A benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe, a preparation method and applications thereof

Abstract

This invention provides a benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe for lipid droplets and lysosomes, its preparation method, and its application, relating to the field of fluorescent probes. The preparation method of the benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe includes the following steps: a primary reaction and a secondary reaction. The benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe of this invention can simultaneously target and monitor two organelles (lipid droplets and lysosomes), and exhibits low cytotoxicity and accurate cell localization in HeLa cell imaging experiments; furthermore, it achieves both good polarity response and effective response to intracellular viscosity, demonstrating excellent response performance.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, and in particular to a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe, its preparation method, and its application. Background Technology

[0002] Intracellular viscosity is a crucial microenvironmental parameter in various biological systems, and abnormal fluctuations in viscosity are closely related to many diseases. Intracellular viscosity plays a vital role in cellular physiology and pathology, significantly impacting substance transport, signal transduction, and organelle function. Abnormal changes in intracellular viscosity can cause numerous adverse effects, such as cellular dysfunction, metabolic disorders, cancer, neurodegenerative disorders, and metabolic diseases. Therefore, the detection and regulation of intracellular viscosity are of paramount importance for maintaining normal function and preventing disease.

[0003] Fluorescent probes are increasingly widely used in clinical medical experiments due to their advantages such as precise in-situ real-time imaging, good biocompatibility, low hazard, and short reaction time. Therefore, developing functional fluorescent probes that respond to viscosity can help to better understand the relationship between changes in intracellular viscosity and disease development, and provide new insights for disease diagnosis and treatment.

[0004] Lipid droplets are lipid-rich subcellular organelles composed of a neutral lipid core and encased in a phospholipid monolayer. They perform important cellular tasks such as membrane transport, fusion, and participation in signal transduction pathways. Existing research has found that lipid droplets play a crucial role in cellular lipid storage and supply; however, abnormal levels of cellular lipid droplets are closely associated with many metabolic diseases, such as obesity, cancer, diabetes, and inflammatory diseases.

[0005] Lysosomes are organelles that exist under acidic conditions. They are involved in a variety of cell biological processes, primarily including endocytosis, degradation reactions, and immunity. Changes in the acidic conditions within lysosomes can lead to cellular dysfunction and consequently, many diseases. Therefore, accurately detecting changes in the acidic environment of lysosomes is crucial for organisms.

[0006] Lipid droplets and lysosomes, as two important organelles within cells, are closely related to intracellular viscosity. Current subcellular viscosity fluorescent probes primarily utilize specific targeting groups for viscosity detection. For example, morpholine groups are used to detect lysosomes alone. However, fluorescent probes capable of simultaneously and effectively detecting both organelles (lipid droplets and lysosomes) to obtain information on intracellular viscosity changes are relatively rare. Using a single fluorescent probe molecule to simultaneously monitor subtle changes in two organelles (lipid droplets and lysosomes) and understand their interactions is of significant technical and research value. Therefore, providing a novel dual-organelle (lipid droplet and lysosome) targeted viscosity-sensitive fluorescent probe is crucial for a better understanding of organelle viscosity and its close relationship with related diseases.

[0007] Furthermore, although existing technologies disclose dual-targeting probes based on benzothiadiazole for lipid droplets and lysosomes, these are limited to responses to polarity and do not respond well to intracellular viscosity, thus failing to achieve an effective response to viscosity. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides a benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe for lipid droplets and lysosomes, capable of simultaneously targeting and monitoring two organelles (lipid droplets and lysosomes). In HeLa cell imaging experiments, it exhibits low cytotoxicity and accurate cell localization; furthermore, it achieves both good polarity response and effective response to intracellular viscosity, demonstrating excellent response performance. This invention also provides a method for preparing the benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe for lipid droplets and lysosomes. Finally, this invention provides an application of the benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe for lipid droplets and lysosomes.

[0009] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0010] A benzothiadiazole-based dual-targeting viscosity-sensitive fluorescent probe targeting both lipid droplets and lysosomes has the following molecular structure:

[0011] .

[0012] A method for preparing a benzothiadiazole-based lipid droplet and lysosomal dual-targeting viscosity-sensitive fluorescent probe, the synthetic route is as follows:

[0013] .

[0014] The method for preparing the benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe includes the following steps: a primary reaction and a secondary reaction;

[0015] The method for the first reaction is as follows: Compound 1 and Compound 2 are added to ethanol, heated to reflux, and kept at this temperature; piperidine and acetic acid are added dropwise, and the mixture is kept at reflux for further reaction; after the reaction is complete, the mixture is purified to obtain Compound 3.

