Fluorescent probe, preparation method and biological application thereof

Abstract

The invention discloses a fluorescent probe as well as a preparation method and biological application thereof. The fluorescent probe disclosed by the invention is simple in synthesis method, cheap in raw material and easy to repeat; the invention relates to the technical field of material synthesis and biological imaging analysis, in particular to a lysosome biosensor, which can realize real-time imaging of living cell lysosome, has high specificity, is positioned in the cell lysosome, is insensitive to pH and polarity response, has strong photobleaching resistance, high biocompatibility and small cytotoxicity, and relates to the technical field of material synthesis and biological imaging analysis. The fluorescent probe as well as the preparation method and the biological application thereof have lysosome targeting specificity, are insensitive to response of pH and polarity, are relatively strong in photobleaching resistance and high in biocompatibility, and basically have no toxicity to cells. Therefore, the fluorescent probe disclosed by the invention is particularly suitable for real-time imaging of the living cell lysosome.

Description

Technical Field

[0001] The invention relates to the technical field of material synthesis and biological imaging analysis, and in particular to a fluorescent probe for locating lysosomes, a preparation method thereof and biological applications thereof. Background Art

[0002] Organelles enable spatial clustering of molecules, thus facilitating their interactions in microenvironments that are well suited for specific complex functions. A well-known function of lysosomes is to degrade and recycle cellular waste products. Extracellular material reaches lysosomes primarily through endocytosis and phagocytosis. Material that reaches lysosomes is degraded by lysosomal hydrolases, and the resulting breakdown products are used to generate new cellular components and energy in response to the nutritional needs of the cell. Lysosomes also participate in an “unconventional” secretory pathway called lysosomal exocytosis, which plays an important role in various physiological processes such as plasma membrane repair, immune response, and bone resorption. The lysosomal lumen has an acidic pH close to 4.5 and contains approximately 60 different soluble hydrolases that are directly involved in the degradation of metabolites. The lysosomal membrane contains proteins such as transporters, ion channels, and SNAREs that mediate different aspects of lysosomal function, as well as the vATPase complex that mediates lysosomal acidification. Notably, lysosomes have also been found to be involved in various common diseases, such as neurodegenerative diseases, infections, obesity, and cancer. Therefore, lysosomes are considered to be important targets for disease diagnosis and treatment, and real-time imaging analysis of their fine structural changes is of great significance for the diagnosis and treatment of lysosomal stress-related diseases.

[0003] At present, organic small molecule imaging has become a hot research topic due to its high biocompatibility, strong resistance to photobleaching, and real-time monitoring of intracellular reactions. It has been applied to many fields such as cell imaging, chemical sensing, and photodynamic therapy. Although various fluorescent probes for observing lysosomal morphology and detecting its components have been reported, the pH stability, photobleaching, and biocompatibility of the currently developed lysosomal fluorescent probes still need to be improved. Therefore, it is very important to develop fluorescent probes for real-time imaging of lysosomes in living cells. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies in the prior art, the present invention provides a fluorescent probe, a preparation method and biological applications thereof, which solve the technical problems of poor photobleaching resistance, low biocompatibility and unstable polarity of the prior fluorescent probes. The present invention provides a fluorescent probe for real-time imaging of lysosomes in living cells, a preparation method of the fluorescent probe and applications of the fluorescent probe in biology.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a fluorescent probe, whose chemical structure is as follows:

[0008]

[0009] In one aspect, the present invention also provides a method for preparing a fluorescent probe, which specifically comprises the following steps:

[0010] S1. Add 1-phenylbutane-1,3-dione and boron trifluoride into a round-bottom flask, use toluene as solvent, and react under heating in a water bath to obtain a white precipitate, namely, intermediate 1;

