Lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, and preparation method and application of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe

Abstract

The invention provides a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole as well as a preparation method and application thereof, and relates to the field of fluorescent probes. The preparation method of the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole comprises the following steps: a primary reaction and a secondary reaction. The lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole disclosed by the invention can simultaneously monitor two organelles (lipid droplet and lysosome) in a targeting manner, and in a HeLa cell imaging experiment, the cytotoxicity is low, and cells can be accurately positioned; in addition, effective response to intracellular viscosity can be realized while good polar response is realized, and the response effect is good.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, and in particular to a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, a preparation method and an application thereof. Background Art

[0002] Intracellular viscosity is an important microenvironment parameter in various biological systems, and abnormal fluctuations in viscosity are closely related to many diseases. Intracellular viscosity plays an important role in cell physiological and pathological processes, and it can play an important role in material transport, signal transduction, organelle function, etc. Abnormal changes in intracellular viscosity can cause many hazards, such as cell dysfunction, metabolic disorders, cancer, neurodegenerative dysfunction, and metabolic diseases. Therefore, the detection and regulation of intracellular viscosity are of great significance for maintaining normal functions and preventing diseases.

[0003] Fluorescent probes have been increasingly used in clinical medical experiments due to their advantages such as accurate in situ real-time imaging, good biocompatibility, low hazard and short reaction time. Therefore, the development of functional fluorescent probes that respond to viscosity will help us to deeply understand the relationship between changes in intracellular viscosity and disease development, and provide new ideas for the diagnosis and treatment of diseases.

[0004] Lipid droplets are lipid-rich subcellular organelles composed of a neutral lipid core and surrounded by a phospholipid monolayer. They have important cellular tasks such as membrane transport, fusion, and participation in signal transduction pathways. Existing studies have found that lipid droplets play a key role in cellular lipid storage and supply, but abnormal levels of cellular lipid droplet content are closely related to many metabolic diseases. For example, obesity, cancer, diabetes, and inflammatory diseases.

[0005] Lysosomes are organelles that exist under acidic conditions. They are related to a variety of cell biological processes, including cell endocytosis, degradation reactions, and immune capabilities. Changes in acidic conditions within lysosomes can lead to cell functional disorders and cause many diseases. Therefore, it is important for organisms to accurately detect changes in the acidic environment of lysosomes.

[0006] As two important organelles in cells, lipid droplets and lysosomes are closely related to intracellular viscosity. In the prior art, the current subcellular viscosity fluorescent probes mainly use specific targeting groups for viscosity detection. For example: using morpholine groups to detect lysosomes alone. However, fluorescent probes that can effectively detect dual organelles (lipid droplets and lysosomes) at the same time and obtain changes in intracellular viscosity are relatively rare in the prior art. By using a fluorescent probe molecule to help monitor slight changes in two organelles (lipid droplets and lysosomes) at the same time, and understanding the interaction between the two organelles through it, it has important technical significance and research value. Therefore, providing a new dual organelle (lipid droplets and lysosomes) targeted viscosity-sensitive fluorescent probe is of great significance for better understanding organelle viscosity and its close relationship with related diseases.

[0007] Furthermore, although the prior art discloses dual-targeting probes for lipid droplets and lysosomes based on benzothiadiazole, they are limited to responding to polarity, and have poor response effects on intracellular viscosity, and cannot achieve effective response to viscosity. Summary of the invention

[0008] In order to solve the technical problems existing in the prior art, the present invention provides a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, which can simultaneously target and monitor two organelles (lipid droplets and lysosomes), and in the HeLa cell imaging experiment, the cytotoxicity is low and the cells can be accurately located; and while achieving a good polarity response, it can achieve an effective response to the intracellular viscosity, and the response effect is good. The present invention also provides a method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole. The present invention also provides an application of a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, the molecular structure is as follows: .

[0010] A method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, the synthetic route is as follows: .

[0011] The method for preparing the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole includes the following steps: a primary reaction, a secondary reaction; The method of the primary reaction is as follows: Compound 1 and Compound 2 are added into ethanol, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the mixture is kept warm and refluxed for reaction; after the reaction is completed, the mixture is purified to obtain Compound 3; The compound 1 is 4-(4-morpholinyl)acetophenone; the compound 2 is malononitrile; The compound 3 has the following molecular structure: ; The secondary reaction method is as follows: Compound 3 and Compound 4 are added into acetonitrile, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the mixture is kept warm and refluxed for reaction; after the reaction is completed, the mixture is purified to obtain a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole; The compound 4 has the following molecular structure: .

