Alkoxythiophene viscosity fluorescence probe, preparation method and application thereof

Abstract

The application belongs to the field of fluorescent probes, and relates to a red fluorescent imaging probe for lysosome targeting cancer cells, in particular to a preparation method and application of a viscosity-sensitive fluorescent probe. The viscosity-sensitive fluorescent probe has a structure of formula (I): the alkoxythiophene viscosity fluorescent probe of the application is applied to multicellular organism imaging and distinguishing normal cells and cancer cells, and has important significance for cancer detection.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes and relates to red fluorescence imaging and multicellular bioimaging probes for lysosomal targeting of cancer cells, particularly a method for preparing and applying an alkoxythiophene-based viscosity fluorescent probe. Background Technology

[0002] Viscosity is a crucial parameter affecting the flow state of proteins, lipids, and polysaccharides within cells, playing a vital role in promoting intracellular biomolecular interactions, chemical signal transduction, and the diffusion of metabolic products. Detecting intracellular viscosity is significant for the early diagnosis of certain diseases. Abnormal cell viscosity can directly cause organelle dysfunction, leading to many diseases such as fatty liver, lysosomal storage diseases, and malignant tumors.

[0003] Traditional viscosity measurement tools, such as capillary viscometers, falling ball viscometers, and rotational viscometers, are only suitable for liquids and cannot be applied to biological systems. In recent years, small-molecule fluorescent probes for viscosity detection have been gradually developed. Compared with traditional measurement tools, fluorescent probes have advantages such as fast response speed, high sensitivity, and simple operation, making them an effective means of detecting viscosity in vivo. Lysosomes are typical acidic organelles that play important roles in intracellular digestion, apoptosis, and autophagy. Currently, our understanding of lysosomal viscosity is still insufficient; therefore, real-time in-situ monitoring of lysosomal viscosity changes is of great significance for understanding lysosomal function and elucidating the pathogenesis of related diseases.

[0004] Patent CN111116539A discloses a fluorescent probe that is dually responsive to the viscosity and pH of lysosomes within cancer cells. However, this probe emits yellow-green light instead of red light, which limits its application due to its inability to penetrate deep tissues and its inability to achieve low background signal. Patents CN112939935A and CN114437010A disclose methods for preparing lysosome-targeted two-photon fluorescent probes, but neither has been applied to distinguish between cancer cells and normal cells. Therefore, it is urgent to develop a fluorescent probe with a molecular excitation wavelength reaching the near-infrared I region (650-900 nm), which has deep tissue penetration, low background signal, avoids cell autofluorescence, and minimizes photodamage. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes an alkoxythiophene-based viscosity fluorescent probe, its preparation method, and its application. This fluorescent probe has a D-π-A structure, is sensitive to viscosity, and has lysosomal targeting capabilities.

[0006] The technical solution of this invention is implemented as follows:

[0007] The present invention provides an alkoxythiophene-based viscosity fluorescent probe having the structure of formula (I):

[0008] .

[0009] The preparation method of the above-mentioned alkoxythiophene-based viscosity fluorescent probe includes the following steps:

[0010] (1) Anhydrous tetrahydrofuran and water (after deoxygenation treatment) were added to a mixture of 5-bromo-4-(6-bromo-1-hexoxythiophene)-2-aldehyde, 4-(diphenylamino)phenylboronic acid, di(tri-tert-butylphosphine)palladium and sodium hydroxide. The mixture was heated to 80-100°C in an oil bath and reacted until complete. The mixture was then extracted, washed with water, and dried to obtain compound 1.

[0011] The technical approach is as follows:

[0012] .

[0013] (2) Dissolve compound 1 in anhydrous acetonitrile, add morpholine, heat in an oil bath to 80~100 ℃, react until complete, wash with water and dry to obtain compound 2;

[0014] The technical approach is as follows:

[0015] .

[0016] (3) Add to compound 2 Anhydrous ethanol and piperidine were heated in an oil bath to 80-100 °C and reacted until complete. After cooling, the precipitate was washed with ethanol and centrifuged to obtain the target compound (I).

[0017] The technical approach is as follows:

[0018] .

[0019] All of the above steps (1) to (3) are carried out under nitrogen protection.