[0011] S2, intermediate 1 and 6-hydroxy-2-naphthaldehyde were mixed and added to a 100 mL three-necked round-bottom flask. A double-row tube was used to evacuate and blow nitrogen three times, and a nitrogen balloon was inserted. Piperidine was added using a syringe under nitrogen environment, and then 40 mL of solvent toluene was added using a syringe. Nitrogen was blown for 30 minutes, and the reaction device was placed in a water bath and heated under reflux to obtain a red precipitate, namely the fluorescent probe;

[0012] S3, filtering the red precipitate obtained in step S2, and washing with ethanol for multiple times to obtain a red solid;

[0013] S4, dissolving the red solid obtained in step S3 with dichloromethane, purifying with column chromatography to obtain a red solution, concentrating the red solution by rotary evaporation, and vacuum drying to obtain a red solid powder, i.e., the fluorescent probe.

[0014] Preferably, in step S1, the molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1:1, the reaction temperature is 80° C., and the reaction time is 10 hours.

[0015] Preferably, in step S2, the molar ratio of intermediate 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1, the reaction temperature is 75° C., and the reaction time is 17 hours.

[0016] Preferably, the eluate during the column chromatography purification process in step S4 is a mixture of petroleum ether and ethyl acetate, and the volume ratio is: V 石油醚 :V 乙酸乙酯 =2:1.

[0017] Preferably, the red precipitate obtained in step S3 is cooled to room temperature and washed with ethanol 2-3 times during suction filtration to obtain a red solid.

[0018] Preferably, in step S2, the amount of piperidine added using a syringe under a nitrogen environment is 0.16 g, 1.86 mmol.

[0019] On the other hand, the present invention also provides an application of a fluorescent probe prepared by the method for preparing a fluorescent probe in real-time imaging of lysosomes in living cells.

[0020] (III) Beneficial effects

[0021] The present invention provides a fluorescent probe, a preparation method and biological application thereof. Compared with the prior art, it has the following beneficial effects:

[0022] (1) The fluorescent probe, preparation method and biological application thereof: The fluorescent probe of the present invention has a simple synthesis method, cheap raw materials and is easy to repeat; and can realize real-time imaging of lysosomes in living cells.

[0023] (2) The fluorescent probe, preparation method and biological application thereof have high specificity, are localized in cell lysosomes, are insensitive to pH and polarity, have strong resistance to photobleaching, high biocompatibility and low cytotoxicity.

[0024] (3) The fluorescent probe, preparation method and biological application thereof have lysosomal targeting specificity, are insensitive to pH and polarity, have strong resistance to photobleaching, high biocompatibility and are substantially non-toxic to cells. Therefore, the fluorescent probe of the present invention is particularly suitable for real-time imaging of lysosomes in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a simplified schematic diagram of the synthesis of the fluorescent probe of the present invention;

[0026] Figure 2 is the mass spectrum of the fluorescent probe of Example 1 of the present invention;

[0027] Figure 3 Schematic diagram of UV-visible spectrum and excitation emission spectrum of the fluorescent probe in Example 4 of the present invention in PBS buffer (0.01M, pH=7.4);

[0028] Figure 4 is a schematic diagram of the UV-visible spectrum and excitation emission spectrum of the fluorescent probe in Example 5 of the present invention in solvents of different polarities;

[0029] Figure 5 is a schematic diagram of the UV-visible spectrum and excitation emission spectrum of the fluorescent probe at different pH values ​​in Example 5 of the present invention;

[0030] Figure 6 is the ratio of the fluorescence intensity of the fluorescent probe at different times to the fluorescence intensity at the initial time under 460 nm laser excitation in Example 5 of the present invention;

[0031] Figure 7This is a schematic diagram of the selective analysis of various analytical substrates contained in cells by the fluorescent probe in Example 6 of the present invention; 1.1mM Cu2SO4, 2.1mM H2S, 3.1mM FeCl3, 4.1mM FeCl2, 5.1mM ZnCl2, 6.1mM MgCl2, 7.1mM NaCl, 8.1mM CaCl2, 9.1mM KCl, 10. Glutathione, 11. Threonine, 12. Cysteine, 13. Glycine, 14. Serine, 15. Glucose, 16. RNA.

