A fluorescent probe for detecting the viscosity of cell microenvironment, a preparation method thereof and a special detection kit

Abstract

The application provides a fluorescent probe for detecting cell microenvironment viscosity change and a preparation method thereof, and the fluorescent probe is 2-((E)-2-(6-((Z)-(4-(dimethylamino)phenyl)azo)-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium. The fluorescent probe can make the electron donor and the electron acceptor freely run around the N=N double bond in a low viscosity environment. When being stimulated by light, the energy of the excited state is released in a non-radiation mode through a TICT mechanism, so that weak fluorescence is displayed. In a high viscosity environment, intramolecular rotation is limited, the energy of the excited state is emitted in a light radiation mode, so that strong fluorescence is generated. The probe has high sensitivity for microenvironment viscosity detection and does not interfere with other common active oxygen, amino acid and enzyme species.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis and biological analysis, and particularly discloses a fluorescent probe for detecting the viscosity of a cell microenvironment, a preparation method thereof and a special detection kit. BACKGROUND

[0002] Abnormal changes in cell microenvironment parameters can lead to normal cell dysfunction and ultimately cause a variety of specific diseases. Viscosity in cells, as a key parameter of the microenvironment, participates in various biological processes, and it can affect the interaction with biomolecules, signal and mass transport, and diffusion of reactive metabolites. For example, the aggregation of proteins starts from the formation of low-viscosity soluble protein oligomers and gradually evolves into high-viscosity insoluble protein aggregates. Abnormal viscosity is always associated with diseases such as cardiovascular disease, Alzheimer's disease and tumors. Therefore, detecting the fluctuation of viscosity in cells is crucial for explaining different pathogenesis of diseases.

[0003] At present, there are many fluorescent probes for detecting the microenvironment reported, such as Chinese patent application No. 202310632865.9, which discloses a BODPIY fluorescent probe for detecting the polarity of the microenvironment. The probe is designed based on intramolecular charge transfer, and can detect the change of the polarity of the microenvironment in lipid droplets to monitor the behavior of lipid droplets and explain related physiological and pathological processes. However, the probe shows visible fluorescence (below 700 nm) detection, which is easily disturbed by biological autofluorescence. In contrast, near-infrared fluorescence (700-900 nm) has minimal light damage to biological samples and can reduce the interference of background autofluorescence, and thus performs well in biological imaging.

[0004] In view of the above problem, the present application provides a near-infrared fluorescent probe with an excitation wavelength of 766 nm and an emission wavelength of 806 nm. It can track the viscosity changes of cells and organisms. The effective response of the probe to viscosity is due to the inhibition of intramolecular charge transfer in high-viscosity solvents. The probe can detect the viscosity changes in cells and observe the rise of cell viscosity when treated with LPS or nystatin. In addition, the probe can also successfully detect the viscosity changes in acute alcoholic liver injury and liver ischemia-reperfusion injury. These research results show that the probe has the potential to be used as a viscosity visualization tool and can be used for rapid detection of diseases caused by viscosity changes. SUMMARY

[0005] In order to solve the above problems, the present application provides a fluorescent probe for detecting the viscosity of a cell microenvironment, a preparation method thereof and a special detection kit.

[0006] The technical scheme of the present application is implemented as follows: a fluorescent probe for detecting cell microenvironment viscosity, which is 2-((E)-2-(6-((Z)-(4-(dimethylamino)phenyl)azo)-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-ol, and its structural formula is shown as formula I.

[0007]

[0008] The present application provides a preparation method of a fluorescent probe for detecting cell microenvironment viscosity, comprising the following steps:

[0009] Step one: under the condition of nitrogen protection at 55 DEG C, 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethenyl)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-ol shown in formula III is mixed with 3-aminobenzenethiol in an organic solvent to react for 12 hours, to obtain a compound shown in formula II,

[0010]

[0011] Step two: under the condition of a catalyst and nitrogen protection at 0 DEG C, (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-ol shown in formula II is mixed with N,N-dimethylaniline in an organic solvent to react for 1 hour, to obtain a compound shown in formula I,

[0012]

[0013]

[0014] Preferably, the catalyst is an organic acid; the organic acid is trifluoroacetic acid or sulfamic acid.

[0015] Preferably, the feeding molar ratio of (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-ol, N,N-dimethylaniline and the catalyst shown in formula II is 1:0.5-2:0.5-2.