[0016] Compound 1 is 4-(4-morpholino)acetophenone; compound 2 is malononitrile;

[0017] Compound 3 has the following molecular structural formula:

[0018] ;

[0019] The secondary reaction method is as follows: Compound 3 and Compound 4 are added to acetonitrile, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the reaction is kept warm under reflux; after the reaction is completed, the mixture is purified to obtain a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe.

[0020] Compound 4 has the following molecular structural formula:

[0021] .

[0022] Preferably, in the primary reaction, the molar amount of compound 2 is controlled to be in excess relative to compound 1.

[0023] More preferably, in the primary reaction, the molar amount of compound 2 is at least 1.2 times the molar amount of compound 1.

[0024] Preferably, in the primary reaction, the molar amount of piperidine is at least 20% of the molar amount of the compound.

[0025] The molar amount of acetic acid is at least 20% of the amount of 1 mole of the compound.

[0026] Preferably, in the primary reaction, the reflux reaction time is 3-6 hours.

[0027] Preferably, in the secondary reaction, the molar amount of compound 3 is controlled to be in excess relative to compound 4.

[0028] More preferably, in the secondary reaction, the molar amount of compound 3 is at least 1.15 times the molar amount of compound 4.

[0029] Preferably, in the secondary reaction, the molar amount of piperidine is at least 20% of the 4 molar amount of the compound;

[0030] The molar amount of acetic acid is at least 20% of the 4 molar amount of the compound.

[0031] Preferably, in the secondary reaction, the reflux reaction time is 10-13 hours.

[0032] The aforementioned benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe is used for the sensing and detection of lipid droplets and lysosomes in biological cells.

[0033] Furthermore, the sensing detection includes: fluorescence detection and cell formation.

[0034] Application of the aforementioned benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe for lipid droplets and lysosomes in viscosity detection within biological cells.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] (1) The benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe of the present invention can simultaneously target and monitor lipid droplets and lysosomes; and can achieve a good polarity response while effectively responding to intracellular viscosity, with good response to cell viscosity, fast cell entry and good stability.

[0037] (2) The preparation method of the benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe of the present invention has a wide range of raw material sources, simple synthesis steps, low production cost, and is conducive to large-scale production.

[0038] (3) The benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe of the present invention has low cytotoxicity in cell imaging experiments and can image lipid droplets and lysosomes in cells.

[0039] (4) The benzothiadiazole-based dual-target 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 to biological cells to obtain changes in intracellular viscosity by simultaneously targeting and monitoring two organelles (lipid droplets and lysosomes). Attached Figure Description

[0040] Figure 1 The probe BTD-CNAr-MOR prepared in Example 1 1 H NMR spectrum.

[0041] Figure 2 The probe BTD-CNAr-MOR prepared in Example 1 13 C NMR spectrum.

[0042] Figure 3 The UV absorption spectra of the probe BTD-CNAr-MOR in different solvents are shown.

[0043] Figure 4The fluorescence spectra of the probe BTD-CNAr-MOR in different solvents are shown.

[0044] Figure 5 Fluorescence spectra of the probe BTD-CNAr-MOR in different ratios of glycerol and methanol.

[0045] Figure 6 The graph shows the linear relationship between the fluorescence intensity of the probe BTD-CNAr-MOR at 633 nm and log η.

[0046] Figure 7 The fluorescence spectra of the probe BTD-CNAr-MOR in PBS buffer at different pH values ​​are shown.

[0047] Figure 8 The graph shows the selectivity test results for the probe BTD-CNAr-MOR.

[0048] Figure 9 The graph shows the time stability test results of the probe BTD-CNAr-MOR.

[0049] Figure 10 MTT assay plot for probe BTD-CNAr-MOR.

[0050] Figure 11 The image shows the lysosomal colocalization experiment of the probe BTD-CNAr-MOR. In the image, (a) is the red channel stained with BTD-CNAr-MOR (10 μM) (λex=405nm, λem=620-670nm); (b) is the green channel stained with Lyso-Tracker Green (500nM) (λex=504nm, λem=500-550nm); (c) is the combined image of (a) and (b); (d) is the bright field image of HeLa cells; (e) is the fluorescence colocalization curve of (a) and (b); and (f) is the average fluorescence intensity graph of (a) and (b).

