Preparation method and application of fluorescent probe for detecting HSO3 <-> / ClO <-> and viscosity

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and property research of a fluorescent probe for detecting HSO3 <-> / ClO <-> and viscosity. The preparation method of the fluorescent probe is simple, the product yield is high, and the fluorescent probe is suitable for large-scale popularization and application. According to the probe (E)-4-(2-(6-(4-(diphenylamino) phenyl)-2, 3-dihydro-1H-xanthene-4-yl) vinyl)-3-ethylbenzothiazole-3-onium iodide (TXET), firstly, when HSO3 <-> is detected, the fluorescence intensity (I500) is also gradually enhanced along with the increase of the concentration of the HSO3 <->; when the concentration of HSO3 <-> is detected to be within 0-0.13 mmol / L, a good linear relation is achieved. When ClO <-> is detected under a low polarity condition, the fluorescence intensity (I530) is gradually enhanced along with the increase of the concentration of ClO <->. Secondly, when the concentration of ClO <-> is detected to be within 40-80 micromole per liter, a good linear relation is achieved. Finally, along with the increase of the viscosity, the fluorescence intensity (I720) is also gradually increased, and a good linear relation is achieved within 7.9-880 centipoises. Biological experiment results show that the probe TXET can monitor HSO3 <-> / ClO <-> and viscosity change level in cells and animal bodies, and can be used as a sensitive tool for identifying liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes and particularly relates to a method for detecting HSO3 - / ClO - Preparation method and application of fluorescent probe for viscosity. Background Art

[0002] Hypochlorous acid (HOCl) is one of the biologically important reactive oxygen species (ROS). Due to its subacidity, HOCl usually undergoes spontaneous hydrolysis in a neutral solution at pH 7.0 to form free ClO - In biology, ClO - The ion is believed to be produced by hydrogen peroxide and chloride ions in activated neutrophils catalyzed by the heme enzyme myeloperoxidase (MPO). However, abnormal production of hypochlorite can lead to tissue damage and diseases such as liver damage, atherosclerosis, lung damage, rheumatoid arthritis, cardiovascular disease, neuronal degeneration, and cancer. Detection of ClO in these diseases is necessary. - The level of sulfur dioxide (SO2) and ClO - It is closely related to the redox balance. SO2 is a reducing substance in cells, mainly produced by the oxidation of sulfur-containing amino acids or H2S. In addition, as an environmental pollutant, SO2 is mainly in the form of SO3 2- / HSO3 - There are two forms. Abnormal SO2 metabolism levels in cells can lead to an imbalance in redox homeostasis, which is closely related to inflammation, ferroptosis, etc. It is crucial to detect the level of SO2 in these diseases.

[0003] Intracellular viscosity is a basic factor in some cellular processes, such as the diffusion of bioactive substances and the transport of signals, which are closely related to intracellular viscosity. Abnormal changes in cell viscosity may be related to certain diseases, such as ferroptosis, kidney damage, liver damage, etc., which can cause increased cell viscosity. Monitoring cell viscosity is also crucial.

[0004] Fluorescent probes have become an indispensable tool in clinical medicine and biomedical research because they can provide dynamic information about the localization and quantity of biomolecules of interest in living cells, tissues, and even animals. To date, many fluorescent probes have been reported for the detection of ClO in biological systems. - or SO2. However, there is no probe that can simultaneously monitor ClO in living organisms. - 、SO2. In summary, the development of a new method to detect HSO3 - / ClO - Fluorescent probe for HSO3 - / ClO - And viscosity testing is very necessary. Summary of the invention

[0005] The present invention aims at a method for detecting HSO3 - / ClO - The preparation method and properties of the fluorescent probe for viscosity and viscosity were studied. (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) has the characteristics of simple synthesis route, good selectivity, high sensitivity, large Stokes shift, etc. It can detect HSO3 under physiological conditions. - / ClO - And viscosity for effective testing.