[0016] Preferably, the feeding molar ratio of (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-ol, N,N-dimethylaniline and the catalyst shown in formula II is 1:2:2.

[0017] Preferably, the organic solvent in step one is N,N-dimethylformamide;

[0018] Preferably, the organic solvent in step two is at least one of dichloromethane and acetonitrile.

[0019] The application also provides a special detection kit, comprising the compound of formula I in claim 1 and a solvent,

[0020] The concentration of the compound of formula I is 1 mM,

[0021] The solvent is ethanol or dimethyl sulfoxide.

[0022] Preferably, a buffer is further included,

[0023] The buffer is a phosphate buffer with a pH value of 6.0-8.0, the phosphate is at least one of Na2HPO4, NaH2PO4 and KH2PO4, and the concentration of the phosphate is 0.01-0.5 M.

[0024] The application provides the use of the microenvironment viscosity detection kit in measuring the microenvironment viscosity, especially in detecting the change of the microenvironment viscosity in a biological system, which all belong to the protection scope of the application.

[0025] The method for measuring the microenvironment viscosity comprises the following steps:

[0026] 1) detecting the viscosity change in the sample to be measured:

[0027] Taking 766 nm as the excitation wavelength, the fluorescence intensity of a series of mixed solutions of water and glycerol with different proportions at an emission wavelength of 806 nm is measured, and a spectrum curve is drawn with the wavelength as the abscissa and the fluorescence intensity as the ordinate.

[0028] In the above detection method, the volume ratio of water to glycerol in the series of mixed solutions of water and glycerol with different proportions is 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in turn.

[0029] The volume of the series of mixed solutions of water and glycerol with different proportions is 1 mL.

[0030] The volume ratio of the reagent stock solution in the viscosity detection kit is 1 mL:10 μL.

[0031] The microenvironment viscosity detection kit provided by the application has the following characteristics:

[0032] 1) It has a long fluorescence emission wavelength (806 nm) and can be detected by fluorescence spectroscopy, and can be used for detecting the change of the microenvironment viscosity of cells under physiological conditions.

[0033] 2) The reaction only occurs in the presence of a high viscosity solvent (glycerol), other common inorganic salts, active nitrogen, active oxygen, amino acids, vitamins, biological thiol species do not produce interference.

[0034] The present application synthesizes a near-infrared fluorescent probe capable of tracking viscosity changes of organisms, wherein N, N-dimethylaniline is selected as an electron donor, and a sulfur-substituted semicyan dye is selected as an electron acceptor, and a D-π-A structure is formed by double bond coupling. The viscosity recognition mechanism of the probe is that the electron donor and the electron acceptor can freely move around the N=N double bond in a low viscosity environment. When stimulated by photons, the energy of the excited state is released in a non-radiation manner through torsional intramolecular charge transfer (TICT), thereby showing weak fluorescence. In a high viscosity environment, intramolecular rotation is limited, and the energy of the excited state is emitted in the form of light radiation, thereby possibly producing strong fluorescence. In a high viscosity solvent, the molecule is limited in the planar configuration of the excited state, thereby greatly improving the possibility of the probe returning to the ground state through fluorescence emission. Therefore, in a high viscosity solvent, the fluorescence emission of the probe will obviously increase. The probe is very simple to make, only two stages are needed, and the process is simple and convenient. The probe has an excitation wavelength (766 nanometers) and an emission wavelength (806 nanometers) in the near-infrared region, and is therefore very suitable for biological imaging. The probe has excellent viscosity sensitivity, and also has advantages such as small cytotoxicity and minimal background interference, the probe is used to evaluate the viscosity of the entire cytoplasmic matrix, and the intracellular viscosity level induced by drugs is observed, the probe can also successfully detect the viscosity changes in acute alcoholic liver injury and liver ischemia-reperfusion injury. Most importantly, the application of the probe is extended to viscosity detection in a mouse alcoholic liver inflammatory injury model, providing solid evidence for subsequent animal-scale comprehensive research. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0036] Figure 1 Chemical reaction equation for preparing the compound represented by formula I in the present application;

[0037] Figure 2 Absorption and fluorescence spectra of the detection kit reacting with different volume ratios of water and glycerol;

[0038] Figure 3 Fluorescence emission spectrum of the detection kit for reaction with various interfering species;

[0039] Figure 4 for a kit for cytotoxicity assay;

[0040] Figure 5 for a kit for detecting viscosity change in drug-induced cells;

