A fluorescent probe with pH and viscosity response function and preparation and application thereof

Abstract

The application discloses a fluorescent probe with pH value and viscosity response functions as well as preparation and application thereof. 24 H 24 NOSI, and a chemical structural formula is as follows: The fluorescent probe has the advantages of simple preparation, good water solubility, strong anti-interference capability and high rotor efficiency; the fluorescent probe provides a visual means for identification of cancer cells and non-alcoholic fatty liver, and provides a design idea for construction of the fluorescent probe.

Description

TECHNICAL FIELD

[0001] The present application relates to a new compound and its preparation and application, in particular to a fluorescent probe with pH value and viscosity response function and its preparation and application. BACKGROUND

[0002] In recent years, due to the increasing incidence and mortality of cancer, cancer has posed a serious threat to human life and health. If it can be detected early and treated in time, it can greatly improve the survival rate of patients. Therefore, it is of great importance to identify tumor markers to achieve early diagnosis and treatment of cancer. Viscosity is an important indicator of normal cell growth, and it is one of the key factors to maintain normal cell function, affecting cell metabolism and differentiation, and having important significance for cell physiological processes. Abnormal cell viscosity is related to many diseases, such as Parkinson's disease, diabetes and sickle cell disease, etc. In addition, the normal operation of cells is also affected by the balance of acid-base balance, and the imbalance of pH homeostasis is related to the occurrence of many diseases, including metabolic diseases, hepatic encephalopathy and hyperchloremia, etc. It is worth noting that cells can sense changes in the surrounding microenvironment, regulate cell morphology and affect physiological processes. Compared with ordinary cells, cancer cells have a unique microenvironment, such as low pH, high viscosity, high active oxygen level, etc. This unique microenvironment is conducive to the survival and metastasis of cancer cells. Viscosity and pH value can be used as important parameters to distinguish cancer cells and normal cells, therefore, real-time monitoring of changes in cell viscosity and pH value is of great significance to understand the normal operation mechanism of cells and elucidate the principle of the occurrence of related diseases.

[0003] Mitochondria is the main place of cell energy production, which controls the signal pathway of longevity and health, and determines the fate of cells. Therefore, the balance of mitochondrial microenvironment is very important, which affects the physiological activities of cells. There are many factors that affect the mitochondrial microenvironment, such as the content of proteases, oxidative factors, pH value and viscosity. Among them, pH and viscosity are particularly important, viscosity can directly affect the signal transduction and energy generation of mitochondria, and pH affects the metabolism of mitochondria.

[0004] Non-alcoholic fatty liver disease includes a series of liver lesions, which is usually caused by the accumulation of fat metabolism in the liver. At the same time, the accumulation of lipid droplets in hepatocytes increases the viscosity of mitochondria, causing abnormal mitochondrial function. Therefore, monitoring the changes of mitochondrial viscosity is of great significance to the study of non-alcoholic fatty liver disease.

[0005] There are many traditional methods to measure viscosity and pH value, such as rotary viscometer method, capillary viscometer method 、Acid-base indicator method and pH meter, etc. However, these detection methods are only suitable for solution detection, and are not suitable for detecting the viscosity and pH value of the internal environment of in vitro cells and the viscosity and pH changes in vivo. Studies have found that fluorescent probes have the characteristics of easy operation, high sensitivity and high resolution, and can be used for biological monitoring. In recent years, many fluorescent probes have been used to monitor the changes of intracellular viscosity and pH value. Mitochondria is the key place of aerobic respiration of cells, and it is particularly important to develop fluorescent probes that can efficiently target mitochondria and sensitively detect the changes of cell viscosity and pH value in understanding the influence of viscosity and pH on mitochondrial diseases. At present, most of the fluorescent probes for monitoring the changes of mitochondrial viscosity are cationic fluorescent probes designed based on twisted intramolecular charge transfer (TICT). Such fluorescent probes have the advantages of fast response and high spatial resolution, and the non-radiative decay of the excited state is affected by the viscosity of the surrounding environment. The rotor of such fluorescent molecules generally has a rotatable conjugated group, so the rotor rotates rapidly in a low-viscosity medium, reduces the conjugation degree of the molecule, consumes a lot of energy, and reduces the fluorescence. However, under high viscosity conditions, rotation is blocked, and fluorescence is enhanced. However, such molecules still have defects, such as poor water solubility, high background interference, complex synthesis, and low rotor efficiency. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a fluorescent probe with pH value and viscosity response function and its preparation and application. The fluorescent probe of the present application has the characteristics of simple preparation, good water solubility, strong anti-interference ability and high rotor efficiency.