[0006] The fluorescent probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) in the present invention has the following molecular structure:

[0007] The fluorescent probe synthesis process of the present invention is as follows:

[0008] The preparation steps of the probe TXET are as follows:

[0009] Chloroform and phosphorus tribromide were added to anhydrous N,N-dimethylformamide (DMF), and the mixture was stirred at 0°C for 1 hour. Cyclohexanone was then added to the chloroform to mix the mixture thoroughly. The mixture was added to the reaction system, vacuumed, and reacted at room temperature for 18 hours. After the reaction was completed, the pH was adjusted to neutral with saturated sodium bicarbonate (NaHCO3), and then the organic phase was extracted with dichloromethane. The organic phase was collected and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column (eluent: dichloromethane: ethanol = 30:1), and a light yellow oily product 2-chloro-1-cyclooctene-1-carbaldehyde (A1, yield 50.4%) was obtained after vacuum drying.

[0010] 4-(Diphenylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde were ultrasonically dissolved in tetrahydrofuran (THF) solution, potassium carbonate aqueous solution and tetrabutylamine bromide were added and ultrasonically dissolved, and stirred at room temperature for 30 minutes, and then tetrakis(triphenylphosphine)palladium was added to the reaction system, ultrasonically dissolved, vacuumed, and heated to reflux for about 6 hours; after the reaction was completed, it was cooled to room temperature and extracted with dichloromethane, the organic phase was collected and the solvent was removed by rotary evaporation, and the silica gel column was purified (the eluent was petroleum ether: dichloromethane = 4:1) to obtain a fluorescent green solid product 4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carboxaldehyde (A2, with a yield of 87.8%).

[0011] A1, A2 and cesium carbonate were dissolved in anhydrous DMF, vacuumized, and then reacted at room temperature for 20 hours. After the reaction, the reaction solution was spin-dried to obtain a crude product, which was separated and purified by column chromatography. The eluent was petroleum ether: ethyl acetate = 15:1. After vacuum drying, a yellow solid product 6-(4-(diphenylamine)phenyl)-2,3-dihydro-1H-xanthene-4-carboxaldehyde (A3, yield 69.6%) was obtained.

[0012] 2-Methylbenzothiazole and iodoethane were added to ethanol and heated under reflux for about 12 hours. When the reaction was finished, the reaction solution was cooled to room temperature, a large amount of precipitate was separated out and filtered and washed, and then dried in a vacuum drying oven to obtain an off-white solid 3-ethyl-2-methylbenzothiazol-3-ium iodide (A4, yield 86.4%).

[0013] A3 and A4 were dissolved in anhydrous ethanol, ultrasonicated to fully dissolve them, and heated to reflux for 8 hours; at the end of the reaction, the reaction stock solution was cooled to room temperature, and a precipitate was generated, which was filtered and washed with ethanol, and dried to obtain a dark blue product (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-oxanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET, yield 62.4%).

[0014] The detection mechanism of the fluorescent probe of the present invention is as follows: Due to the photoinduced electron transfer effect of the probe molecule itself, the fluorescent molecule TXET exhibits fluorescence quenching. - When the probe reacts with ClO, the Michael addition reaction occurs, which makes the photoinduced electron transfer effect of the fluorescent molecule disappear and emits strong green fluorescence. - After the response, the carbon-carbon double bond breaks, generating free fluorophores, which make the probe show strong yellow fluorescence in low-polarity solvents, while the red fluorescence is quenched under high viscosity. Based on the above functions, a new type of HSO3 detection - / ClO - A fluorescent probe based on the viscosity of HSO3 was designed and synthesized to detect the - / ClO - and viscosity.

[0015] Figure 3 The probe TXET (3×10 -6 mol / L) in different solvents. The UV-visible absorption spectra of the probe in different solvents have no obvious differences.