[0051] Figure 12 The images show the lipid droplet colocalization experiment of the probe BTD-CNAr-MOR; (a) is the red channel stained with BTD-CNAr-MOR (10 μM) (λex=405nm, λem=620-670nm); (b) is the green channel stained with BODIPY (500nM) (λex=493nm, λem=470-520nm); (c) is the combined image of (a) and (b); (d) is the bright field image of HeLa cells; (e) is the fluorescence colocalization curve of (a) and (b); and (f) is the average fluorescence intensity graph of (a) and (b). Detailed Implementation

[0052] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0053] In each embodiment, probe BTD-CNAr-MOR represents a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe, and the compound numbers in the embodiments correspond to the compound numbers in the synthetic route of the above invention.

[0054] Example 1

[0055] This embodiment provides a benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe, BTD-CNAr-MOR, with the following molecular structure:

[0056] .

[0057] This embodiment also provides a method for preparing BTD-CNAr-MOR, a benzothiadiazole-based lipid droplet and lysosomal dual-targeting viscosity-sensitive fluorescent probe. The specific synthetic route is as follows:

[0058] .

[0059] The specific steps are as follows:

[0060] 1. A single reaction (preparation of compound 3)

[0061] Compound 1 (4.105 g, 20 mmol) and compound 2 (1.58 g, 24 mmol) were dissolved in ethanol (20 mL) at room temperature; 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 to catalyze the reaction, and the reaction was maintained under reflux for 4 h; after the reaction was completed as monitored by TLC, the product was purified by silica gel column chromatography using a mixture of petroleum ether / ethyl acetate (volume ratio of 5:1) as eluent to obtain compound 3 (3.201 g, yield 64%).

[0062] Compound 1 is 4-(4-morpholino)acetophenone, CAS number 39910-98-0, which was purchased through commercial channels.

[0063] Compound 2 is malononitrile, CAS number 109-77-3, which was obtained through commercial channels.

[0064] 2. Secondary reaction (preparation of BTD-CNAr-MOR)

[0065] 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 to catalyze the reaction, which was then refluxed for 12 h; after the reaction was completed by TLC monitoring, the product was purified by silica gel column chromatography using a mixture of petroleum ether / ethyl acetate (5:1 v / v) as the eluent to obtain BTD-CNAr-MOR (84 mg, yield 43%), a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe.

[0066] Compound 4 can be prepared using existing methods.

[0067] The specific preparation method of compound 4 in this embodiment is referenced in: Wang Lin, Zhang Ning, He Yuan, et al. A Fluorescent Probe with Large Stokes Shift Based on Benzotisadiazole Scaffolds for Selective Detection of Hg 2+ in Lysosomes and ItsApplication in Biological Imaging. Chem AsianJ. 2024, e202401067. DOI:10.1002 / asia.202401067.

[0068] The specific preparation method of compound 4 is as follows: 7-bromo-4-aldehyde benzo[C][1,2,5]thiadiazole (CAS No. 1071224-34-4) (121.0 mg, 5.0 mmol) was dissolved in morpholine (5.0 mL) aqueous solution at 135 °C and reacted for 6 h. After the reaction was completed, the compound 4 was purified by silica gel column chromatography (petroleum ether:ethyl acetate volume ratio 2:1) to obtain compound 4. The specific synthetic route is as follows:

[0069] .

[0070] The aforementioned preparation method yielded a benzothiadiazole-based lipid droplet and lysosomal dual-targeting viscosity-sensitive fluorescent probe, BTD-CNAr-MOR. 1 H NMR spectrum as follows Figure 1 As shown, 13 C NMR spectra as follows Figure 2 As shown.

[0071] The molecular formula of the benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe BTD-CNAr-MOR is: C 26 H 24 N6O2S.

[0072] This embodiment also provides the application of the aforementioned benzothiadiazole-based dual-target viscosity-sensitive fluorescent probe BTD-CNAr-MOR for the detection of lipid droplets and lysosomes in biological cells, for the application of viscosity detection in biological cells.

[0073] Example 2

[0074] Spectroscopic properties of BTD-CNAr-MOR, a viscosity-sensitive fluorescent probe based on benzothiadiazole targeting both lipid droplets and lysosomes:

[0075] First, the fluorescent probe BTD-CNAr-MOR was dissolved in DMSO to prepare a stock solution with a concentration of 1 mmol / L. Then, by adding 20 μL of the stock solution to 2.0 mL of solvents with different polarities, probes with a concentration of 10 μmol / L were obtained. This 10 μmol / L probe was used in all photophysics experiments, and UV absorption measurements were performed in different solvents (ethyl acetate, 1,4-dioxane, ethanol, methanol, acetonitrile, dimethyl sulfoxide, and PBS buffer at pH 7). Figure 3 As shown, its absorption peak is around 500 nm.