[0032] Figure 8 is a schematic diagram of the fluorescent probe cytotoxicity analysis in Example 7 of the present invention;

[0033] Fig. 9 This is a schematic diagram of real-time imaging of the fluorescent probe entering cells in Example 7 of the present invention;

[0034] Fig.10 is a schematic diagram of real-time imaging of fluorescent probes of different concentrations entering cells in Example 7 of the present invention;

[0035] Fig.11 is a schematic diagram of co-localization imaging analysis of fluorescent probes in HeLa in Example 7 of the present invention;

[0036] Fig.12 This is the hydrogen spectrum of the fluorescent probe of Example 1 of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The raw materials in the following examples are described:

[0039] 1-Phenylbutane-1,3-dione, boron trifluoride, 6-hydroxy-2-naphthaldehyde, toluene, ethanol, piperidine, PBS buffer (0.01 M, pH = 7.4), dimethyl sulfoxide, methanol, dimethylformamide, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, Cu2SO4, H2S, FeCl3, FeCl2, ZnCl2, MgCl2, NaCl, CaCl2, and KCl were all purchased from Aladdin Reagent Shanghai Co., Ltd.

[0040] Glutathione, threonine, cysteine, glycine, serine, glucose, and RNA were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.

[0041] See also Figure 1-12 , a fluorescent probe, preparation method and biological application thereof specifically include the following embodiments:

[0042] Example 1: Preparation of fluorescent probe

[0043] The fluorescent probe of the present invention is a fluorescent probe for real-time imaging of lysosomes in living cells. The fluorescent probe is obtained by reacting 1-phenylbutane-1,3-dione, boron trifluoride and 6-hydroxy-2-naphthaldehyde as raw materials. The specific preparation process is shown in Example 2.

[0044] The fluorescent probe of the present invention has lysosome targeting specificity, strong anti-photobleaching performance, is substantially non-toxic to cells, has high biocompatibility, and is insensitive to pH and polarity responses. Therefore, the fluorescent probe of the present invention is particularly suitable for real-time imaging of lysosomes in living cells.

[0045] Example 2: Preparation of fluorescent probe

[0046] (1) Please refer to Figure 1 , which is a simplified schematic diagram of the synthesis of the fluorescent probe of the present invention. The preparation method of the fluorescent probe comprises the following steps:

[0047] 1-Phenylbutane-1,3-dione and boron trifluoride were added to a round-bottom flask, and toluene was used as a solvent. The reaction was carried out under heating in a water bath to obtain a white precipitate, namely, intermediate 1. Intermediate 1 was then mixed with 6-hydroxy-2-naphthaldehyde, and piperidine (0.16 g, 1.86 mmol) was added using a syringe under a nitrogen environment, followed by the addition of 40 mL of solvent toluene using a syringe; nitrogen was blown for 30 minutes, and the reaction device was placed in a water bath and heated under reflux to obtain a red precipitate, namely, the fluorescent probe.

[0048] The molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1: 1. The reaction is carried out in a water bath, the reaction temperature is preferably 80° C., and the reaction time is preferably 10 hours.

[0049] The molar ratio of intermediate product 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1. For example, if 0.2 g of 6-hydroxy-2-naphthaldehyde is used, 0.37 g of intermediate product 1 is required. The reaction is carried out in a water bath, the reaction temperature is preferably 75° C., and the reaction time is preferably 17 hours.

[0050] (2) The red precipitate obtained in step (1) is filtered and washed with ethanol several times to obtain a red solid.

[0051] (3) The red solid obtained in step (2) is dissolved in dichloromethane and purified by column chromatography to obtain a red solution. The obtained orange solution is concentrated by rotary evaporation and vacuum dried to obtain a red solid powder, i.e., the fluorescent probe.

[0052] The eluent during column chromatography purification is a mixture of petroleum ether and ethyl acetate (V 石油醚 :V 乙酸乙酯 =2:1)

[0053] Example 3: Preparation of fluorescent probe

[0054] The preparation method of the fluorescent probe of Example 2 is described in detail by way of example in this example.