[0007] The technical scheme of the present application is as follows:

[0008] A fluorescent probe with pH value and viscosity response function, its molecular formula is C 24 H 24 NOSI, its chemical structural formula is as follows:

[0009]

[0010] A preparation method of the aforementioned fluorescent probe with pH value and viscosity response function, comprising the following steps:

[0011] S1. Dissolve 5-bromothiophene-2-formaldehyde, p-hydroxyphenyl boronic acid and tetrakis(triphenylphosphine) palladium in tetrahydrofuran, then add potassium carbonate aqueous solution to obtain a mixed solution;

[0012] S2. Under the protection of nitrogen, heat the mixed solution to reflux and stir at 70-90℃ for 10-20 hours, then quench with water, extract the water layer with dichloromethane, and concentrate the extract under reduced pressure to obtain yellow oil;

[0013] S3. Purify the yellow oil by column chromatography to obtain compound 1;

[0014] S4. Compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide are mixed in ethanol, heated to reflux for 5-12 hours, then cooled to room temperature, and a purple solid is precipitated, which is filtered off and washed with ethanol to obtain a fluorescent probe with pH value and viscosity response functions.

[0015] Preferably, in the preparation method of the aforementioned fluorescent probe with pH value and viscosity response functions, the mass ratio of 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium is 9-10:6-8:1.

[0016] Preferably, in the preparation method of the aforementioned fluorescent probe with pH value and viscosity response functions, the mass concentration of potassium carbonate in the aqueous potassium carbonate solution is 20-25%, and the mass ratio of potassium carbonate to tetrakis(triphenylphosphine)palladium is 40-50:1.

[0017] Preferably, in the preparation method of the aforementioned fluorescent probe with pH value and viscosity response functions, when the column chromatography purification is performed, the mobile phase is petroleum ether / ethyl acetate=1 / 1, v / v.

[0018] Preferably, in the preparation method of the aforementioned fluorescent probe with pH value and viscosity response functions, the mass ratio of compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide is 8:12-13.

[0019] The application of the aforementioned fluorescent probe with pH value and viscosity response functions in the preparation of a mitochondrial probe.

[0020] The application of the aforementioned fluorescent probe with pH value and viscosity response functions in monitoring the changes of pH value and viscosity in a solution.

[0021] A fluorescent probe for monitoring the changes of pH value and viscosity in cells, comprising the aforementioned fluorescent probe with pH value and viscosity response functions.

[0022] A fluorescent probe for identifying non-alcoholic fatty liver, comprising the aforementioned fluorescent probe with pH value and viscosity response functions.

[0023] The beneficial effects of the present application are as follows:

[0024] 1. The preparation process of the fluorescent probe of the present application is simple and easy to implement in industrialization.

[0025] 2. This invention uses phenol as an electron donor (D), thiophene groups as electron donors and partial π-bridges, and introduces them into a traditional hemicyanine molecule to construct a fluorescent probe HTC with a D-D'(π)-π-A configuration. HTC carries a positive charge, can target mitochondria, and contains phenolic hydroxyl groups, which can increase the probe's water solubility and responsiveness to changes in pH.

[0026] 3. More importantly, the fluorescent probe HTC of this invention provides two viscosity recognition sites: a single bond between the benzene ring and the thiophene ring, and a double bond between the thiophene ring and the indole ring. This effectively improves sensitivity and anti-interference capability. In low-viscosity media, the HTC molecule rotates freely within its molecules, leading to an increased non-radiative decay rate and almost no fluorescence. However, in high-viscosity media, the intramolecular rotation of the probe is hindered, resulting in enhanced fluorescence. Because the probe contains Ar-OH, it is sensitive to changes in environmental pH.

