reflux with stirring for 1 h. After the reaction is completed, quench with saturated Na2S2O3 solution, concentrate the product by rotary evaporation, extract with ethyl acetate, wash twice with brine, combine the organic phases, add MgSO4 for drying, evaporate the solvent, and purify by column chromatography (volume ratio of CH2Cl2:MeOH is 10:1) to obtain a yellowish-white solid 5-iodo-2'-deoxy-2'-fluorouridine (79%, 2.40 g).

[0085] 2. Synthesis of 2-(4-Ethynylphenyl)-1,2,2-triphenylethylene

[0086] Under ice bath conditions, dissolve diphenylmethane (3.7 g, 22 mmol, 1.1 equiv.) in anhydrous THF (40 mL), and slowly add n-butyllithium (n-BuLi, 13.75 mL) dropwise, and stir for 1 h. Subsequently, slowly add THF (10 mL) containing 4-bromobenzophenone (5.222 g, 20 mmol, 1 equiv.) to the reaction system, and stir at room temperature for 6 h. After the reaction is complete, quench the reaction with saturated NH4Cl solution. Then extract the mixed solution with dichloromethane, combine the organic phases, wash with water and brine, dry with anhydrous Na2SO4, evaporate the solvent to obtain a white crude product. Dissolve the crude product and a catalytic amount of p-toluenesulfonic acid (PTSA) in toluene (20 mL), heat under reflux overnight, concentrate the reactant, and purify by column chromatography (PE) to obtain a white solid 1-(4-bromophenyl)-1,2,2-triphenylethylene (5.58 g, 68%).

[0087] Under nitrogen protection, PdCl2(PPh3)2 (70.2 mg, 0.1 mmol), CuI (38.1 mg, 0.2 mmol), triphenylphosphine (78.7 mg, 0.3 mmol) and 1-(4-bromophenyl)-1,2,2-triphenylethylene (2.057 g, 5 mmol, 1 equiv.) were added to a flask, and then 100 mL of a mixed solution of THF / Et3N / piperidine (volume ratio 5:4:1) was added. After the catalyst was completely dissolved, trimethylsilylacetylene (0.85 mL, 6 mmol, 1.2 equiv.) was injected into the flask, and the mixture was stirred overnight at 50 °C. After the reaction was monitored by TLC to be complete, the formed solid was filtered off, the solvent was evaporated, and the residue was extracted with ethyl acetate, washed with water and brine, the organic phases were combined, and anhydrous Na2SO4 was added for drying. The solvent was evaporated, and the crude product was purified by column chromatography (PE) to obtain a pale yellow solid 1-[4-(2-trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene (1.41 g, 65.9%).

[0088] 1-[4-(2-Trimethylsilylethynyl)phenyl]-1,2,2-triphenylethylene (1.28 g, 3 mmol, 1 equiv.) and tetrabutylammonium fluoride (1.57 g, 6 mmol, 2 equiv.) were dissolved in 50 mL of THF, and the reaction was stirred at room temperature for 2 h. The crude product was purified by column chromatography (PE) to obtain a white solid 1-(4-ethynylphenyl)-1,2,2-triphenylethylene (0.94 g, 87.9%).

[0089] 3. Synthesis of Fluorescent Probe (TPE-FdU)

[0090] 2-(4-Ethynylphenyl)-1,2,2-triphenylethylene (0.428 g, 1.2 mmol, 2 equiv.), 5-iodo-2'-deoxy-2'-fluorouridine (0.223 g, 0.60 mmol, 1 equiv.), Pd(PPh3)4 (0.069 g, 0.06 mmol), and CuI (0.022 g, 0.12 mmol) were dissolved in 10 mL of DMF, 0.3 mL of Et3N was added, and the mixture was stirred overnight at room temperature. After the crude product was evaporated to dryness, it was separated and purified by column chromatography (volume ratio of PE:EA 1:1) to obtain a pale yellow solid TPE-FdU (0.227 g, 63%).

[0091] Test 1

[0092] The fluorescent probe was dissolved in different solvents for UV-visible absorption testing.

[0093] Test 2

[0094] The fluorescence intensity of the fluorescent probe was tested using RNAs at different concentrations.

[0095] The test results are as follows:

[0096] As Figure 1 , two absorption peaks appeared in the absorption spectra of TPE-FdU (5 μM) in methanol and DMSO solvents, respectively. In the DMSO solution, TPE-FdU had absorption at wavelengths of 250 nm and 335 nm. In the methanol solution, TPE-FdU had absorption at wavelengths of 215 nm and 335 nm.

[0097] As Figure 2 , as the concentration of RNA in the system increased, the fluorescence intensity of the whole system increased. And when the concentration of TPE-FdU was relatively low, strong fluorescence intensity could be generated, indicating that TPE-FdU has excellent fluorescence properties.