[0041] Figure 6 for a kit for detecting viscosity change in alcoholic fatty liver cells;

[0042] Figure 7 for a kit for detecting viscosity change in ischemia-reperfusion cells;

[0043] Figure 8 for a kit for detecting viscosity change in acute alcoholic liver injury mice;

[0044] Figure 9 for a hydrogen spectrum of Formula II (400 MHz, DMSO-d6, 298 K);

[0045] Figure 10 for a carbon spectrum of Formula II (125 MHz, CDCl3, 298 K);

[0046] Figure 11 for a hydrogen spectrum of Formula I (400 MHz, CDCl3, 298 K);

[0047] Figure 12 for a carbon spectrum of Formula I (125 MHz, CD3OD, 298 K). DETAILED DESCRIPTION

[0048] The present application is described below by way of specific examples, but the present application is not limited thereto.

[0049] The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biological materials described are all commercially available unless otherwise specified.

[0050] Example 1, Preparation of 2-((E)-2-(6-((Z)-(4-(dimethylamino)phenyl)azo)-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-olate shown in Formula I. According to the procedure described in Figure 1The chemical reaction scheme shown was prepared by the following procedure: 3- aminothiophenol (40 mg, 0.32 mmol) and NaH (7 mg, 0.3 mmol) were mixed in a flask containing N,N-dimethylformamide (5 mL) and stirred at room temperature under nitrogen atmosphere for 10 min. Then, a solution of 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3- trimethylindolin-2-ylidene)ethylidene)cyclohex-1 -en-1 -yl)vinyl)-1,3,3-trimethyl-3H- indolium (96.8 mg, 0.16 mmol) in N,N-dimethylformamide (1 mL) was added to the mixture and the reaction mixture was heated at 55 °C for 12 h. After cooling, the solvent was concentrated under reduced pressure to give a crude product which was purified by column chromatography using dichloromethane / methanol (10:1, v / v) as eluent to give product II 37 mg. The hydrogen spectrum, carbon spectrum of product II are shown in Figures 1 and 2, respectively. Figure 9 、 10

[0051] The structural characterization data of this product II are as follows:

[0052] 1 H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 13.9 Hz, 1 H), 7.69 (d, J = 7.4 Hz, 1 H), 7.54 (d, J = 7.9 Hz, 1 H), 7.48 (t, J = 7.4, 3H), 7.37 (t, J = 8.2 Hz, 1 H), 6.90 (s, 1 H), 6.80 (d, J = 8.6 Hz, 1 H), 6.68 (s, 2H), 6.43 (d, J = 14.0 Hz, 1 H), 3.75 (s, 3H), 2.73 (s, 2H), 2.67 (s, 2H), 1.83 (s, 2H), 1.71 (s, 6H);

[0053] 13 C NMR (125 MHz, CDCI3) δ 101.24, 96.14, 95.02, 94.59, 93.68, 93.25, 92.39, 91.55, 90.44, 90.1 1, 89.22, 88.47, 86.89, 86.05, 85.56, 83.31, 71.21, 70.12, 65.72, 65.02, 64.76, 64.41, 63.45, 63.02, 60.63.

[0054] ​The product II (23 mg, 0.057 mmol) was put into acetonitrile / dichloromethane / trifluoroacetic acid (volume ratio 1:4:0.01, total volume 5 mL), sodium nitrite (8 mg, 0.12 mmol) was added, the solution was stirred for 15 min, amidosulfonic acid (12 mg, 0.12 mmol) was added, and the solution was stirred for 7 min. Subsequently, N,N-dimethylaniline (46 μL, 361 μmol) dissolved in acetonitrile (1 mL) was added to the reaction mixture, which was stirred under N2atmosphere at 0°C for 1 h. After the reaction was completed, the solvent was concentrated under reduced pressure after cooling, and the crude product was purified by column chromatography using dichloromethane / methanol (10:1, v / v) as the eluent to obtain 21.9 mg of product.