[0027] 4. The fluorescent probe of the present invention has pH-responsive and viscosity-responsive properties, can be used for cell fluorescence imaging, can target mitochondria, and can distinguish between normal cells and cancer cells. In addition, the fluorescent probe of the present invention can also identify non-alcoholic fatty liver disease.

[0028] In summary, the fluorescent probe of the present invention has the advantages of simple preparation, good water solubility, strong anti-interference ability, and high rotor efficiency; the fluorescent probe of the present invention provides a visualization means for the identification of cancer cells and the differentiation of non-alcoholic fatty liver disease, and provides design ideas for the construction of fluorescent probes. Attached Figure Description

[0029] Appendix Figure 1 The crystal structure ellipsoid diagram of the fluorescent probe HTC of this invention (ellipsoid probability is 30%) is shown.

[0030] Appendix Figure 2 The fluorescent probe HTC of this invention forms a one-dimensional helical structure through atypical C12—H12···O1 and C23—H23A···O1 hydrogen bonds;

[0031] Appendix Figure 3 The absorbance of the fluorescent probe HTC of this invention in water and glycerol;

[0032] Appendix Figure 4 The fluorescence changes of the fluorescent probe HTC in different viscosity systems are shown in this invention.

[0033] Appendix Figure 5 This is a linear correlation diagram of the viscosity of the fluorescent probe HTC of this invention;

[0034] Appendix Figure 6 The fluorescence spectra of the fluorescent probe HTC of this invention in solutions of different polarities are shown below.

[0035] Appendix Figure 7Quantum yield of the fluorescent probe HTC of the present application;

[0036] Figure 1 Figure 8 Specificity detection graph of the fluorescent probe HTC of the present application responding to viscosity;

[0037] Figure 2 Figure 9 Spectrum response graph of the fluorescent probe HTC of the present application when pH value is 7-10.5;

[0038] Figure 3 Figure 10 Toxicity graph of the fluorescent probe HTC of the present application in LO2, HepG2, and 4T1;

[0039] Figure 4 Figure 11 Fluorescence imaging of the fluorescent probe HTC of the present application when cells were pre-incubated with serum-free medium for 4h, then co-stained with HTC and Mito-Tracker Green (200 μM). Green Channel: fluorescence image of Mito-Tracker Green (200 μM) (λex= 488 nm, λem= 500-550 nm). Red Channel: fluorescence image of HTC (5 μM) (λex= 520 nm, λem= 610-630 nm). Merge: fluorescence image of Green Channel and Red Channel merged.

[0040] Figure 5 Figure 12 Confocal imaging of PBS-induced HepG2 cells at different pH values (A). Scale bar: 10 μM. Quantitative fluorescence intensity of red channel (B).

[0041] Figure 6 Figure 13 Fluorescence image of viscosity change of the fluorescent probe HTC (5 μM) in HepG2. (A) Control group: co-incubate HTC and cells for 2h. HBSS group: pre-incubate cells with serum-free HBSS medium for 2h, then incubate with HTC for 2h. Nys group: first incubate HTC and HepG2 for 2h, then incubate with Nys (10 μM) for 30 min. (B) Relative fluorescence pixel intensity. λ ex = 520 nm, λ em = 610-630 nm; scale bar: 10 μM.

[0042] Figure 7 Figure 14(A) Serum ALT and AST levels in normal and fatty liver mice. (B) Relative pixel intensity of (D). (C) HE staining of mouse liver tissue sections, scale bar: 10 μM. (D) Fluorescence imaging of normal and fatty liver mice after injection of HTC (10 μM). (E) Fluorescence imaging of major organs in normal and fatty liver mice: 1. Heart, 2. Lung, 3. Kidney, 4. Spleen, 5. Liver; λ ex =520nm,λ em =610-630nm. (F) Urine imaging images of normal mice and fatty liver mice collected after 8h. (G) Metabolic fluorescence images of the probe in mice from 0h to 8h after injection of HTC (10μM).

