Fluorescent probe with pH value and viscosity response function and preparation and application thereof

Abstract

The invention discloses a fluorescent probe with a pH value and viscosity response function as well as preparation and application of the fluorescent probe. The molecular formula of the fluorescent probe is C24H24NOSI, and the chemical structural formula of the fluorescent probe is as follows: # imgabs0 #. The fluorescent probe disclosed by the invention has the advantages of simplicity in preparation, good water solubility, strong anti-interference capability and high rotor efficiency; the fluorescent probe provided by the invention provides a visual means for identification of cancer cells and identification of the non-alcoholic fatty liver, and provides a design thought for construction of the fluorescent probe.

Description

Technical Field

[0001] The invention 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 Art

[0002] In recent years, due to the increasing incidence and mortality of cancer, cancer has become a serious threat to human life and health. If it can be detected and treated as early as possible, the survival rate of patients can be greatly improved. Therefore, it is crucial to identify tumor markers and achieve early diagnosis and treatment of cancer for human health. Viscosity is an important indicator of normal cell growth. It is one of the key factors to maintain normal cell function, affecting cell metabolism and differentiation, and is of great significance to the physiological process of cells. Abnormal cell viscosity is associated with a variety of diseases, such as Parkinson's disease, diabetes, and sickle cell disease. In addition, the normal functioning of cells is also affected by acid-base balance. pH homeostasis imbalance is related to the occurrence of a variety of diseases, including metabolic diseases, hepatic encephalopathy, and hyperchloremia. 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, and high reactive oxygen levels. This unique microenvironment is conducive to the survival and metastasis of cancer cells. Viscosity and pH can be used as important parameters to distinguish cancer cells from normal cells. Therefore, real-time monitoring of changes in cell viscosity and pH is of great significance for understanding the normal operation mechanism of cells and clarifying the causes of related diseases.

[0003] Mitochondria are the main site of cellular energy production. They control the signaling pathways for longevity and health and determine the fate of cells. Therefore, the balance of the mitochondrial microenvironment is very important, as it affects the physiological activities of cells. There are many factors that affect the mitochondrial microenvironment, such as the content of proteases, oxidative factors, pH, and viscosity. Among them, pH and viscosity are particularly important. Viscosity can directly affect mitochondrial signal transduction and energy generation, while pH affects mitochondrial metabolism.

[0004] Non-alcoholic fatty liver disease includes a series of liver lesions, which are usually caused by the metabolic accumulation of fat in the liver. At the same time, the accumulation of lipid droplets in hepatocytes increases the viscosity of mitochondria and causes mitochondrial dysfunction. Therefore, monitoring changes in mitochondrial viscosity is of great significance for the study of non-alcoholic fatty liver disease.

[0005] There are many traditional methods for measuring viscosity and pH, such as rotational 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 cells in vitro and the changes in viscosity and pH 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 changes in intracellular viscosity and pH. Mitochondria are the key sites of cellular aerobic respiration. The development of fluorescent probes that can both efficiently target mitochondria and sensitively detect changes in cell viscosity and pH is particularly important in understanding the effects of viscosity and pH on mitochondrial diseases. At present, most of the fluorescent probes for monitoring changes in mitochondrial viscosity are cationic fluorescent probes based on twisted intramolecular charge transfer (TICT) design. This type of fluorescent probe has the advantages of fast response and high spatial resolution, and its excited state non-radiative decay is affected by the viscosity of the surrounding environment. This type of fluorescent molecular rotor generally has a rotatable conjugated group, so the molecular rotor rotates rapidly in a low-viscosity medium, reducing the degree of conjugation of the molecule, consuming a lot of energy, and weakening the fluorescence. However, under high viscosity conditions, rotation is hindered and fluorescence is enhanced. However, this type of molecule still has defects, such as poor water solubility, high background interference, complex synthesis, and low rotor efficiency. Summary of the invention

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

[0007] The technical solution of the present invention:

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

[0009]

[0010] A method for preparing the aforementioned fluorescent probe having pH and viscosity response functions comprises the following steps:

[0011] S1. dissolving 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboric acid and tetrakis(triphenylphosphine)palladium in tetrahydrofuran, and then adding an aqueous solution of potassium carbonate to obtain a mixed solution;

[0012] S2. Under nitrogen protection, the mixture was heated to reflux at 70-90 ° C with stirring for 10-20 hours, then quenched with water, the aqueous layer was extracted with dichloromethane, and the extract was concentrated under reduced pressure to obtain a yellow oil;

[0013] S3. The yellow oil was purified by column chromatography to obtain compound 1;

[0014] S4. Compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide were mixed in ethanol, heated under reflux for 5-12 hours, and then cooled to room temperature to precipitate a purple solid. The solid was filtered out and washed with ethanol to obtain a fluorescent probe with pH and viscosity response functions.