[0055] The structural characterization data of the product are as follows:

[0056] 1 H NMR (400 MHz, CDC13) δ 8.35 (d, J = 14.4 Hz, 1H), 7.90 (t, J = 10.0 Hz, 3H), 7.83 (s, 1H), 7.54-7.49 (m, 4H), 7.46-7.43 (m, 2H), 7.11 (d, J = 14.5 Hz, 1H), 7.05 (s, 1H), 6.77 (d, J = 8.6 Hz, 2H), 4.24 (s, 3H), 3.14 (s, 6H), 2.90 (s, 2H), 2.78 (s, 2H), 1.99 (s, 2H), 1.81 (s, 6H);

[0057] 13 C NMR (151 MHz, CD3OD) δ 158.12, 153.68, 144.06, 143.93, 142.73, 141.29, 139.70, 134.66, 131.42, 129.74, 127.10, 126.63, 126.46, 126.34, 123.42, 123.20, 117.99, 112.56, 111.91, 108.21, 102.85, 33.26, 32.12, 31.75, 28.75, 28.17, 28.16, 28.10, 27.48, 27.48, 26.42, 21.92.

[0058] From the above Figure 8 , Figure 9 it can be known that the product is correct in structure, which is 2-((E)-2-(6-((Z)-(4-(dimethylamino)phenyl)diazenyl)-2,3-dihydro-1H-thioxin-4-yl)vinyl)-1,3,3-trimethyl-3H-indolium-1-olate, formula I, the hydrogen spectrum and carbon spectrum of formula I are as follows: Figure 11 ,12 as shown.

[0059] Example 2: Spectral properties of the compound shown in formula I reacting with different volume ratios of water and glycerol mixed solution

[0060] 10 μL of the above mother liquor was added to 10 mM phosphate buffer solution, then different volume ratios of water and glycerol mixed solution were added, and then 10 mM phosphate buffer solution was added to 1 mL. After 20 min of reaction at 37°C, the UV-visible absorption spectrum and fluorescence emission spectrum were measured. The fluorescence emission spectrum was measured at 766 nm excitation; the excitation and emission slit width was 5 nm; the voltage was 700 V.

[0061] Figure 2 Fluorescence spectrum of reagent 1 reacting with different volume ratios of water and glycerol mixed solution

[0062] Figure 2 The results show that the reagent 1 in the present application has the following characteristics:

[0063] 1) The probe is light green in solution and has low fluorescence, but as the volume ratio of glycerol increases, the probe produces absorption at about 790 nm and red fluorescence at 806 nm;

[0064] 2) The intensity of UV-visible absorption and fluorescence intensity increases with the increase of the volume ratio of glycerol;

[0065] 3) When 10 μM of reagent 1 is used, the fluorescence enhancement is linearly related to the volume ratio of glycerol.

[0066] Example 3: Reaction of the compound shown in formula I with other species

[0067] Various substances were added to the buffer solution: 1. blank, 2. Ca 2+ (100 μM), 3. K + (100 μM), 4. Mg 2+ (100 μM), 5. Cu 2+ (100 μM), 6. Cr 3+ (100 μM), 7. Ni + (100 μM), 8. Hg 2+ (100 μM), 9. Pb 2+ (100 μM), 10. Ag + (100 μM), 11. Tyrosinase (5 U / mL), 12. NaClO (10 μM), 13. ONOO -(10 μM), 14. GSH (10 μM), 15. Cys (10 μM), 16. Hcy (10 μM), 17. Ser (100 μM), 18. HSO3 - (10 μM), 19. Ala (10 μM), 20. NTR (5 μg / mL), 21. Chymotrypsin (100 μg / mL), 22. HSA (10 μg / mL), 23. BSA (10 μg / mL), 24. β-Gal (4 U / mL), 25. ALDH (10 U / mL), 26. · OH (10 μM), 27. 1 O2(10 μM), 28. Glycerol (20% water content). After 20 min of reaction at 37 °C, the fluorescence emission spectrum was measured. The excitation wavelength was 766 nm; the excitation and emission slit widths were 5 nm; and the voltage was 700 V.

[0068] 10 μL of reagent 1 stock solution (1 mM) was added to 1 mL of the solution containing the above-mentioned various substances.

[0069] Figure 3 The fluorescence emission spectrum obtained when reagent 1 (10 μM) was mixed with various other substances.

[0070] The results of the experiment showed that only glycerol could cause reagent 1 to produce a significant optical signal response, proving that the reagent has a high degree of selectivity for high-viscosity solvents (glycerol), and the presence of other species does not interfere with the determination of viscosity.

[0071] Example 4: Quantitative determination of changes in the intracellular microenvironment viscosity under physiological conditions using a kit.