[0055] 0.2g 1-phenylbutane-1,3-dione and 0.082g boron trifluoride were added to a round-bottom flask, with toluene as solvent, and the mixture was reacted for 10 hours under water bath heating to obtain a white precipitate, which was cooled to room temperature and filtered to obtain a white precipitate, namely intermediate product 1. 0.37g intermediate 1 was then mixed with 0.2g 6-hydroxy-2-naphthaldehyde, and piperidine (0.16g, 1.86mmol) was added using a syringe under a nitrogen environment, followed by the addition of 40mL of solvent toluene using a syringe; nitrogen was blown for 30 minutes, and a double-row tube was used to evacuate and blow nitrogen three times, and a nitrogen balloon was inserted; the reaction device was placed in a water bath and heated under reflux for 17 hours to obtain a red precipitate, namely the fluorescent probe. The red precipitate was naturally cooled to room temperature, filtered, and the filter cake was washed with ethanol several times to obtain a red solid. The red solid obtained by filtration was dissolved in dichloromethane and purified by column chromatography. The eluate was a mixture of petroleum ether and ethyl acetate (V 石油醚 :V 乙酸乙酯 =2:1) ​​to obtain a red solution, which is concentrated by rotary evaporation and then vacuum dried to obtain a red solid powder, i.e., the fluorescent probe.

[0056] For the characterization of the fluorescent probe prepared in this example, please refer to Figure 2 and Fig.12

[0057] Example 4: UV-Visible Spectra and Excitation-Emission Spectra of Fluorescent Probes

[0058] See also Figure 3 ,like Figure 3 As shown in a, the fluorescent probe has a maximum absorption peak at 465nm in PBS buffer (0.01M, pH=7.4). Figure 3 b, When the excitation wavelength is 460 nm in PBS buffer, the fluorescent probe has the maximum fluorescence intensity at 650 nm.

[0059] Embodiment 5:

[0060] In this embodiment, various investigations were conducted on the fluorescent probes in the above embodiments.

[0061] 1. Investigation of the polarity response of fluorescent probes

[0062] 20 μL of 10 -3 M small molecule fluorescent probe, prepared to obtain fluorescent probe solutions with the same concentration (10μM) in solvents of different polarity. Please refer to 4a. In the absorption range of 300nm-700nm, the UV-visible absorption spectrum of the small molecule fluorescent probe does not change with the solvent polarity, indicating that the small molecule fluorescent probe has no obvious solvation effect and is a polarity-insensitive molecule. It also further proves that the fluorescent probe is a small molecule with good stability. Please refer to Figure 4 b. When the excitation is 460nm, it can be seen that the small molecule fluorescent probe shows stronger fluorescence intensity in different solvents than in PBS buffer, but the fluorescence emission spectrum does not move with the change of solvent polarity and does not show polarity dependence, which further supports the results of UV-visible absorption spectrum. The results of the measurement of solvent effect by UV-visible absorption spectrum and fluorescence spectrum show that the small molecule fluorescent probe is a molecule with no obvious solvent effect and is relatively stable.

[0063] 2. Investigation of the response of fluorescent probes to different pH values

[0064] Different proportions of sodium hydroxide solution and sulfuric acid solution were used to prepare buffer solutions of different pH values ​​(pH4, pH4.5, pH5, pH5.5, pH6, pH6.5, pH7, pH7.5, pH8, pH8.5, pH9, pH9.5, pH10). 2 mL of buffer solutions of different pH values ​​were added to 20 μL of 10 -3 M small molecule fluorescent probe, prepared with small molecule fluorescent probe solutions containing the same concentration (10μM) at different pH. Figure 5 a. The UV-visible absorption spectra of the small molecule fluorescent probe in different pH conditions do not show any pH-dependent changes, indicating that the small molecule fluorescent probe has no obvious pH effect and is a pH-insensitive molecule. This also further proves that the fluorescent probe is a small molecule with good stability. Figure 5 b. Under room temperature conditions, by exciting with a 460nm laser light source, we can see that the absorption intensity of the small molecule fluorescent probe is different at different pH values, but it does not show polarity dependence, which further supports the results of the UV-visible absorption spectrum. The results of the measurement of the pH effect by UV-visible absorption and fluorescence spectra show that the small molecule fluorescent probe is a relatively stable molecule with no obvious pH effect.