[0015] Preferably, in the aforementioned method for preparing a fluorescent probe having pH and viscosity responsive functions, the mass ratio of 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboric acid and tetrakis(triphenylphosphine)palladium is 9-10:6-8:1.

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

[0017] Preferably, in the aforementioned method for preparing the fluorescent probe with pH and viscosity responsive functions, during the column chromatography purification, the mobile phase is petroleum ether / ethyl acetate = 1 / 1, v / v.

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

[0019] An application of the aforementioned fluorescent probe with pH and viscosity response functions in the preparation of mitochondrial probes.

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

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

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

[0023] Beneficial effects of the present invention:

[0024] 1. The preparation process of the fluorescent probe of the present invention is simple and easy to be promoted and implemented industrially.

[0025] 2. The present invention uses phenol as an electron donor (D), and thiophene groups as electron donors and partial π bridges, which are introduced into traditional hemicyanine molecules to construct a fluorescent probe HTC with a D-D'(π)-π-A configuration. HTC has a positive charge and can target mitochondria. The presence of phenolic hydroxyl groups can increase the water solubility of the probe and respond to changes in pH.

[0026] 3. More importantly, the fluorescent probe HTC of the present invention provides two viscosity recognition sites, the single bond between the benzene ring and the thiophene ring and the double bond between the thiophene ring and the indole ring, which can effectively improve the sensitivity and anti-interference ability; in low-viscosity media, the HTC molecule rotates freely, resulting in an increase in the non-radiative attenuation rate and almost no fluorescence, while in high-viscosity media, the intramolecular rotation of the probe is hindered and the fluorescence is enhanced. Because the probe contains Ar-OH, it is sensitive to changes in the pH value of the environment.

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

[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 non-alcoholic fatty liver disease, and provides a design idea for the construction of fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Attached Figure 1 This is the crystal structure ellipsoid diagram of the fluorescent probe HTC of the present invention (ellipsoid probability is 30%);

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

[0031] Attached Figure 3 is the absorbance of the fluorescent probe HTC of the present invention in water and glycerol;

[0032] Attached Figure 4 The fluorescence change of the fluorescent probe HTC of the present invention in different viscosity systems;

[0033] Attached Figure 5 is the viscosity linear correlation diagram of the fluorescent probe HTC of the present invention;

[0034] Attached Figure 6 is the fluorescence spectrum of the fluorescent probe HTC of the present invention in solutions of different polarities;

[0035] Attached Figure 7is the quantum yield of the fluorescent probe HTC of the present invention;

[0036] Attached Figure 8 This is a specific detection diagram of the fluorescent probe HTC of the present invention in response to viscosity;

[0037] Attached Fig. 9 This is the spectral response diagram of the fluorescent probe HTC of the present invention when the pH value is 7 to 10.5;

[0038] Attached Fig.10 The toxicity diagram of the fluorescent probe HTC of the present invention in LO2, HepG2, and 4T1;

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

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

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

[0042] Attached Fig.14(A) ALT and AST levels in serum of normal mice and mice with fatty liver. (B) Relative pixel intensity of (D). (C) HE staining of mouse liver tissue sections, scale: 10μM. (D) Fluorescence imaging of normal mice and mice with fatty liver after injection of HTC (10μM). (E) Fluorescence imaging of major organs of normal mice and mice with fatty liver, 1, heart, 2, lung, 3, kidney, 4, spleen, 5, liver; λ ex =520nm,λ em =610-630nm. (F) Urine imaging of normal mice and mice with fatty liver collected after 8h. (G) Metabolic fluorescence image of the probe in the mice from 0h to 8h after HTC (10μM) was injected into mice with fatty liver.