[0072] 1) Cell culture:

[0073] Cancer cells HepG2, pancreatic cancer cells Panc02-H7 and normal liver cells L02 were grown in glass-bottomed culture dishes (Corning Inc.) in DMEM (Dulbecco's moditied eagle media) liquid medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, at an environmental temperature of 37 °C and a carbon dioxide concentration of 5%.

[0074] 2) Kit toxicity determination

[0075] Different concentrations (0 μmol / L, 1 μmol / L, 2 μmol / L, 2.5 μmol / L, 3 μmol / L, 3.5 μmol / L, 4 μmol / L, 4.5 μmol / L, 5 μmol / L, 7.5 μmol / L) of the kit were incubated with liver cancer cells HepG2, pancreatic cancer cells Panc02-H7 and normal liver cells L02, respectively, 5 x 10 3 cells per well, and cultured at 37°C and 5% CO2 for 24 hours. After the cells were completely adhered to the wall, 6 replicate wells were prepared for each concentration, and cultured for another 12 hours. Then 100 μL of MTT solution (diluted to 0.5 mg / mL with DMEM) was added to each well. After 4 hours, the MTT solution was removed, 100 μL of DMSO was added to each well and shaken for 10 minutes. Then the absorbance at 490 nm was measured on a microplate reader to detect the toxicity of the kit, as Figure 4 .

[0076] 3) The kit detects the changes in cell viscosity induced by drugs.

[0077] The cells were treated as follows: the control group was continued to be cultured with the culture medium, the experimental group was divided into 2 groups, and 0.2 μg / mL and 0.7 μg / mL lipopolysaccharide were incubated with the cells for 12 hours, respectively, 3 replicate wells were prepared for each concentration, then washed with PBS for 3 times, and then incubated with the kit (5 μmol / L) and the nuclear tracer Hoechst 33342 (5 μmol / L) for 30 minutes. The culture medium was discarded, washed with PBS, and finally 100 μL of DMEM was added for high-content cell imaging analysis, as Figure 5 The cell imaging experiment showed that the HepG2 cells treated with the kit only showed weak red fluorescence. In the experimental group of cells pretreated with LPS, the cells showed stronger red fluorescence, and the fluorescence intensity increased with the concentration. This indicates that even under the condition of lipopolysaccharide stimulation, the kit can achieve clear imaging effect on HepG2 cells.

[0078] 4) The kit detects the changes in viscosity in alcoholic fatty liver

[0079] The cells were treated as follows: the control group was continued to be cultured with the culture medium, the experimental group was divided into 3 groups, and 200 μmol / L oleic acid, 2% ethanol, and 200 μmol / L oleic acid + 2% ethanol were incubated with the cells for 1 hour, respectively, 3 replicate wells were prepared for each concentration, then washed with PBS for 3 times, and the imaging steps were the same as step 3), as Figure 6, imaging showed that cells treated with the kit alone exhibited weak fluorescence, while cells treated with oleic acid or ethanol or both oleic acid and ethanol followed by treatment with the kit showed a significant increase in fluorescence in all three treatment groups. Overall, these findings confirmed that the kit we designed was able to distinguish between normal and alcoholic fatty liver cells and revealed that there was a change in viscosity in alcoholic fatty liver cells.

[0080] 5) Kit for detecting changes in viscosity in cells subjected to ischemia-reperfusion

[0081] The cells were treated as follows: the control group was continued to be cultured with the culture medium, the experimental group was divided into three groups, and each group was incubated with serum-free low-glucose DMEM medium for 30 minutes, and then reperfused with serum-containing high-glucose DMEM medium for 30 minutes, and each concentration had three replicate wells, then washed with PBS for 3 times, and the imaging step was the same as step 3), as follows: Figure 7 , imaging showed that the red fluorescence of the control group stained only with the kit was weak, the fluorescence of the ischemia 30-minute group was enhanced, and the reperfusion 30-minute group showed a stronger red fluorescence signal enhancement, indicating that the cell viscosity of the ischemia-reperfusion liver cells was significantly higher than that of normal liver cells.

[0082] 6) Kit for detecting changes in viscosity in acute alcoholic liver injury mice.

[0083] Control group: PBS-treated mice group; AALI group: mice orally administered Chinese liquor (20 mL / kg) at 8:30 am and 8:30 pm for 3 consecutive days. The ethanol content of Chinese liquor was 52%. Before imaging, the mice were injected with 100 μM probe through the tail vein, and the images were collected 30 minutes later. Then, fluorescence imaging was performed using the IVIS Spectrum in vivo fluorescence imaging system. After anesthesia, the kidneys and organs such as heart, liver, spleen, and lungs were washed with PBS (pH 7.4), and image analysis of organ distribution was performed. All isolated organs were fixed in 10% paraformaldehyde for histological staining. The samples were dehydrated, embedded, sectioned, and stained with hematoxylin and eosin, as follows: Figure 8 .