[0098] Example 2

[0099] Cell imaging of the TPE-FdU fluorescent probe (synthesized in Example 1)

[0100] 1. Cytotoxicity experiment

[0101] In the cultured cells, 100 μL of TPE-FdU solutions with different concentrations prepared with culture medium were added respectively, and the concentrations were: 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, 640 μM. Then they were placed in an incubator for 24 h. The old culture medium was discarded, 80 μL of new culture medium was added to each well, and then under light-shielded conditions, 20 μL of MTT solution with a concentration of 5 mg / mL was added to each well. They were incubated in the incubator for another 4 h. The liquid in the wells was aspirated, 150 μL of DMSO was added to each well, shaken on a shaker for 15 min, and the absorbance OD value was measured at 590 nm with an enzyme-labeling instrument. The cell survival rate (%) = OD of the drug-treated group / OD of the normal control group × 100%.

[0102] 2. Laser confocal imaging

[0103] (1) Different concentrations: When the cells grew to the logarithmic phase, they were digested with trypsin to prepare a cell suspension, and then the cell count was performed. The cell concentration was adjusted to 1×10 5Reserve at cell / mL. Add 2 mL of culture medium into a bottom glass culture dish, place it in the incubator for 15 min, aspirate the culture medium, and add 500 μL of cell-containing culture medium into the bottom well. Place it in the incubator for 2 h to allow the cells to settle and adhere to the wall. Carefully add 2 mL of cell-free culture medium to provide sufficient culture medium for the cells and at the same time reduce the change in osmotic pressure caused by water evaporation. Place it in the incubator for 24 h. Take out the culture dish, discard the old culture medium, carefully add 1 mL of PBS to wash the cells 3 times, and add 1 mL of TPE-FdU solutions with different concentrations (5 μM, 10 μM, 20 μM) prepared with the culture medium in advance to the three culture dishes respectively, and incubate for 4 h. Perform confocal imaging under 405 nm excitation light. The experiment was repeated three times.

[0104] (2) Different times: The cell seeding and culture steps are the same as above. Culture for 24 h, discard the old culture medium, carefully add 1 mL of PBS to wash the cells 3 times, add the same concentration of TPE-FdU solution (10 μM) to the four culture media respectively, and incubate for 30 min, 2 h, 4 h, and 6 h respectively. Perform confocal imaging under 405 nm excitation light. The experiment was repeated three times.

[0105] 3. Co-localization experiment

[0106] The cell seeding and culture steps are the same as above. Culture for 24 h, discard the old culture medium, carefully add 1 mL of PBS to wash the cells 3 times, add the same concentration of TPE-FdU solution (10 μM) to the two culture dishes, incubate for 4 h, carefully add 1 mL of PBS to wash the cells 3 times, add the commercial mitochondrial fluorescent probe Mito-Tracker Green and the commercial lysosomal fluorescent probe Lyso-Tracker Green respectively, incubate for 30 min, and then wash the cells 3 times with 1 mL of PBS solution and add 1 mL of PBS solution to the culture dish. Perform confocal imaging under 405 nm and 488 nm excitation lights. The experiment was repeated three times.

[0107] Test 3

[0108] Detection of the cytotoxicity of this fluorescent probe.

[0109] Test 4

[0110] Laser confocal imaging of this fluorescent probe.

[0111] Test 5

[0112] Co-localization detection of this fluorescent probe with commercial mitochondria and lysosomes.

[0113] The test results are as follows:

[0114] As Figure 3, when the concentration of TPE-FdU is 5 - 20 μM, the cell survival rate is very high. When the concentrations are 40 μM and 80 μM, the cell survival rate is also above 90%. However, when the compound concentration rises to 160 μM, the cell survival rate significantly decreases, indicating that cells are not suitable for survival at this drug concentration. When the concentration rises to 320 μM, the cell survival rate has dropped below 50%, indicating that TPE-FdU has a cytotoxic effect on cells.

[0115] As Figure 4 , A is the imaging of the bright field, B is the imaging of the blue channel, C is the fused imaging of the bright field and the blue channel. The TPE-FdU fluorescent probe shows obvious cell imaging effects at E x = 405 nm, and the imaging is outside the cell nucleus.

[0116] As Figure 5 , when the concentration of TPE-FdU is 5 μM, cell imaging can be carried out, and with the increase of the compound concentration, the fluorescence effect of cell imaging also becomes stronger.

[0117] As Figure 6 , when using a lower concentration of TPE-FdU to co-incubate with NIH-3T3 cells for 30 min, the cells are already stained. As the incubation time becomes longer, the fluorescence intensity becomes stronger and the imaging effect becomes clearer. Thus, it can be seen that TPE-FdU is a very sensitive and strong-fluorescence-producing fluorescent probe, showing excellent performance in cell imaging.