[0065] 3. Photostability experiment of fluorescent probe

[0066] See also Figure 6, Figure 6 It is the ratio of the fluorescence intensity of the small molecule probe after continuous ultraviolet light (365nm) irradiation for 5min, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, and 100min to the initial fluorescence intensity of the probe. There is no obvious decrease in the fluorescence intensity, which indicates that the small molecule fluorescent probe has excellent photostability.

[0067] Example 6: Probe selectivity exploration

[0068] Fluorescence response of the fluorescent probe prepared in Test Example 1 to common substances in organisms

[0069] In order to test the selectivity of fluorescent probes for biological molecules contained in cells, such as inorganic salts, amino acids, peptides, and other proteins or enzymes, fluorescent probes (10μM) were added to PBS buffer solution (0.01M, pH 7.4), and then common biological molecules in cells, such as Cu2SO4, H2S, FeCl3, FeCl2, ZnCl2, MgCl2, NaCl, CaCl2, KCl, glutathione, threonine, cysteine, glycine, serine, glucose, and RNA solutions were added to test the changes in fluorescence intensity after reaction with the probes. Please refer to Figure 7 ,Depend on Figure 7 It can be seen that under 460nm excitation, the fluorescence intensity of the fluorescent probe in PBS buffer solution (0.01M, pH 7.4) containing different biomolecules does not change significantly, indicating that the probe has good stability in vivo.

[0070] Example 7: Application of probes in cell imaging

[0071] 1. Cytotoxicity Analysis

[0072] Mouse embryonic fibroblasts (3T3-L1) and human cervical cancer cells (HeLa) were cultured at 1×10 4 Cells were inoculated into a 96-well plate, and the 96-well plate was placed in a cell culture incubator under the following culture conditions: 37°C, 5% CO2, saturated humidity, and cultured for 24 hours to allow them to completely adhere to the wall. After that, fresh culture medium was replaced, and 10 μL of different concentrations of fluorescent probe dispersion was added. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well, and the incubation continued for 4 hours. Then, 100 μL of DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance (OD value) of the solution in each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD 待测组 -OD 空白组 ) / (OD 细胞组 -OD空白组 ) × 100%. Please refer to Figure 8 ,from Figure 8 As can be seen from the figure, the fluorescent probe has almost no cytotoxicity in the range of 0-60 μM.

[0073] 2. Real-time imaging of the process of fluorescent probes entering cells.

[0074] Human cervical cancer cells (HeLa) were seeded into glass-bottomed culture dishes and cultured for 2 days at 37°C, 5% CO2, and saturated humidity. Subsequently, the probe (5 μM) was added to the cells and imaged using a laser confocal microscope. Fig. 9 As shown, when the excitation wavelength is 470nm and the emission wavelength is 490nm-650nm, the fluorescent probe begins to enter the cell within one minute, and the fluorescence intensity reaches the highest after 30 minutes.

[0075] 3. Real-time imaging of different concentrations of fluorescent probes entering cells.

[0076] Human cervical cancer cells (HeLa) were inoculated into glass-bottomed culture dishes and cultured for 2 days at 37°C, 5% CO2, and saturated humidity. Subsequently, small molecule fluorescent probes of different concentrations (0.5 μM, 1 μM, 2 μM, 4 μM, and 5 μM) were added to the HeLa cells, incubated in an incubator for 30 minutes, and then imaged using a laser confocal microscope. Fig.10 As shown, when the excitation wavelength is 470nm and the emission wavelength is 490nm-650nm, there is weak fluorescence in the cells when the concentration of the fluorescent probe is 0.5μM. As the concentration of the probe increases, the fluorescence intensity gradually increases.