[0084] PBS-pretreated mice (control group) and acute alcoholic liver injury (AALI) group. Then, the dissected mouse liver organs were imaged, and the liver tissue of the control mice showed weak red fluorescence. In the case of AALI mice, the red fluorescence in the liver tissue was significantly increased, which meant that the viscosity change in the alcohol-damaged tissue was significantly greater. In addition, the dissected mouse organs were imaged in vitro, which clearly showed that the liver fluorescence was significantly higher than that of other organs such as heart, spleen, lung, and kidney. This indicated that the kit could be used to detect viscosity changes in acute alcoholic mice in vivo.

[0085] The liver tissues of control group and AALI mice were treated with hematoxylin and eosin (H&E) staining. The liver cells of control group mice were normal in morphology, and the arrangement of liver cord was neat, radial, and clear in distribution. We can see that the sections of control group showed no obvious pathological changes and inflammatory changes, while the liver tissues of AALI group showed cell swelling and inflammatory cell infiltration. The results of H&E staining clearly showed that the AALI mouse group experienced more severe liver injury compared with the control group, which was consistent with the results of fluorescence imaging. There were no obvious pathological changes in other organs of the two groups of mice.

[0086] Finally, it should be noted that the above examples are only given with reagent 1 as the fluorescent reagent, and the concentrations of 1 and phosphate buffer in the reaction system are 10 μM and 10 mM, respectively, and the reaction time is 20 min. The results of other fluorescent reagent concentrations and reaction times are not listed one by one, but they are not intended to limit the present application. Any person skilled in the art should be able to make various modifications and changes without departing from the spirit and scope of the present application.

Claims

1. A fluorescent probe for detecting the viscosity of a cell microenvironment, characterized in that: The fluorescent probe is 2-((E)-2-(6-((Z)-(4-(dimethylamino)phenyl)azo)-2,3-dihydro-1H-thioxanthen-4-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium, and its structural formula is shown in Formula I:

2. The method for preparing a fluorescent probe for detecting cell microenvironment viscosity according to claim 1, wherein: The following steps are involved: Step 1: Under nitrogen protection at 55°C, 2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium represented by Formula III is mixed and reacted with 3-aminothiophenol in an organic solvent for 12 hours to obtain a compound represented by Formula II. Step 2: Under the conditions of a catalyst and nitrogen protection at 0°C, (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ammonium represented by Formula II and N,N-dimethylaniline are mixed and reacted in an organic solvent for 1 hour to obtain the compound represented by Formula I.

3. The method for preparing a fluorescent probe for detecting cell microenvironment viscosity according to claim 2, wherein: The molar ratio of (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-ammonium, N,N-dimethylaniline and catalyst described in formula II is 1:0.5~2:0.5~2.

4. The method for preparing a fluorescent probe for detecting viscosity of a cell microenvironment according to claim 3, wherein: The molar ratio of (E)-2-(2-(6-amino-2,3-dihydro-1H-thioxanthene-4-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ammonium, N,N-dimethylaniline and the catalyst described in formula II is 1:2:

2.

5. The method for preparing a fluorescent probe for detecting viscosity of a cell microenvironment according to claim 2, wherein: The catalyst is an organic acid; the organic acid is trifluoroacetic acid or aminosulfonic acid.

6. The method for preparing a fluorescent probe for detecting viscosity of a cell microenvironment according to claim 2, wherein: The organic solvent in step 1 is N,N-dimethylformamide; The organic solvent in step 2 is at least one of dichloromethane and acetonitrile.

7. A special detection kit, characterized in that: Comprising the compound represented by formula I in claim 1 and a solvent, The concentration of the compound represented by formula I is 1 mM, The solvent is ethanol or dimethyl sulfoxide.

8. A special detection kit according to claim 7, characterized in that, Also includes buffer, The buffer solution is a phosphate buffer solution with a pH value of 6.0 to 8.0, wherein the phosphate is selected from at least one of Na2HPO4, NaH2PO4 and KH2PO4; and the concentration of the phosphate is 0.01 to 0.5M.

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