[0020] In step (1) above, the molar ratio of 5-bromo-4-(6-bromo-1-hexoxythiophene)-2-aldehyde, 4-(diphenylamino)phenylboronic acid, di(tri-tert-butylphosphine)palladium to sodium hydroxide is 1:1~2:0.03~0.05:2.

[0021] In step (2) above, the molar ratio of compound 1 to morpholine is 1~5:100.

[0022] In step (3) above, compound 2 and The molar ratio of the substances is 1:1~2, and the volume ratio of anhydrous ethanol to piperidine is 200~300:1.

[0023] The aforementioned alkoxythiophene-based viscosity fluorescent probes are viscosity-sensitive and lysosomal-targeting.

[0024] The above-mentioned alkoxythiophene-based viscosity fluorescent probes are used in the preparation of chemical reagents to distinguish between normal cells and cancer cells.

[0025] The above-mentioned alkoxythiophene-based viscosity fluorescent probes are used in the preparation of reagents for multi-cell bioimaging.

[0026] The present invention has the following beneficial effects:

[0027] 1. This invention provides a novel fluorescent probe for lysosome-targeted cancer cell imaging and multi-cell bioimaging. The synthesis method of this probe is simple and easy to operate, with a high yield (≥80%), which is conducive to its commercial application.

[0028] 2. The fluorescent probe provided by this invention has a morpholine group in its molecule that easily enters acidic lysosomes, exhibiting lysosomal targeting. Simultaneously, the molecule contains multiple molecular rotors, such as pyridine, cyano, and triphenylamine. The distorted intramolecular charge transfer (TICT) process significantly affects the fluorescence molecule's viscosity, resulting in a 3.5-fold increase in fluorescence intensity compared to the control group. Therefore, the fluorescent probe of this application belongs to the viscosity-sensitive near-infrared fluorescent probe category.

[0029] 3. When distinguishing between cancer cells and normal cells, the fluorescent probe of this application uses an excitation wavelength of 600-700 nm, which is located in the near-infrared I region. It has the function of penetrating deep tissues and having a low background signal. Under this condition, normal cells show very weak fluorescence after being stained with the probe, while tumor cells show strong red fluorescence. The significant difference can be used to distinguish between the two types of cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 For fluorescent probe (I) 1 H NMR spectrum.

[0032] Figure 2 For fluorescent probe (I) 1 C10 NMR spectrum.

[0033] Figure 3 This is a high-resolution mass spectrum of the fluorescent probe (I).

[0034] Figure 4The fluorescence spectrum of the fluorescent probe (I) in a methanol-glycerol system with different viscosities is shown. The excitation wavelength is 495 nm and the slit width is 2.5 nm.

[0035] Figure 5 The linear relationship between the fluorescence intensity of the fluorescent probe (I) and its viscosity in the methanol-glycerol system is shown.

[0036] Figure 6 The image shows the fluorescent probe (I) in cancer cells and normal cells. The excitation wavelength was 514 nm, the scale bar was 25 μm, the concentration was 10 μM, and the P value was 0.0001.

[0037] Figure 7 The viscosity response of the fluorescent probe (I) in SMMC-7721 cells was shown. The excitation wavelength was 514 nm and the scale bar was 10 μm. The concentrations of nystatin and the probe were both 10 μM.

[0038] Figure 8 The image shows the colocalization fluorescence pattern and colocalization coefficient of the fluorescent probe (I) in lysosomes of SMMC-7721 cells. The excitation wavelength was 514 nm, the scale bar was 10 μm, and the concentration of the commercial probe and probe (I) was 10 μM.

[0039] Figure 9 Imaging of Caenorhabditis elegans with fluorescent probe (I), probe (10 μM), nystatin (50 μM), scale bar 100 μm. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] The preparation method of alkoxythiophene-based viscosity fluorescent probes includes the following steps:

[0043] (1) 5-Bromo-4-(6-bromo-1-hexyloxythiophene)-2-aldehyde (119.1 mg), 4-(diphenylamino)phenylboronic acid (113.1 mg), di(tri-tert-butylphosphine)palladium (4.9 mg), and sodium hydroxide (25.7 mg) were added to 50 ml of Schlenk. The mixture was dried under vacuum. Under nitrogen protection, 6 mL of anhydrous tetrahydrofuran and 2 mL of water (after deoxygenation) were added. The Schlenk was placed in an oil bath and heated to 80 °C. After reacting for 4 h, the reaction solution was transferred to a separatory funnel, extracted with dichloromethane, washed with water, and dried with anhydrous MgSO4 to obtain the crude product. The crude product was then subjected to column chromatography to obtain compound 1 (169.2 mg), with a yield of 98%.