[0012] Preferably, in the primary reaction, the molar amount of compound 2 is controlled to be in excess relative to compound 1.

[0013] More preferably, in the one reaction, the molar amount of compound 2 is at least 1.2 times the molar amount of compound 1.

[0014] Preferably, in the one reaction, the molar amount of piperidine is at least 20% of the molar amount of compound 1; The molar amount of acetic acid is at least 20% of 1 mole of the compound.

[0015] Preferably, in the primary reaction, the insulation reflux reaction time is 3-6 hours.

[0016] Preferably, in the secondary reaction, the molar amount of compound 3 is controlled to be in excess relative to compound 4.

[0017] More preferably, in the secondary reaction, the molar amount of compound 3 is at least 1.15 times the molar amount of compound 4.

[0018] Preferably, in the secondary reaction, the molar amount of piperidine is at least 20% of the molar amount of compound 4; The molar amount of acetic acid is at least 20% of the molar amount of compound 4.

[0019] Preferably, in the secondary reaction, the heat preservation reflux reaction time is 10-13h.

[0020] An application of the aforementioned benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe in the sensing detection of lipid droplets and lysosomes in biological cells.

[0021] Furthermore, the sensing detection includes: fluorescence detection and cell molding.

[0022] An application of the aforementioned benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe in the detection of viscosity within biological cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe of the present invention can simultaneously target and monitor lipid droplets and lysosomes; and can achieve an effective response to intracellular viscosity while achieving a good polarity response, has a good response effect to cell viscosity, enters cells quickly, and has good stability.

[0024] (2) The preparation method of the benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe of the present invention has a wide source of raw materials, simple synthesis steps, low production cost, and is conducive to large-scale production.

[0025] (3) The benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe of the present invention has low cytotoxicity in cell imaging experiments and can image lipid droplets and lysosomes in cells.

[0026] (4) The benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe of the present invention has the characteristics of low cost, high sensitivity, good targeting effect on lipid droplets and lysosomes, and good viscosity response effect. It can be effectively applied in biological cells to obtain changes in intracellular viscosity by simultaneously targeting and monitoring two organelles (lipid droplets and lysosomes). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The probe BTD-CNAr-MOR prepared in Example 1 1 H NMR spectrum.

[0028] Figure 2 The probe BTD-CNAr-MOR prepared in Example 1 13 C NMR spectrum.

[0029] Figure 3 The UV absorption spectra of probe BTD-CNAr-MOR in different solvents.

[0030] Figure 4 Fluorescence spectra of probe BTD-CNAr-MOR in different solvents.

[0031] Figure 5 The fluorescence spectra of the probe BTD-CNAr-MOR in different ratios of glycerol and methanol.

[0032] Figure 6 The linear plot between the fluorescence intensity of the probe BTD-CNAr-MOR at 633 nm and log η.

[0033] Figure 7 The fluorescence spectra of the probe BTD-CNAr-MOR in PBS buffers of different pH values.

[0034] Figure 8 This is the selectivity test result of probe BTD-CNAr-MOR.

[0035] Fig. 9 This is the time stability test result of probe BTD-CNAr-MOR.

[0036] Fig.10 This is the MTT analysis diagram of the probe BTD-CNAr-MOR.

[0037] Fig.11 The lysosomal co-localization experiment diagram of the probe BTD-CNAr-MOR; in the figure, (a) is the red channel stained with BTD-CNAr-MOR (10μM) (λex=405nm, λem=620-670nm); (b) is the green channel stained with Lyso-Tracker Green (500nM) (λex=504nm, λem=500-550nm); (c) is the merged image of (a) and (b); (d) is the bright field of HeLa cells; (e) is the fluorescence co-localization curve of (a) and (b); (f) is the average fluorescence intensity diagram of (a) and (b).

[0038] Fig.12 The lipid droplet co-localization experiment diagram of the probe BTD-CNAr-MOR; (a) is the red channel stained with BTD-CNAr-MOR (10μM) (λex=405nm, λem=620-670nm); (b) is the green channel stained with BODIPY (500nM) (λex=493nm, λem=470-520nm); (c) is the merged image of (a) and (b); (d) is the bright field of HeLa cells; (e) is the fluorescence co-localization curve of (a) and (b); (f) is the average fluorescence intensity diagram of (a) and (b). DETAILED DESCRIPTION

[0039] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0040] The probe BTD-CNAr-MOR in each example represents a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, and the compound numbers in the examples correspond to the compound numbers in the synthesis route of the above invention.