[0043] Attached Fig.15 It is a high-resolution mass spectrum of the fluorescent probe HTC of the present invention;

[0044] Attached Fig.16 The fluorescent probe HTC of the present invention 1 H NMR spectrum;

[0045] Attached Fig.17 The fluorescent probe HTC of the present invention 13 C NMR spectrum;

[0046] Figure 1 and Figure 2 The crystal structure of HTC shows that the C=C double bond connects the indole ring and the thiophene ring, and C=C is in an "E" configuration. The thiophene ring and the benzene ring are basically parallel, and the dihedral angle between them is 3.30°. The dihedral angle between the indole ring and the thiophene ring is 27.19°. The phenolic hydroxyl group and I - The ions form intermolecular O1-H1···I1 hydrogen bonds. Due to the existence of hydrogen bonds, it is difficult for the hydrogen of the phenolic hydroxyl group to leave, so the pKa of the probe is relatively large. The probe HTC expands through the intermolecular C12-H12···O1 and C23-H23A···O1 atypical hydrogen bonds to form a one-dimensional helical structure. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0048] Embodiments of the present invention

[0049] Example 1

[0050] A method for preparing a fluorescent probe with pH and viscosity response functions, the steps are as follows:

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

[0052] S2. Under nitrogen protection, the mixture was refluxed with stirring for 12 hours, then quenched with water, the aqueous layer was extracted with dichloromethane, and the extract was concentrated under reduced pressure to obtain a yellow oil;

[0053] S3. The yellow oil was purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0054] S4. Compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.62 g) were mixed in ethanol, heated to 80°C and refluxed for 8 hours, then cooled to room temperature to precipitate a purple solid. The solid was filtered out and washed three times with ethanol to obtain a fluorescent probe with pH and viscosity response functions.

[0055] Example 2

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

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

[0058] S2. Under nitrogen protection, the mixture was refluxed with stirring for 10 hours, then quenched with water, the aqueous layer was extracted with dichloromethane, and the extract was concentrated under reduced pressure to obtain a yellow oil;

[0059] S3. The yellow oil was purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0060] S4. Compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.60 g) were mixed in ethanol, heated to 70°C and refluxed for 5 hours, then cooled to room temperature to precipitate a purple solid. The solid was filtered out and washed three times with ethanol to obtain a fluorescent probe with pH and viscosity response functions.

[0061] Example 3

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

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

[0064] S2. Under nitrogen protection, the mixture was refluxed with stirring for 20 hours, then quenched with water, the aqueous layer was extracted with dichloromethane, and the extract was concentrated under reduced pressure to obtain a yellow oil;

[0065] S3. The yellow oil was purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain compound 1;

[0066] S4. Compound 1 (0.40 g) and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide (0.65 g) were mixed in ethanol, heated to 90°C and refluxed for 12 hours, then cooled to room temperature to precipitate a purple solid. The solid was filtered out and washed three times with ethanol 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 and viscosity of the fluorescent probe HTC of the present invention, we tested the absorbance of the probe in water and glycerol. The results are as follows: Figure 3 As shown, different ratios of glycerol and pure water were used as the viscosity test system.

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

[0071] like Figure 5 As shown, according to LogI 627nm =0.396Logη+1.819 The equation of LogI 627nm There is an obvious linear correlation between R 2 =0.989).

[0072] We investigated the fluorescence spectra of HTC in solutions of different polarities (the results are shown in Figure 6), the position of the maximum emission peak of HTC in different solvents is not greatly affected by the solvents, and is around 627nm. The fluorescence in glycerol is the strongest, and the fluorescence in other solvents is very weak. This is because in the low viscosity medium, Ar-OH and thiophene in HTC act as electron donors, and the C=N + As an electron acceptor, a push-pull electron structure is formed. At the same time, since 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, TICT is promoted and the excited state energy of HTC is consumed in the non-radiative pathway, so the fluorescence is very weak. In a high viscosity medium, the free rotation of the molecule is hindered, which inhibits the non-radiative energy consumption and enhances the fluorescence.

[0073] We examined the quantum yield of the probe, e.g. Figure 7 As shown, the quantum yield of the probe in water is only 1.5%, while in glycerol it is 32.5%, an increase of 22 times.