[0043] Appendix Figure 15 This is the high-resolution mass spectrum of the fluorescent probe HTC of this invention;

[0044] Appendix Figure 16 The fluorescent probe HTC of this invention 1 H NMR spectrum;

[0045] Appendix Figure 17 The fluorescent probe HTC of this invention 13 C NMR spectrum;

[0046] Figure 1 and Figure 2 The crystal structure of HTC shows that a C=C double bond connects the indole and thiophene rings, with the C=C rings in an "E" configuration. The thiophene and benzene rings are essentially parallel, with a dihedral angle of 3.30° between them. The dihedral angle between the indole and thiophene rings is 27.19°. The probe HTC's phenolic hydroxyl group and I... - Ions form intermolecular O1—H1···I1 hydrogen bonds. Due to the presence of hydrogen bonds, it is difficult for hydrogen to leave the phenolic hydroxyl group, so the probe has a large pKa. The probe HTC extends to form a one-dimensional helical structure through atypical intermolecular C12—H12···O1 and C23—H23A···O1 hydrogen bonds. Detailed Implementation

[0047] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0048] Embodiments of the present invention

[0049] Example 1

[0050] A method for preparing a fluorescent probe with pH and viscosity responsiveness, comprising the following steps:

[0051] S1. Dissolve 5-bromothiophene-2-carboxaldehyde (0.95 g), p-hydroxyphenylboronic acid (0.70 g) and tetrakis(triphenylphosphine)palladium (0.10 g) in 50 ml of tetrahydrofuran, then quickly add 20 ml of aqueous potassium carbonate solution (22 wt%) to obtain a mixed solution;

[0052] S2. Stir the mixed solution under nitrogen protection at reflux for 12 hours, then quench with water, extract the water layer with dichloromethane, and concentrate the extract under reduced pressure to obtain a yellow oil;

[0053] S3. Purify the yellow oil by column chromatography (the mobile phase is petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0054] S4. Mix compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.62 g) in ethanol, heat to 80°C and reflux for 8 hours, then cool to room temperature, precipitate a purple solid, filter the solid and wash with ethanol 3 times to obtain a fluorescent probe with pH value and viscosity response functions.

[0055] Example 2

[0056] A method for preparing a fluorescent probe with pH value and viscosity response functions, the steps are as follows:

[0057] S1. Dissolve 5-bromothiophene-2-carboxaldehyde (0.90 g), p-hydroxyphenylboronic acid (0.60 g) and tetrakis(triphenylphosphine)palladium (0.10 g) in 50 ml of tetrahydrofuran, then quickly add 20 ml of aqueous potassium carbonate solution (20 wt%) to obtain a mixed solution;

[0058] S2. Stir the mixed solution under nitrogen protection at reflux for 10 hours, then quench with water, extract the water layer with dichloromethane, and concentrate the extract under reduced pressure to obtain a yellow oil;

[0059] S3. Purify the yellow oil by column chromatography (the mobile phase is petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0060] S4. Mix compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.60 g) in ethanol, heat to 70°C and reflux for 5 hours, then cool to room temperature, precipitate a purple solid, filter the solid and wash with ethanol 3 times to obtain a fluorescent probe with pH value and viscosity response functions.

[0061] Example 3

[0062] A method for preparing a fluorescent probe with pH value and viscosity response functions, the steps are as follows:

[0063] S1. Dissolve 5-bromothiophene-2-carboxaldehyde (1.0 g), p-hydroxyphenylboronic acid (0.80 g) and tetrakis(triphenylphosphine)palladium (0.10 g) in 50 ml of tetrahydrofuran, then quickly add 20 ml of aqueous potassium carbonate solution (25 wt%) to obtain a mixed solution;

[0064] S2. Stir the mixed solution under reflux for 20 hours under nitrogen protection, then quench with water, extract the water layer with dichloromethane, and concentrate the extract under reduced pressure to obtain a yellow oil;

[0065] S3. Purify the yellow oil by column chromatography (the mobile phase is petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0066] S4. Mix compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.65 g) in ethanol, heat to 90°C and reflux for 12 hours, then cool to room temperature, precipitate a purple solid, filter the solid and wash with ethanol 3 times to obtain a fluorescent probe with pH and viscosity response functions.