[0041] Example 1 This embodiment provides a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole, which has the following molecular structure: .

[0042] This example also provides a method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole. The specific synthesis route is as follows: .

[0043] The specific steps are as follows: 1. Primary reaction (preparation of compound 3) At room temperature, compound 1 (4.105 g, 20 mmol) and compound 2 (1.58 g, 24 mmol) were dissolved in ethanol (20 mL); the temperature was raised to 100 ° C, and piperidine (0.21 g, 4 mmol) and acetic acid (0.24 g, 4 mmol) were added dropwise under reflux conditions for catalysis, and the reaction was refluxed for 4 hours; after the reaction was completed as monitored by TLC, a mixture of petroleum ether / ethyl acetate (volume ratio of 5:1) was used as an eluent, and the product was purified by silica gel chromatography to obtain compound 3 (3.201 g, yield 64%).

[0044] Wherein, compound 1 is 4-(4-morpholinyl)acetophenone, CAS No. 39910-98-0, which is purchased from commercial sources.

[0045] Compound 2 is malononitrile, CAS No. 109-77-3, and is purchased from commercial sources.

[0046] 2. Secondary reaction (preparation of BTD-CNAr-MOR) At room temperature, compound 3 (120 mg, 0.47 mmol) and compound 4 (100 mg, 0.4 mmol) were dissolved in acetonitrile (7 mL); the temperature was raised to 105 °C, and piperidine (10 μL) and acetic acid (5 μL) were added dropwise under reflux conditions for catalysis, and the reaction was refluxed for 12 h; after the reaction was completed as monitored by TLC, a mixture of petroleum ether / ethyl acetate (volume ratio of 5:1) was used as an eluent, and the product was purified by silica gel chromatography to obtain a benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR (84 mg, yield 43%).

[0047] Among them, compound 4 can be prepared by existing methods.

[0048] The specific preparation method of compound 4 in this example is referenced in: Wang Lin, Zhang Ning, He Yuan, et al. A Fluorescent Probe with Large Stokes Shift Based on Benzothiadiazole Scaffolds for Selective Detection of Hg 2+ in Lysosomes and ItsApplication in Biological Imaging. Chem AsianJ. 2024, e202401067. DOI:10.1002 / asia.202401067.

[0049] The specific preparation method of compound 4 is as follows: 7-bromo-4-formaldehydebenzo[C][1,2,5]thiadiazole (CAS No. 1071224-34-4) (121.0 mg, 5.0 mmol) was dissolved in a morpholine (5.0 mL) aqueous solution at 135°C and kept warm for 6 hours. After the reaction, it was purified by silica gel column (petroleum ether: ethyl acetate volume ratio 2:1) to obtain compound 4. The specific synthesis route is as follows: .

[0050] The lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole prepared by the above preparation method 1 H NMR spectrum Figure 1 As shown, 13 C NMR spectrum Figure 2 shown.

[0051] The molecular formula of the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole is: 26 H 24 N6O2S.

[0052] This embodiment also provides the application of the aforementioned benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR in the detection of lipid droplets and lysosomes in biological cells, so as to detect viscosity in biological cells.

[0053] Example 2 Spectral property test of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: First, the fluorescent probe BTD-CNAr-MOR was dissolved in DMSO to prepare a stock solution with a concentration of 1 mmol / L. By adding 20 μL of the stock solution to 2.0 mL of solvents of different polarities, a probe with a concentration of 10 μmol / L was obtained. This probe with a concentration of 10 μmol / L was used in all photophysical experiments, and UV absorption tests were performed in different solvents (ethyl acetate, 1,4-dioxane, ethanol, methanol, acetonitrile, dimethyl sulfoxide, pH=7 PBS buffer). Figure 3 As shown, it was found that its absorption peak is around 500nm.