[0076] 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, and PBS buffer at pH 7) for fluorescence testing. Measure the fluorescence emission intensity using an excitation wavelength of 500 nm. Figure 4 As shown, the fluorescence intensity of probe BTD-CNAr-MOR in lipophilic solvents such as 1,4-dioxane and ethyl acetate was significantly stronger than that in water-soluble solvents such as methanol and ethanol. This indicates that it is a lipophilic sensitive fluorophore. Morpholine has already been proven to be effective for lysosomal targeting. The above data demonstrate that probe BTD-CNAr-MOR can serve as a novel, polarly sensitive probe targeting both lipid droplets and lysosomes.

[0077] Example 3

[0078] Viscosity response testing of BTD-CNAr-MOR, a benzothiadiazole-based lipid droplet and lysosomal dual-target viscosity-sensitive fluorescent probe:

[0079] Take 20 mL of the probe stock solution prepared in Example 2 and add it to 2 mL of a glycerol / methanol mixed solvent with different volume ratios to test the sensitivity of the probe BTD-CNAr-MOR for viscosity detection. Figure 5 As shown, the probe BTD-CNAr-MOR exhibits almost no fluorescence in pure methanol (0%); however, in pure glycerol (100%), the maximum fluorescence emission peak of the probe BTD-CNAr-MOR appears at 630 nm. The fluorescence intensity at 630 nm in pure glycerol is approximately 100 times that in pure methanol. This indicates that the probe BTD-CNAr-MOR is a fluorescent probe for viscosity recognition.

[0080] Based on relevant literature, the viscosity parameter (logη) of the methanol / glycerol system from pure methanol to pure glycerol at room temperature was determined. Within the viscosity range of 1-998 cP, the linear relationship between the fluorescence intensity of the probe BTD-CNAr-MOR and the viscosity parameter (logI 650 nm) and viscosity (logη) was plotted. Figure 6 As shown. According to the Forster-Hoffmann equation, a good linear relationship was found between the probe BTD-CNAr-MOR and the MOR, indicating that the probe BTD-CNAr-MOR can accurately quantify the viscosity of the methanol / glycerol mixture.

[0081] Example 4

[0082] pH stability, anti-interference and time stability tests of BTD-CNAr-MOR, a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe:

[0083] Take 20 mL of the probe stock solution prepared in Example 2 and place it in 2 mL of PBS buffer at different pH values ​​to achieve a final probe concentration of 10 μmol / L in the test solution. Test the fluorescence change of the probe BTD-CNAr-MOR in buffer solutions at different pH values. Figure 7 As shown, the probe exhibited no significant fluorescence change within a pH range of 4-10. These results indicate that the BTD-CNAr-MOR probe possesses good pH stability under normal physiological conditions, which is beneficial for its application in cell imaging.

[0084] Take 20 mL of the probe stock solution prepared in Example 2 and add it to 2 mL of PBS buffer (pH=7) to make the final concentration of the probe in the test solution 10 μmol / L. Then add 20 mL of different interference solutions with a concentration of 1 mmol / L to each solution. Figure 8 As shown, the fluorescence intensity of the fluorescent probe hardly changed after the addition of different ions. These results indicate that the probe BTD-CNAr-MOR has good anti-interference capabilities.

[0085] The solutes in the aforementioned interfering solutions are as follows: 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.

[0086] 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, to make the final concentration of the probe in the test solution 10 μmol / L. These are used as the two solvents to detect the time stability of the probe, to determine the time stability of the probe under different viscosity conditions in low-viscosity and high-viscosity environments. Figure 9 As shown, the probe BTD-CNAr-MOR exhibits good stability at both low viscosity (PBS buffer) and high viscosity (glycerol). This indicates that the probe BTD-CNAr-MOR maintains good stability at different viscosities.