[0067] Experimental Example

[0068] 1. Spectral response to viscosity

[0069] In order to verify the relationship between the fluorescence intensity of the fluorescent probe HTC of the present application and the viscosity, we detected the absorbance of the probe in water and glycerol, and the results are shown in Figure 3 , using different proportions of glycerol and pure water as the viscosity detection system.

[0070] As shown in Figure 4 , by adjusting the volume ratio of glycerol, the viscosity of the solution gradually increases from 1.2 cp (pure water) to 499.5 cp (glycerol), and the fluorescence of the probe at 627 nm increases with the increase of the viscosity of the solution, and the fluorescence intensity increases by 20 times.

[0071] As shown in Figure 5 , according to the equation LogI 627nm = 0.396 Logη + 1.819 , there is a clear linear correlation between LogI 627nm and Log(viscosity) (R 2 = 0.989).

[0072] We examined the fluorescence spectrum of HTC in different polar solutions (the results are shown in Figure 6), the position of the maximum emission peak of HTC in different solvents is not affected by the solvent, all around 627 nm, the fluorescence in glycerol is the strongest, and the fluorescence in other solvents is weak, which is due to the Ar-OH and thiophene in HTC as electron donors, the C=N + as electron acceptor, forming a push-pull electron structure, at the same time, due to the single bond between the benzene ring and the thiophene ring and the double bond between the thiophene ring and the indole ring can rotate freely, thus promoting TICT and consuming the excited state energy of HTC in the non-radiative path, so the fluorescence is very weak. In high viscosity medium, the free rotation of molecules is hindered, the non-radiative energy consumption is inhibited, and the fluorescence is enhanced.

[0073] We detected the quantum yield of the probe, as shown in Figure 7 , the quantum yield of the probe in water is only 1.5%, and the quantum yield of the probe in glycerol is 32.5%, which is increased by 22 times.

[0074] In order to study the specificity of the probe to viscosity, we added analytes to the probe and detected the fluorescence intensity at 627 nm, the relative fluorescence intensity of HTC (10 μM) with various analytes (1: Blank; 2: Cu 2+ ; 3: Ca 2+ ; 4: H2O2; 5: Ag + ; 6: Na + ; 7: SO3 2- ; 8: Zn + ; 9: Br - ; 10: Cl - ; 11: F - ; 12: I - ; 13: CO3 2- ; 14: SO4 2- ; 15: GSH; 16: NO3 - ; 17: ClO - ; 18: C2O4 2- ; 19: Cys; 20: TBHP; 21: SNP; 22: Gly) at 627 nm, the results are shown in Figure 8 , which shows that the probe still has a high selective response to viscosity.

[0075] 2. Spectral response of the probe to pH value

[0076] Since the probe of the present application has Ar-OH, it is sensitive to pH value change, so we investigate the influence of pH value on the spectral properties of the probe. In the range of pH value from 7 to 10.5, with the increase of pH value, the absorption peak at 520 nm gradually decreases, the absorption peak at 600 nm gradually increases, and the isosbestic points at 424 nm and 535 nm appear (the results are shown in Figure 9(As shown in Figure A). This is because in alkaline solution, the phenolic hydroxyl group gradually loses a proton, leading to an enhanced ICT effect.

[0077] Similarly, Figure 9 The fluorescence spectrum of B showed that, under 520 nm excitation, the fluorescence intensity of the probe at 627 nm gradually decreased with increasing pH. Under 600 nm excitation, the fluorescence intensity of the probe at 720 nm gradually increased. Figure 9 (C). This matches the UV-Vis absorption spectrum changes of HTC, further confirming our hypothesis. Finally, we used the fluorescence intensity ratio of the probe at 621 nm and 720 nm (I0.05). 627nm / I 720nm Plotting the pH values ​​yields... Figure 9 As shown in D, the linear relationship is good, R 2 =0.99. According to the Henderson-Hasselbalch equation (pH = pKa + lg[(I... max -I) / (II min The pKa of the probe was found to be 8.6. This is consistent with the crystal structure we obtained, which shows phenolic hydroxyl groups and I... - Ions form intermolecular O1—H1···I1 hydrogen bonds, and the hydrogen in the phenolic hydroxyl group is difficult to leave, so the pKa is relatively large.