[0054] Take 20 μL of the above stock solution and dissolve it in 2 mL of different solvents (ethyl acetate, 1,4-dioxane, ethanol, methanol, acetonitrile, dimethyl sulfoxide, pH=7 PBS buffer) for fluorescence testing. Test the fluorescence emission intensity at an excitation wavelength of 500 nm. Figure 4 As shown in the figure, it was found that the fluorescence intensity of the probe BTD-CNAr-MOR in fat-soluble solvents such as 1,4-dioxane and ethyl acetate was significantly stronger than that in water-soluble solvents such as methanol and ethanol. Therefore, it can be shown that it is a lipophilic sensitive fluorophore. Morpholine has been proven to be able to be used for lysosome targeting. The above data show that the probe BTD-CNAr-MOR can be used as a new polarity-sensitive probe for dual targeting of lipid droplets and lysosomes.

[0055] Example 3 Viscosity response test of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: 20 mL of the probe stock solution prepared in Example 2 was added to 2 mL of a glycerol / methanol mixed solvent of different volume ratios to test the sensitivity of the probe BTD-CNAr-MOR to viscosity detection. Figure 5 As shown in the figure, in pure methanol (0%), the probe BTD-CNAr-MOR has almost no fluorescence; in pure glycerol (100%), the maximum fluorescence emission peak of the probe BTD-CNAr-MOR appears at 630nm. The fluorescence intensity at 630nm in pure glycerol is about 100 times that in pure methanol. This shows that the probe BTD-CNAr-MOR is a fluorescent probe that recognizes viscosity.

[0056] According to the relevant literature, the viscosity parameter (logη) of the methanol / glycerol system from pure methanol to pure glycerol at room temperature was determined. In the viscosity range of 1-998 cP, the linear relationship between the fluorescence intensity value of the probe BTD-CNAr-MOR and the viscosity parameter (logI 650nm) and viscosity (logη) was plotted, as shown in Figure 2. Figure 6According to the Forster-Hoffmann equation, it was found that the probe BTD-CNAr-MOR had a good linear relationship, indicating that the probe BTD-CNAr-MOR can accurately and quantitatively detect the viscosity of the methanol / glycerol mixed system.

[0057] Example 4 pH stability, anti-interference and time stability test of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: 20 mL of the probe stock solution prepared in Example 2 was taken and placed in 2 mL of PBS buffer solutions of different pH values, so that the final concentration of the probe in the test solution was 10 μmol / L. The fluorescence changes of the probe BTD-CNAr-MOR in buffer solutions of different pH values ​​were tested. Figure 7 As shown, the probe has no obvious fluorescence change in the pH range of 4 to 10. The results show that the probe BTD-CNAr-MOR has good pH stability in a general physiological environment, which is beneficial to the cell imaging application of the probe.

[0058] Take 20 mL of the probe stock solution prepared in Example 2 and add it to 2 mL of PBS buffer (pH=7) so that the final concentration of the probe in the test solution is 10 μmol / L, and then add 20 mL of different interference solutions with a concentration of 1 mmol / L. Figure 8 As shown in the figure, when different ions are added to it, the fluorescence intensity of the fluorescent probe hardly changes. The above results show that the probe BTD-CNAr-MOR has good anti-interference ability.

[0059] The solutes in the above-mentioned interfering solutions are: 1. glycine Gly, 2. Fe2(SO4)3, 3. AgCl, 4. CaCl2, 5. MgSO4, 6. NaCl, 7. Al(NO3)3, 8. FeSO4, 9. cysteine ​​Cys, 10. NaClO, 11. CuSO4, 12. ZnCl2.

[0060] Take 20 mL of the probe stock solution prepared in Example 2 and add it to 2 mL of glycerol and PBS buffer (pH=7) respectively, so that the final concentration of the probe in the test solution is 10 μmol / L. Use these two solvents to detect the temporal stability of the probe to determine the temporal stability of the probe under different viscosity conditions in low viscosity and high viscosity environments. Fig. 9 As shown in the figure, the probe BTD-CNAr-MOR has good stability at low viscosity (PBS buffer) and high viscosity (propylene glycol), indicating that the probe BTD-CNAr-MOR can maintain good stability at different viscosities.

[0061] Example 5 Biological toxicity detection of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: Before cell imaging, we need to conduct a cytotoxicity experiment on BTD-CNAr-MOR. The standard MTT colorimetric method is used to detect the cytotoxicity of the probe BTD-CNAr-MOR to HeLa cells in vitro. First, 2×10 5 Cells at 100 cells / mL were seeded in a 96-well plate and then treated with different concentrations of BTD-CNAr-MOR (0, 2, 5, 10, 15, and 20 μM) for 24 h. Subsequently, 10 μL of MTT (5 mg / mL) was added to each well and incubated for another 3 h. Finally, the culture medium was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The plate was gently shaken for about 10 min and the absorbance was measured using a microplate reader. Fig.10 The MTT assay of the probe BTD-CNAr-MOR in HeLa cells showed a viability of over 80%, indicating that this probe can be used as a practical tool for lipid droplet and lysosome labeling in complex biological environments.