[0077] IV. Intracellular Colocalization Imaging

[0078] In the co-localization experiment, human cervical cancer cells (HeLa) were incubated with fluorescent probes (5μM excitation wavelength 470nm, emission wavelength 490-590) for 30min, and then incubated with 1μM lysosomal stain (Lyso-Tracker Red DND-99 excitation wavelength 577nm, emission wavelength 590nm-630nm) and 1μM mitochondrial stain (Mito-Tracker Deep Red excitation wavelength 644nm, emission wavelength 660nm-690nm) reagents for 10 minutes. Finally, the cells were washed twice with PBS buffer solution (0.01M pH 7.4) and imaged with a laser confocal microscope. The results are shown in Figure 2. Fig.11 As shown, the Pearson overlap coefficient of the green channel of the fluorescent probe and the red channel of the lysosome commercial dye is 88.82%, and the Pearson overlap coefficient with the mitochondria is 62%, proving that it is well localized in the lysosome organelles.

[0079] In summary, the fluorescent probe of the present invention is a kind of real-time imaging for living cell lysosomes. The fluorescent probe of the present invention can be selectively located in the cell lysosome. The fluorescent probe of the present invention overcomes the photobleaching of ordinary lysosomal probes, and at the same time, other lysosomal fluorescent probes imaging bring serious cytotoxicity, biocompatibility, polarity sensitivity, pH sensitivity and other problems.

[0080] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0081] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fluorescent probe, characterized in that: Its chemical structure is as follows:

2. The method for preparing a fluorescent probe according to claim 1, characterized in that: The specific steps include: S1. Add 1-phenylbutane-1,3-dione and boron trifluoride into a round-bottom flask, use toluene as solvent, and react under heating in a water bath to obtain a white precipitate, namely, intermediate 1; S2, intermediate 1 and 6-hydroxy-2-naphthaldehyde were mixed and added to a 100 mL three-necked round-bottom flask. A double-row tube was used to evacuate and blow nitrogen three times, and a nitrogen balloon was inserted. Piperidine was added using a syringe under nitrogen environment, and then 40 mL of solvent toluene was added using a syringe. Nitrogen was blown for 30 minutes, and the reaction device was placed in a water bath and heated under reflux to obtain a red precipitate, namely the fluorescent probe; S3, filtering the red precipitate obtained in step S2, and washing with ethanol for multiple times to obtain a red solid; S4, dissolving the red solid obtained in step S3 with dichloromethane, purifying with column chromatography to obtain a red solution, concentrating the red solution by rotary evaporation, and vacuum drying to obtain a red solid powder, i.e., the fluorescent probe.

3. The method for preparing a fluorescent probe according to claim 2, characterized in that: In step S1, the molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1:1, the reaction temperature is 80° C., and the reaction time is 10 hours.

4. The method for preparing a fluorescent probe according to claim 2, characterized in that: In step S2, the molar ratio of intermediate 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1, the reaction temperature is 75° C., and the reaction time is 17 hours.

5. The method for preparing a fluorescent probe according to claim 2, characterized in that: The eluate during column chromatography purification in step S4 is a mixture of petroleum ether and ethyl acetate, and the volume ratio is: V 石油醚 :V 乙酸乙酯 =2:

1.

6. The method for preparing a fluorescent probe according to claim 2, characterized in that: The red precipitate obtained in step S3 is cooled to room temperature and washed with ethanol 2-3 times during suction filtration to obtain a red solid.

7. The method for preparing a fluorescent probe according to claim 2, characterized in that: In the step S2, the amount of piperidine added using a syringe under a nitrogen environment is 0.16 g, 1.86 mmol.

8. Use of a fluorescent probe prepared by the method for preparing a fluorescent probe according to any one of claims 2 to 7 in real-time imaging of lysosomes in living cells.

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