[0044] (2) Dissolve compound 1 (123.0 mg) in 2 mL of anhydrous acetonitrile, transfer to a Schlenk container, add morpholine (2 mL) under N2 protection, heat to 90 °C in an oil bath and react for 8 h. Transfer the reaction solution to a separatory funnel, wash thoroughly with water to obtain product 2 (113.8 mg), yield 92%;

[0045] (3) Add 50 mL of Schlenk Compound 2 (56.5 mg), anhydrous ethanol (4 mL), and piperidine (0.02 mL) were added under nitrogen protection. The mixture was heated to 85 °C in an oil bath and reacted for 24 h. After cooling, a precipitate was formed. The precipitate was washed with ethanol and centrifuged to obtain probe (I) (59.6 mg), yield 80%.

[0046] Its characteristics are as follows:

[0047] 13 C NMR (100 MHz, CDCl3) Figure 2 ) δ153.57, 150.56, 147.71, 147.21,141.59, 135.97, 129.41, 124.96, 123.56, 122.59, 122.20, 119.29, 117.57,103.28, 77.35,77.24, 77.03, 76.72, 71.82, 66.95, 58.99, 53.78, 29.39, 27.17,26.43, 25.98.

[0048] HRMS (ESI) m / z: [M+H] + ( Figure 3 )calcd for: C 40 H 41 N4O2S 641.2950; found641.2941.

[0049] Example 2

[0050] The preparation method of alkoxythiophene-based viscosity fluorescent probes includes the following steps:

[0051] (1) 5-Bromo-4-(6-bromo-1-hexyloxythiophene)-2-aldehyde (119.1 mg), 4-(diphenylamino)phenylboronic acid (139.2 mg), di(tri-tert-butylphosphine)palladium (5.49 mg), and sodium hydroxide (25.7 mg) were added to 50 mL of Schlenk. The mixture was dried under vacuum. Under nitrogen protection, 4 mL of anhydrous tetrahydrofuran and 4 mL of water (after deoxygenation) were added. The Schlenk was placed in an oil bath and heated to 100 °C. After reacting for 4 h, the reaction solution was transferred to a separatory funnel, extracted with dichloromethane, washed with water, and dried with anhydrous MgSO4 to obtain the crude product. The crude product was then subjected to column chromatography to obtain compound 1 (160.8 mg), with a yield of 94%.

[0052] (2) Compound 1 (183.0 mg) was dissolved in 2 mL of anhydrous acetonitrile, transferred to a Schlenk container, and morpholine (2 mL) was added under N2 protection. The mixture was heated to 90 °C in an oil bath and reacted for 8 h. The reaction solution was then transferred to a separatory funnel and washed thoroughly with water to obtain product 2 (110 mg), with a yield of 88%.

[0053] (3) Add 50 mL of Schlenk Compound 2 (56.5 mg), anhydrous ethanol (6 mL), and piperidine (0.02 mL) were added under nitrogen protection. The mixture was heated to 85 °C in an oil bath and reacted for 24 h. After cooling, a precipitate formed. The precipitate was washed with ethanol and centrifuged to obtain probe (I) (63.9 mg), with a yield of 85%.

[0054] (4) Under N2 protection, compound 3 (31.5 mg), anhydrous acetonitrile (3 mL), and iodomethane (0.14 mL) were added to 50 mL of Schlenk. The mixture was heated to 90 °C in an oil bath and reacted for 10 h. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain probe (I) (33.5 mg), with a yield of 89%.

[0055] Its characteristics are as follows:

[0056] 13 C NMR (100 MHz, CDCl3) Figure 2) δ153.57, 150.56, 147.71, 147.21,141.59, 135.97, 129.41, 124.96, 123.56, 122.59, 122.20, 119.29, 117.57,103.28, 77.35,77.24, 77.03, 76.72, 71.82, 66.95, 58.99, 53.78, 29.39, 27.17,26.43, 25.98.