[0062] Example 6 Lysosome co-localization imaging test of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: The probe BTD-CNAr-MOR was used to perform cell co-localization imaging of lysosomes. Specifically, HeLa cells, Lyso-Tracker Green (500nM) and BTD-CNAr-MOR (10μΜ) were incubated for 20min, the cells were washed three times with sterile PBS buffer, and then cell co-localization imaging was performed. Fig.11 As shown in a, there is obvious red fluorescence on the cells (λex=405nm, λem=620-670nm), and Fig.11 In b, fluorescence imaging was collected by the commercial organic dye Lyso-Tracker Green (λex=504nm, λem=500-550nm), showing obvious green fluorescence. Fig.11 The colocalization plots showed that the green and red plots overlapped substantially, with a Pearson coefficient of up to 99.8%. All of the above features indicate that the probe BTD-CNAr-MOR can be used for cell lysosome labeling.

[0063] Example 7 Lipid droplet co-localization imaging test of lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe BTD-CNAr-MOR based on benzothiadiazole: The probe BTD-CNAr-MOR was used to perform cell co-localization imaging of lipid droplets. Specifically, HeLa cells, BODIPY (500nM) and BTD-CNAr-MOR (10μΜ) were incubated for 20min, the cells were washed three times with sterile PBS buffer, and then cell co-localization imaging was performed. Fig.12 As shown in a, there is obvious red fluorescence on the cell lipid droplets (λex=405nm, λem=620-670nm), while Fig.12 In b, fluorescence imaging was collected by the commercial organic dye BODIPY (λex = 493 nm, λem = 470-520 nm), showing obvious green fluorescence. Fig.12 The colocalization plots showed that the green and red plots overlapped substantially, with a Pearson coefficient of up to 85%. All of the above features indicate that the probe BTD-CNAr-MOR can be used for cell lipid droplet labeling.

[0064] Unless otherwise specified, all percentages used in the present invention are by mass.

[0065] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, characterized in that: It has the following molecular structure: 。 2. A method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole, characterized in that: The method comprises the following steps: primary reaction, secondary reaction; The method of the primary reaction is as follows: Compound 1 and Compound 2 are added into ethanol, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the mixture is kept warm for reflux reaction; After the reaction is completed, the product is purified to obtain compound 3; The compound 1 is 4-(4-morpholinyl)acetophenone; the compound 2 is malononitrile; The compound 3 has the following molecular structure: ； The secondary reaction method is as follows: Compound 3 and Compound 4 are added into acetonitrile, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the mixture is kept warm and refluxed for reaction; after the reaction is completed, the mixture is purified to obtain a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole; The compound 4 has the following molecular structure: 。 3. The method for preparing the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the primary reaction, the molar amount of compound 2 is controlled to be excessive relative to compound 1.

4. The method for preparing the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the one reaction, the molar amount of piperidine is at least 20% of the molar amount of the compound; The molar amount of acetic acid is at least 20% of 1 mole of the compound.

5. The method for preparing the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the primary reaction, the heat preservation and reflux reaction time is 3-6h.

6. The method for preparing the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the secondary reaction, the molar amount of compound 3 is controlled to be in excess relative to compound 4.

7. The method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the secondary reaction, the molar amount of piperidine is at least 20% of the molar amount of compound 4; The molar amount of acetic acid is at least 20% of the molar amount of compound 4.

8. The method for preparing a lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 2, characterized in that: In the secondary reaction, the heat preservation and reflux reaction time is 10-13h.

9. An application of the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 1, characterized in that: The benzothiadiazole-based lipid droplet and lysosome dual-targeted viscosity-sensitive fluorescent probe is used in sensing and detecting lipid droplets and lysosomes in biological cells.

10. An application of the lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe based on benzothiadiazole according to claim 1, characterized in that: The benzothiadiazole-based lipid droplet and lysosome dual-targeted viscosity-sensitive fluorescent probe is used in the detection of viscosity in biological cells.

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