[0074] To investigate the specificity of the probe's response to viscosity, we added analytes to the probe and detected the fluorescence intensity at 627 nm. HTC (10 μM) was compared 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 627nm relative fluorescence intensity, the results are as follows Figure 8 As shown, it 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 invention has Ar-OH and is sensitive to pH changes, we investigated the effect of pH on the spectral properties of the probe. In the pH range of 7 to 10.5, as the pH value increases, the absorption peak at 520nm gradually decreases, the absorption peak at 600nm gradually increases, and isosbestic points appear at 424nm and 535nm (the results are shown in Figure 2). Fig. 9This is because in alkaline solution, the phenolic hydroxyl group gradually loses protons, resulting in an enhanced ICT effect.

[0077] Likewise, Fig. 9 The fluorescence spectrum of B shows that under 520nm excitation, the fluorescence intensity of the probe at 627nm gradually decreases with the increase of pH value. Under 600nm excitation, the fluorescence intensity of the probe at 720nm gradually increases ( Fig. 9 C). The results are consistent with the changes in the UV-visible absorption spectrum of HTC, further proving our speculation. Finally, we used the fluorescence intensity ratio of the probe at 621nm and 720nm (I 627nm / I 720nm ) versus pH, we get Fig. 9 D shows that the linear relationship is good, R 2 =0.99. By Henderson-Hasselbalch equation (pH = pKa + lg [(I max -I) / (II min )]), the pKa of the probe was 8.6. This is consistent with the crystal structure we obtained, which shows that the phenolic hydroxyl group and I - The ions form intermolecular O1—H1···I1 hydrogen bonds, and the hydrogen of the phenolic hydroxyl group is difficult to leave, so the pKa is larger.

[0078] 3. Detection of mitochondrial targeting ability

[0079] The biocompatibility of the probe is an important indicator of the biological application of the probe, which is crucial to the practical application of the probe. Since the fluorescent probe of the present invention has pH response and viscosity response, we tried to detect pH and fluorescence imaging in living cells.

[0080] First, we investigated the cytotoxicity of the probe. The CCK8 assay showed that after incubation of cells with probes of different concentrations (0-10 μM) for 24 hours, the survival rate of all cells was above 80% ( Fig.10 ), indicating that the probe has low toxicity to cells and good biocompatibility.

[0081] Next, the probe's targeting of organelles was investigated. Mitochondria are double-membrane organelles with negative membrane potential. Therefore, the positively charged probe HTC can easily enter the mitochondrial matrix and is expected to target mitochondria. Cell co-localization imaging experiments showed that the probe can target mitochondria for cell fluorescence imaging (Pearson coefficient is 0.91, Fig.11 ), indicating that the probe can target mitochondria for cellular fluorescence imaging.

[0082] 4. Response to pH in cells

[0083] Laser confocal microscopy was used to investigate the fluorescence imaging of HepG2 living cells in an environment with a pH value of 6.0 to 8.0. At pH = 6 or 7, the fluorescence intensity in the red channel was strong. At pH = 8, the red light in the red channel was quenched. The fluorescence intensity at pH = 6 or 7 was 4 times that of pH = 8 ( Fig.12 ). This result is consistent with the fluorescence change at 627nm in solution, indicating that the probe HTC can detect changes in pH value in cells.

[0084] 5. Detection of changes in viability of living cells

[0085] In order to investigate whether the probe of the present invention can be used to detect changes in viscosity in living cells, we selected HepG2 as a cell model, used nystatin and serum-free HBSS culture medium as external and internal factors to increase the mitochondrial viscosity of cells, and then performed cell fluorescence imaging. The results showed that only when the probe HTC was incubated with cells, a weak red fluorescence could be seen. As the cell mitochondrial viscosity increased, the red light was significantly enhanced ( Fig.13 ). The results showed that HTC can monitor the changes in cell viscosity induced by endogenous and exogenous factors in living cells.