[0057] HRMS (ESI) m / z: [M+H] + ( Figure 3 )calcd for: C 40 H 41 N4O2S 641.2950; found641.2941.

[0058] Example 3

[0059] The preparation method of alkoxythiophene-based viscosity fluorescent probes includes the following steps:

[0060] (1) 5-bromo-4-(6-bromo-1-hexyloxythiophene)-2-aldehyde (119.1 mg), 4-(diphenylamino)phenylboronic acid (167 mg), di(tri-tert-butylphosphine)palladium (6.27 mg), and sodium hydroxide (25.7 mg) were added to 50 mL of Schlenk. The mixture was dried under vacuum. Under nitrogen protection, 4 mL of anhydrous tetrahydrofuran and 4 mL of water (after deoxygenation treatment) were added. The Schlenk was placed in an oil bath and heated to 100 °C. After reacting for 4 h, the reaction solution was transferred to a separatory funnel, extracted with dichloromethane, washed with water, and dried with anhydrous MgSO4 to obtain the crude product. The crude product was then subjected to column chromatography to obtain compound 1 (164.3 mg) with a yield of 96%. The reaction was repeated once, and a total of 328.6 mg was prepared.

[0061] (2) Dissolve compound 1 (244.0 mg) in 2 mL of anhydrous acetonitrile, transfer to a Schlenk container, add morpholine (2 mL) under N2 protection, heat to 90 °C in an oil bath, react for 8 h, transfer the reaction solution to a separatory funnel, wash thoroughly with water to obtain product 2 (112.5 mg), yield 90%;

[0062] (3) Add 50ml of Schlenk Compound 2 (56.5 mg), anhydrous ethanol (6 mL), and piperidine (0.02 mL) were added under nitrogen protection. The mixture was heated to 100 °C in an oil bath and reacted for 24 h. After cooling, a precipitate was formed. The precipitate was washed with ethanol and centrifuged to obtain probe (I) (62.4 mg), with a yield of 83%.

[0063] Its characteristics are as follows:

[0064] 13 C NMR (100 MHz, CDCl3) Figure 2 ) δ153.57, 150.56, 147.71, 147.21,141.59, 135.97, 129.41, 124.96, 123.56, 122.59, 122.20, 119.29, 117.57,103.28, 77.35,77.24, 77.03, 76.72, 71.82, 66.95, 58.99, 53.78, 29.39, 27.17,26.43, 25.98.

[0065] HRMS (ESI) m / z: [M+H] + ( Figure 3 )calcd for: C 40 H 41 N4O2S 641.2950; found641.2941.

[0066] Example 4

[0067] The preparation method of alkoxythiophene-based viscosity fluorescent probes includes the following steps:

[0068] (1) 5-Bromo-4-(6-bromo-1-hexyloxythiophene)-2-aldehyde (119.1 mg), 4-(diphenylamino)phenylboronic acid (185.6 mg), di(tri-tert-butylphosphine)palladium (7.84 mg), and sodium hydroxide (25.7 mg) were added to 50 mL of Schlenk. The mixture was dried under vacuum. Under nitrogen protection, 4 mL of anhydrous tetrahydrofuran and 4 mL of water (after deoxygenation) were added. The Schlenk was placed in an oil bath and heated to 90 °C. After reacting for 4 h, the reaction solution was transferred to a separatory funnel, extracted with dichloromethane, washed with water, and dried with anhydrous MgSO4 to obtain the crude product. The crude product was then subjected to column chromatography to obtain compound 1 (166 mg), with a yield of 97%.

[0069] (2) Dissolve compound 1 (366.0 mg) in 2 mL of anhydrous acetonitrile, transfer to a Schlenk container, add morpholine (2 mL) under N2 protection, heat to 90 °C in an oil bath and react for 8 h. Transfer the reaction solution to a separatory funnel, wash thoroughly with water to obtain product 2 (116.3 mg), yield 93%;

[0070] (3) Add 50 mL of Schlenk Compound 2 (56.5 mg), anhydrous ethanol (6 mL), and piperidine (0.02 mL) were added under nitrogen protection. The mixture was heated to 90 °C in an oil bath and reacted for 24 h. After cooling, a precipitate was formed. The precipitate was washed with ethanol and centrifuged to obtain probe (I) (62.4 mg), with a yield of 83%.