[0078] 3. Detection of mitochondrial targeting capabilities

[0079] Biocompatibility of probes is an important indicator for the application of probe biology and is crucial for the practical application of probes. Since the fluorescent probe of this invention has pH response and viscosity response, we attempt to detect pH and fluorescence imaging in living cells.

[0080] First, we examined the cytotoxicity of the probe. The CCK8 assay showed that after co-incubating cells with different concentrations (0-10 μM) of the probe for 24 hours, the survival rate of all cells was over 80%. Figure 10 This indicates that the probe has low toxicity to cells and good biocompatibility.

[0081] Next, the targeting ability of the probe to organelles was investigated. Mitochondria are double-membrane organelles with a negative membrane potential; therefore, the positively charged probe HTC can easily enter the mitochondrial matrix and is expected to target mitochondria. Cellular colocalization imaging experiments showed that the probe can target mitochondria for cellular fluorescence imaging (Pearson coefficient 0.91). Figure 11 This indicates that the probe can target mitochondria for cell fluorescence imaging.

[0082] 4. Intracellular pH response

[0083] The fluorescence imaging of HepG2 living cells under the environment of pH 6.0-8.0 was investigated by laser confocal microscope. The fluorescence intensity under the red channel was strong at pH 6 or 7, the red light under the red channel was quenched at pH 8, and the fluorescence intensity at pH 6 or 7 was 4 times of that at pH 8 ( Figure 12 ). The result was consistent with the fluorescence change at 627 nm in solution state, indicating that the probe HTC could detect the change of pH value in cells.

[0084] 5. Detecting the change of viscosity of living cells

[0085] In order to investigate whether the probe of the application could be used to detect the change of viscosity in living cells, HepG2 was selected as a cell model, and nystatin and serum-free HBSS medium were used as external and internal factors to enhance the mitochondrial viscosity of cells, and then the fluorescence imaging of cells was carried out. The results showed that only when the probe HTC was incubated with cells, weak red fluorescence could be observed. With the increase of mitochondrial viscosity of cells, the red light was obviously enhanced ( Figure 13 ). The results proved that HTC could monitor the change of cell viscosity induced by exogenous factors in living cells.

[0086] 6. Detecting NAFLD mouse model

[0087] It is known that fatty liver tissue has high viscosity. Therefore, we tried to identify the high viscosity organs in mice. High-fat diet was used to feed mice and CCl4-containing olive oil was injected intraperitoneally to establish a mouse model of non-alcoholic fatty liver. The mice were taken eyeball blood, and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice were measured, and the results are shown in Figure 14 A, the ALT and AST of fatty liver mice were higher than those of normal mice, indicating that the non-alcoholic fatty liver model was successfully established. Then, HTC was injected intraperitoneally into normal mice and fatty liver mice, respectively, and then fluorescence imaging was carried out by small animal imaging instrument, and the results are shown in Figure 14 C, the fluorescence of normal mice was weak, and the fluorescence of fatty liver mice was strong. The fluorescence of mouse abdomen was quantified ( Figure 14 D), and the fluorescence intensity of fatty liver mice was about 2 times of that of normal mice. It is proved that HTC can distinguish normal mice and fatty liver mice. Then, the mice were killed, and important organs were taken out for fluorescence imaging, and the results are shown in Figure 14 E, the fluorescence of liver of normal mice was weak, and the fluorescence of fatty liver model mice was strong, and the fluorescence of other organs was weak, which indicated that the probe was mainly enriched in liver. Subsequently, the liver sections of normal group and model group mice were stained by HE. As shown in Figure 14As shown in Figure B, normal mouse liver tissue showed no obvious morphological changes, while hepatocytes in fatty liver mice exhibited vacuolation. Tissue sections and serum measurements indicate that fatty liver occurs concurrently with liver damage. To observe the metabolic pathway of the probe in the model mice, we monitored the fluorescence changes of the probe in mice from 0 to 8 hours. Figure 14 (F) It was found that the probe fluorescence was strongest at 4 h, and gradually weakened from 4 to 8 h. Urine samples were collected from mice from 0 to 8 h. Figure 14 Fluorescence imaging using a small animal imaging system (G) revealed that the urine emitted a reddish glow, indicating that the probe was rapidly metabolized and excreted in the urine after entering the mouse body. These experimental results demonstrate that the HTC probe can assist in the detection of non-alcoholic fatty liver disease in mice, providing a visual means for the diagnosis of NALD.