[0086] 6. Testing NAFLD mouse model

[0087] It is well known that fatty liver tissue has high viscosity. Therefore, we tried to identify high viscosity organs in mice. We established a mouse model of non-alcoholic fatty liver disease by feeding mice with a high-fat diet and intraperitoneally injecting olive oil containing CCl4. Blood was collected from the mouse eyeballs and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum were measured. Fig.14 As shown in A, the ALT and AST of mice with fatty liver were higher than those of normal mice, indicating that the non-alcoholic fatty liver model was successfully established. Then, HTC was intraperitoneally injected into normal mice and mice with fatty liver, and fluorescence imaging was performed using a small animal imager. The results are shown in Fig.14 As shown in C, the fluorescence of normal mice is weak, while the fluorescence of fatty liver mice is strong. Quantification of the fluorescence in the abdomen of mice ( Fig.14 D), the fluorescence intensity of the fatty liver mice was about 2 times that of the normal mice. This shows that HTC can distinguish between normal mice and fatty liver mice. Next, we killed the mice, dissected out the important organs, and performed fluorescence imaging on the organs. The results are as follows Fig.14 As shown in E, the fluorescence of the liver of normal mice is weak, the fluorescence of the fatty liver model mice is strong, and the fluorescence of other organs is weak, which indicates that the probe is mainly enriched in the liver. Subsequently, the liver sections of the normal group and the model group mice were stained with HE. Fig.14As shown in B, normal mouse liver tissue has no obvious morphological changes, while hepatocytes of fatty liver mice show vacuolation. From the tissue sections and serum measurement indicators, it can be seen that fatty liver is accompanied by liver damage. In order to observe the metabolic pathway of the probe in the model mice, we monitored the fluorescence changes of the probe in the mice from 0 to 8 hours ( Fig.14 F), and found that the probe fluorescence was strongest at 4h, and the fluorescence gradually weakened at 4-8h. The urine of mice was collected at 0-8h ( Fig.14 G), using a small animal imager for fluorescence imaging, the results showed that the urine glowed red, indicating that the probe was excreted with urine after entering the mouse body and metabolized quickly. The above experimental results prove that the probe HTC can assist in the detection of non-alcoholic fatty liver disease in mice and provide a visual means for the diagnosis of non-alcoholic fatty liver disease.

[0088] In summary, we used phenol as an electron donor (D), thiophene groups as electron donors and partial π bridges to construct a hemicyanine fluorescent probe HTC with a D-D'(π)-π-A configuration. Fluorescent HTC is positively charged and can target mitochondria in cells. The Pearson coefficient for cell co-localization fluorescence imaging with commercial mitochondrial probes is 0.91. In solution, the probe has pH-responsive and viscosity-responsive properties. In cells, the probe can monitor changes in pH and viscosity in cells and distinguish normal cells from cancer cells. Taking advantage of the viscosity-responsive property of the probe, we used the probe to identify the formation of non-alcoholic fatty liver disease in mice. In addition, the probe is easily metabolized in mice and has good biocompatibility. This experiment provides a visualization method for the identification of cancer cells and non-alcoholic fatty liver disease, and provides new ideas for the design of fluorescent probes.

[0089] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A fluorescent probe with pH and viscosity response function, characterized in that: Its molecular formula is C 24 H 24 NOSI, its chemical structure is as follows:

2. A method for preparing a fluorescent probe having pH and viscosity response functions according to claim 1, characterized in that: The steps include: S1. dissolving 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboric acid and tetrakis(triphenylphosphine)palladium in tetrahydrofuran, and then adding an aqueous solution of potassium carbonate to obtain a mixed solution; S2. Under nitrogen protection, the mixture was refluxed with stirring for 10-20 hours, then quenched with water, the aqueous layer was extracted with dichloromethane, and the extract was concentrated under reduced pressure to obtain a yellow oil; S3. The yellow oil was purified by column chromatography to obtain compound 1; S4. Compound 1 and 1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide were mixed in ethanol, heated under reflux at 70-90°C for 5-12 hours, and then cooled to room temperature to precipitate a purple solid. The solid was filtered out and washed with ethanol to obtain a fluorescent probe with pH and viscosity response functions.

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

1.

4. The method for preparing a fluorescent probe having pH and viscosity response functions according to claim 2, characterized in that: The mass concentration of potassium carbonate in the potassium carbonate aqueous 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 having pH and viscosity response functions according to claim 2, characterized in that: During the column chromatography purification, the mobile phase was petroleum ether / ethyl acetate = 1 / 1, v / v.

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

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

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

9. A fluorescent probe for monitoring changes in pH and viscosity in cells, characterized in that: The fluorescent probe having pH value and viscosity response function as claimed in claim 1 is included.

10. A fluorescent probe for identifying non-alcoholic fatty liver disease, characterized in that: The fluorescent probe having pH value and viscosity response function as claimed in claim 1 is included.

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