[0071] Its characteristics are as follows:

[0072] 13 C NMR (100 MHz, CDCl3) Figure 2 ) δ153.57, 150.56, 147.71, 147.21,141.59, 135.97, 129.41, 124.96, 123.56, 122.59, 122.20, 119.29, 117.57,103.28, 77.35,77.24, 77.03, 76.72, 71.82, 66.95, 58.99, 53.78, 29.39, 27.17,26.43, 25.98.

[0073] HRMS (ESI) m / z: [M+H] + ( Figure 3 )calcd for: C 40 H 41 N4O2S 641.2950; found641.2941.

[0074] Application Example 1

[0075] The fluorescent probe (I) prepared in Example 1 was placed in a colorimetric tube, and a mixed solvent of methanol-glycerol in different proportions was added to bring the volume to 5 mL, with a final concentration of 10 μM. Figure 4 The fluorescence spectra of fluorescent probe (I) in methanol-glycerol systems of different viscosities are shown. The excitation wavelength was 495 nm, and the slit width was 2.5 nm. Figure 4 The wavelength is the emission wavelength; this graph shows the spectrum obtained through excitation at 495 nm. Figure 4It can be seen that as the content of glycerol increases, the viscosity of the mixed solvent also increases, and the fluorescence intensity of the probe gradually increases. Figure 5 This represents the linear relationship between the fluorescence intensity log I of the probe (I) and the viscosity log η of the solvent (η represents the viscosity value). From... Figure 5 It can be seen that the two exhibit a good linear relationship, with the linear equation being logI = 0.2715logη + 5.2372 (R²). 2 =0.972).

[0076] Application Example 2

[0077] The probe prepared in Example 1 was used to distinguish between cancer cells and normal cells:

[0078] The probe was co-incubated with normal cells (HL-7702 and RAW264.7) and cancer cells (HeLa and SMMC-7721) for imaging. Cells were seeded into confocal microscope dishes and cultured for 24 h. After cell attachment, 10 μM of the probe was added and incubated for 20 min. The culture medium was discarded, and the cells were washed with PBS and then 500 μL of cell fixative was added. Imaging was observed under a confocal microscope, with the probe excited at 514 nm and light collected at 600–700 nm. Figure 6 As shown, under the same conditions, normal cells exhibited very weak fluorescence after probe staining, while tumor cells showed strong red fluorescence, with a significant difference between the two (P<0.0001).

[0079] Application Example 3

[0080] Viscosity response was performed using the probe prepared in Example 1:

[0081] Nystatin can cause abnormal cell function and increase cell viscosity. SMMC-7721 cells were seeded into confocal microscopy dishes and cultured for 24 h. After cell adhesion, the cells were washed once with PBS. The control group was incubated with the prepared probe (10 μM) for 30 min, while the experimental group was incubated with nystatin (10 μM) for 30 min, followed by incubation with the probe (10 μM) for 30 min. The culture medium was then discarded, excess probe was washed away with PBS, and 500 μL of cell fixation medium was added. Confocal microscopy was used for imaging, with the probe excited at 514 nm and light collected at 600–700 nm. Figure 7 As shown, the fluorescence intensity of the experimental group was 3.5 times that of the control group, indicating that the probe can monitor changes in lysosomal viscosity under drug stimulation.

[0082] Application Example 4

[0083] Lysosomal targeting assay was performed using the probe prepared in Example 1:

[0084] SMMC-7721 cells were seeded into confocal microscopy dishes and cultured for 24 h. After cell adhesion, 10 μM of probe was added and incubated for 20 min. The culture medium was discarded, and excess probe was washed away with PBS. Commercial lysosomal green probe (Lyso-Tracker Green), mitochondrial green probe (Mito-Tracker Green), and endoplasmic reticulum blue probe (ER-Tracker Blue-White DPX) were added, and incubation continued for 20 min. Cells were washed twice with PBS, and 500 μL of cell fixative was added. Confocal microscopy was used for imaging, with the probes excited at 514 nm and light collected at 600–700 nm. Figure 8 As shown, the probe can be localized to the lysosomes of SMMC-7721 cells.