[0088] In summary, we constructed a hemicyanine fluorescent probe, HTC, with a D-D'(π)-π-A configuration, using phenol as the electron donor (D), thiophene groups as the electron donor and a partial π-bridge. The fluorescent HTC carries a positive charge and can target mitochondria in cells, exhibiting a Pearson coefficient of 0.91 for co-localization fluorescence imaging with commercial mitochondrial probes. In solution, the probe exhibits pH and viscosity responsiveness. In cells, the probe can monitor changes in pH and viscosity, distinguishing between normal and cancer cells. Utilizing the viscosity-responsive property, we used the probe to identify the formation of non-alcoholic fatty liver disease in mice. Furthermore, the probe is readily metabolized in mice and exhibits good biocompatibility. This experiment provides a visual means for the identification of cancer cells and the differentiation of non-alcoholic fatty liver disease, offering new insights for the design of fluorescent probes.

[0089] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fluorescent probe having pH and viscosity response functions, characterized in that, The molecular formula of which is C 24 H 24 NOSI, the chemical structural formula of which is as follows:

2. A method for preparing the fluorescent probe having pH and viscosity response functions according to claim 1, characterized by, Comprising the following steps: S1. Dissolving 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium in tetrahydrofuran, then adding an aqueous potassium carbonate solution to obtain a mixed solution; S2. Refluxing and stirring the mixed solution under nitrogen protection for 10-20 hours, then quenching with water, extracting the water layer with dichloromethane, and concentrating the extract under reduced pressure to obtain a yellow oil; S3. Purifying the yellow oil by column chromatography to obtain compound 1; S4. Mixing compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide in ethanol, heating and refluxing at 70-90℃ for 5-12 hours, then cooling to room temperature to precipitate a purple solid, filtering the solid and washing with ethanol to obtain a fluorescent probe with pH value and viscosity response functions.

3. The method for preparing the fluorescent probe having pH and viscosity response function according to claim 2, characterized in that: The mass ratio of the 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium is 9-10:6-8:

1.

4. The method for preparing a fluorescent probe with pH and viscosity response functions according to claim 2, characterized in that: The mass concentration of potassium carbonate in the aqueous potassium carbonate solution is 20-25%, and the mass ratio of potassium carbonate to tetrakis(triphenylphosphine)palladium is 40-50:

1.

5. The method for preparing a fluorescent probe with pH and viscosity response functions according to claim 2, characterized in that: When the column chromatography purification is performed, the mobile phase is petroleum ether / ethyl acetate=1 / 1, v / v.

6. The method for preparing a fluorescent probe with pH and viscosity response functions according to claim 2, characterized in that: The mass ratio of the compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide is 8:12-13.

7. Use of the fluorescent probe with pH value and viscosity response functions according to claim 1 in preparing a mitochondrial probe.

8. Use of the fluorescent probe with pH value and viscosity response functions according to claim 1 in monitoring pH value and viscosity changes in a solution.

9. A fluorescent probe for monitoring changes in pH and viscosity in cells, characterized by: The fluorescent probe with pH value and viscosity response functions according to claim 1.

10. A fluorescent probe for discriminating non-alcoholic fatty liver, characterized by: The fluorescent probe with pH value and viscosity response functions according to claim 1.

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