[0087] Example 5

[0088] Biotoxicity assay of BTD-CNAr-MOR, a viscosity-sensitive fluorescent probe based on benzothiadiazole targeting both lipid droplets and lysosomes:

[0089] Before performing cell imaging, we need to conduct cytotoxicity experiments on BTD-CNAr-MOR, using the standard MTT assay to detect the cytotoxicity of the probe BTD-CNAr-MOR against HeLa cells in vitro. First, 2×10⁻⁶ cells were prepared... 5 Cells were seeded at 100 cells / mL in 96-well plates and 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 plates were gently shaken for approximately 10 min, and the absorbance was measured using a microplate reader. Figure 10 As shown in the figure, MTT analysis of the probe BTD-CNAr-MOR in HeLa cells showed a survival rate of over 80%, indicating that this probe can serve as a practical tool for labeling lipid droplets and lysosomes in complex biological environments.

[0090] Example 6

[0091] Lysosomal colocalization imaging assay using BTD-CNAr-MOR, a benzothiadiazole-based lipid droplet and lysosomal dual-target viscosity-sensitive fluorescent probe:

[0092] The probe BTD-CNAr-MOR was used for lysosomal colocalization imaging. Specifically, HeLa cells were incubated with Lyso-Tracker Green (500 nM) and BTD-CNAr-MOR (10 μM) for 20 min, washed three times with sterile PBS buffer, and then colocalization imaging was performed. Figure 11 As shown in Figure a, the cells exhibit distinct red fluorescence (λex = 405 nm, λem = 620-670 nm), while... Figure 11 In image b, fluorescence imaging was performed using the commercial organic dye Lyso-Tracker Green (λex = 504 nm, λem = 500-550 nm), exhibiting distinct green fluorescence. Furthermore, as... Figure 11 The colocalization map shows that the green and red maps largely overlap, with a Pearson coefficient as high as 99.8%. All of the above characteristics indicate that the probe BTD-CNAr-MOR can be used for lysosomal labeling.

[0093] Example 7

[0094] Lipid droplet colocalization imaging assay using the benzothiadiazole-based lipid droplet and lysosomal dual-target viscosity-sensitive fluorescent probe BTD-CNAr-MOR:

[0095] Cell colocalization imaging of lipid droplets was performed using the probe BTD-CNAr-MOR. Specifically, HeLa cells, BODIPY (500 nM), and BTD-CNAr-MOR (10 μM) were incubated for 20 min. Cells were washed three times with sterile PBS buffer, followed by cell colocalization imaging. Figure 12 As shown in Figure a, there is obvious red fluorescence on the cell lipid droplets (λex=405nm, λem=620-670nm), while... Figure 12 In image b, fluorescence imaging was performed using the commercial organic dye BODIPY (λex = 493 nm, λem = 470-520 nm), exhibiting distinct green fluorescence. Furthermore, as... Figure 12 The colocalization plots show that the green and red plots largely overlap, with a Pearson coefficient as high as 85%. All of the above characteristics indicate that the probe BTD-CNAr-MOR can be used for cellular lipid droplet labeling.

[0096] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0097] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe, characterized in that, It has the following molecular structural formula: 。 2. A method for preparing a benzothiadiazole-based lipid droplet and lysosomal dual-targeting viscosity-sensitive fluorescent probe, characterized in that, It includes the following steps: primary reaction, secondary reaction; The method for the first reaction is as follows: Compound 1 and Compound 2 are added to ethanol, heated to reflux, and kept at the temperature; piperidine and acetic acid are added dropwise, and the reaction is kept at the temperature and refluxed. After the reaction was complete, the mixture was purified to obtain compound 3. Compound 1 is 4-(4-morpholino)acetophenone; compound 2 is malononitrile; Compound 3 has the following molecular structural formula: ； The secondary reaction method is as follows: Compound 3 and Compound 4 are added to acetonitrile, heated to reflux, and kept warm; piperidine and acetic acid are added dropwise, and the reaction is kept warm under reflux; after the reaction is completed, the mixture is purified to obtain a benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe. Compound 4 has the following molecular structural formula: 。 3. The method for preparing a benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe according to claim 2, characterized in that, In the aforementioned reaction, the molar amount of compound 2 is controlled to be in excess relative to compound 1.

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

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

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

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

9. The application of the benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe as described in claim 1, characterized in that, The application of the benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe in the preparation of products for sensing and detecting lipid droplets and lysosomes in biological cells.

10. The application of the benzothiadiazole-based lipid droplet and lysosome dual-targeting viscosity-sensitive fluorescent probe as described in claim 1, characterized in that, The application of the benzothiadiazole-based lipid droplet and lysosome dual-target viscosity-sensitive fluorescent probe in the preparation of products for viscosity detection in biological cells.

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