[0085] Application Example 5

[0086] Imaging of nematodes using the probe prepared in Example 1:

[0087] *C. elegans* is an optically transparent, unique multicellular organism. To test the potential of probe (I) in bioimaging, the control group was incubated with probe (I) (10 μM), while the experimental group was incubated with probe (I) (10 μM) and nystatin (50 μM). Imaging was observed under a fluorescence microscope. Figure 9 As shown, the red fluorescence observed in nematodes incubated with probe (I) was weak; however, when nematodes were simultaneously incubated with probe (I) and nystatin, bright red fluorescence was observed in the nematodes. This indicates that probe (I) can be successfully ingested in large quantities with food and accumulate in the nematodes, and that nystatin can increase the viscosity of the nematodes, further demonstrating that probe (I) is sensitive to viscosity and suitable for bioimaging.

[0088] The above demonstrates that the alkoxythiophene probes synthesized in Example 1 can be used for red fluorescence imaging of cancer cells and multicellular biological imaging, showing promising application prospects.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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. The application of an alkoxythiophene-based viscosity fluorescent probe in the preparation of chemical reagents for distinguishing between normal cells and cancer cells, characterized in that: The cancer cells are HeLa cells or SMMC-7721 cells; the structural formula of the alkoxythiophene-based viscosity fluorescent probe is as shown in formula (I): 。 2. A method for preparing an alkoxythiophene-based viscosity fluorescent probe, characterized in that, The structural formula of the alkoxythiophene-based viscosity fluorescent probe is shown in formula (I): ； The preparation steps are as follows: (1) Solvent I was added to a mixture of 5-bromo-4-(6-bromo-1-hexyloxythiophene)-2-aldehyde, 4-(diphenylamino)phenylboronic acid, di(tri-tert-butylphosphine)palladium and sodium hydroxide. The mixture was heated in an oil bath to 80-100°C and reacted until complete. The resulting reactants were washed with water, dried, and subjected to column chromatography to obtain compound 1. The structural formula of compound 1 is: ; (2) Compound 1 was dissolved in anhydrous acetonitrile, then morpholine was added, and the mixture was heated in an oil bath to 80-120°C until complete. The resulting reactant was washed with water to obtain compound 2. The structural formula of compound 2 is: ; (3) Add to compound 2 The target compound (I), namely an alkoxythiophene viscosity fluorescent probe, was obtained by reacting with solvent II in an oil bath at 80-100°C until complete, followed by cooling, centrifugation, and washing with ethanol.

3. The method for preparing the alkoxythiophene-based viscosity fluorescent probe according to claim 2, characterized in that: All steps (1) to (3) are performed under nitrogen protection.

4. The method for preparing the alkoxythiophene-based viscosity fluorescent probe according to claim 3, characterized in that: In step (1), solvent I is a mixed solution of anhydrous tetrahydrofuran and water with a volume ratio of 1 to 3:1, wherein the water is water that has undergone deoxygenation treatment.

5. The method for preparing an alkoxythiophene-based viscosity fluorescent probe according to claim 3 or 4, characterized in that: The molar ratio of 5-bromo-4-(6-bromo-1-hexoxythiophene)-2-aldehyde, 4-(diphenylamino)phenylboronic acid, di(tri-tert-butylphosphine)palladium to sodium hydroxide is 1:1~2:0.03~0.05:

2.

6. The method for preparing the alkoxythiophene-based viscosity fluorescent probe according to claim 5, characterized in that: In step (2), the molar ratio of compound 1 to morpholine is 1~5:

100.

7. The method for preparing the alkoxythiophene-based viscosity fluorescent probe according to claim 6, characterized in that: In step (3), solvent II is a mixed solution of anhydrous ethanol and piperidine with a volume ratio of 200~300:

1.

8. The method for preparing the alkoxythiophene-based viscosity fluorescent probe according to claim 7, characterized in that: The compound 2 and The molar ratio of the substances is 1:1~2.

9. The application of the alkoxythiophene-based viscosity fluorescent probe prepared by the preparation method according to any one of claims 2-8 in the preparation of an imaging reagent for Caenorhabditis elegans.

Images