[0118] As Figure 7 and Figure 8 , using commercial lysosome and mitochondrial probes to co-stain NIH-3T3 cells with TPE-FdU, Figure 7 - Figure 8 In, A is the imaging of the blue channel, B is the imaging of the green channel, C is the fused imaging of the blue and green channels. It can be found that the Pearson correlation coefficient Pr between the lysosome fluorescent probe Lyso-Tracker Green (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) and TPE-FdU is only 62%, but the Pearson correlation coefficient Pr between the mitochondrial fluorescent probe Mito-Tracker Green (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) and TPE-FdU is as high as 85%, indicating that the TPE-FdU fluorescent probe can be localized in the mitochondria of NIH-3T3 cells, and compared with the co-localization experiment with lysosomes, it shows that the TPE-FdU fluorescent probe has a certain specificity for targeting cell mitochondria.

[0119] Example 3

[0120] pH response of the TPE-FdU fluorescent probe (synthesized in Example 1)

[0121] A series of solutions with different pH values ranging from 2 to 12 were prepared using sodium hydroxide and hydrochloric acid solutions. 10 μL of the TPE-FdU solution (1 mM) with the same concentration was added to 1 mL of the solutions with different pH values, and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0122] Test 6

[0123] pH spectral titration diagram of TPE-FdU.

[0124] The test results are as follows:

[0125] As Figure 9 , the fluorescence intensity reached the peak at pH = 4, and significant fluorescence quenching occurred after pH = 9.5.

[0126] Example 4

[0127] Ion response of the TPE-FdU fluorescent probe (synthesized in Example 1)

[0128] 1. Ion response experiment

[0129] An appropriate amount of compounds containing different metal ions was weighed and dissolved in water to prepare stock solutions of 10 mM different ions, which were then diluted to 100 μM. 10 μL of the TPE-FdU solution (1 mM) with the same concentration was added to 1 mL of the solutions with different ions respectively, and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0130] 2. Ion sensitivity experiment

[0131] A series of mercury ion solutions with different concentrations ranging from 0.01 μM to 100 μM were prepared. 10 μL of the TPE-FdU solution (1 mM) with the same concentration was added to 1 mL of the solutions containing different mercury ion concentrations, and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0132] 3. Ion selectivity experiment

[0133] 500 μL of the solution with different interfering ions (100 μM) was added with 500 μL of the mercury ion solution (100 μM) and 10 μL of the TPE-FdU solution (1 mM) with the same concentration, and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0134] Test 7

[0135] Metal ion selectivity diagram of TPE-FdU.

[0136] Test 8

[0137] Mercury ion sensitivity diagram of TPE-FdU.

[0138] Test 9

[0139] Mercury ion selectivity graph of TPE-FdU.

[0140] The test results are as follows:

[0141] As Figure 10 , when mercury ions are present in the solution, the fluorescence intensity of TPE-FdU is significantly quenched.

[0142] As Figure 11 , when the concentration of mercury ions is 0.01 μM, the fluorescence intensity of TPE-FdU has been effectively quenched, and as the concentration of mercury ions increases, the fluorescence intensity weakens more. When the concentration of mercury ions is 100 μM, the fluorescence intensity decays to 2%, indicating that low concentrations of mercury ions can cause quenching of the fluorescence intensity of TPE-FdU. That is to say, this probe has high sensitivity as a mercury ion indicator.

[0143] As Figure 12 , when both mercury ions and other metal ions are present in the solution, the fluorescence intensity of TPE-FdU is also significantly quenched, indicating that this probe has high selectivity for mercury ions and is not easily interfered by other metal ions.

[0144] In summary, a fluorescent probe provided by the present invention, its preparation method and application. The fluorescent probe has strong fluorescence efficiency, and when using a low concentration of the fluorescent probe to co-incubate with NIH-3T3 cells, cell imaging can be performed in only 30 minutes, and it can specifically bind to mitochondria. The design of the fluorescent probe in the present invention well solves the interference of poor biocompatibility. At the same time, under the influence of pH, the fluorescence intensity of the fluorescent probe will change accordingly, so the change of environmental pH can be effectively monitored. In addition, the fluorescent probe can also sensitively detect the presence of mercury ions in the environment. The preparation method of the fluorescent probe of the present invention is simple and efficient. The fluorescent probe of the present invention has good selectivity, high biocompatibility, and convenient use of related instruments. It is a detection method with relatively high sensitivity in current chemiluminescence detection methods and has important potential application value.

[0145] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all these improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. Use of a fluorescent probe in pH detection for non-disease diagnosis or treatment purposes, the structural formula of the fluorescent probe is as follows:

2. Use of a fluorescent probe in the detection of mercury ions for non-disease diagnosis or treatment purposes, and the structural formula of the fluorescent probe is as follows:

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