[0016] Figure 4 The probe TXET (3×10 -6mol / L) in different solvents at an excitation wavelength of 620 nm. The probe has fluorescence in solvents with lower polarity and fluorescence quenching in solvents with higher polarity

[0017] Figure 5 The probe TXET (3×10 -5 mol / L) were added to the DMF-PBS system with different DMF contents at an excitation wavelength of 400 nm. - The fluorescence intensity change diagram before and after. When the volume percentage of the probe in DMF is 50%, 0.13mmol / L HSO3 is added - The fluorescence intensity changes before and after are obvious.

[0018] Figure 6 The probe TXET (3×10 -5 mol / L) were added to the mixed solvent of PBS and DMF in equal proportions. - The fluorescence intensity before and after the probe is added with HSO3 - After 15 minutes, the fluorescence intensity did not change significantly with time.

[0019] Figure 7 The probe TXET (3×10 -5 mol / L) were added with different concentrations of HSO3 in a mixed solvent of PBS and DMF in equal proportions. - UV-visible absorption spectrum. With the HSO3 - With the increase of concentration, a new absorption peak appears at 400 nanometers and gradually increases, while the absorption peak at 600 nanometers decreases.

[0020] Figure 8 The probe TXET (3×10 -5 mol / L) were added to the mixed solvent of PBS and DMF at an excitation wavelength of 400 nm with different concentrations of HSO3 - Fluorescence emission spectrum of. The fluorescence intensity increases with the increase of HSO3 - increased with increasing concentration.

[0021] Fig. 9 The probe TXET (3×10 -5 mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The concentration range is 0mmol / L-0.13mmol / L 500 The linear relationship diagram shows a good linear relationship.

[0022] Fig.10 The probe TXET (3×10 -5mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The fluorescence intensity before and after changes with pH value. The probe detects HSO3 under neutral and alkaline conditions - The effect is better.

[0023] Fig.11 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules.

[0024] Fig.12 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0025] Fig.13 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3- 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0026] Fig.14 The probe TXET (3×10 -5 mol / L) were added to the THF-PBS system with different contents of tetrahydrofuran (THF) at an excitation wavelength of 400 nm. - The fluorescence intensity change diagram before and after. When the volume percentage of THF was 80%, 80 μmol / L ClO - The fluorescence intensity changes before and after are obvious.

[0027] Fig.15 The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF at different concentrations of ClO - The fluorescence intensity before and after the probe was added with ClO - After 3 minutes, the fluorescence intensity did not change significantly with time.

[0028] Fig.16 The probe TXET (3×10 -5 mol / L) were added into THF-PBS mixed solvent containing 80% THF with different concentrations of ClO - UV-visible absorption spectrum. - As the concentration increases, the absorption peak at 400 nanometers gradually decreases, and the absorption peak at 600 nanometers also gradually decreases.

[0029] Fig.17 The probe TXET (3×10 -5 mol / L) were added to different concentrations of ClO in a THF-PBS mixed solvent containing 80% THF at an excitation wavelength of 400 nm. - Fluorescence emission spectrum of. Fluorescence intensity I 530 With ClO - increased with increasing concentration.

[0030] Fig.18 The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF. -The concentration range is 40μmol / L-80μmol / L 530 The linear relationship diagram shows a good linear relationship.

[0031] Fig.19 The probe TXET (3×10 -5 mol / L) was added to a THF-PBS mixed solvent containing 80% THF. - The fluorescence intensity changes with pH before and after. The probe detects ClO under neutral and alkaline conditions - The effect is better.

[0032] Fig. 20 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules.

[0033] Fig.21 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0034] Fig. 22 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS- 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0035] Fig.23 The probe TXET (3×10 -5 mol / L) in different ratios of PBS and glycerol mixed solvents. As the volume of glycerol increases, the absorption peak at 620 nm gradually increases.

[0036] Fig.24 The probe TXET (3×10 -5 mol / L) in different proportions of PBS and glycerol mixed solvents at an excitation wavelength of 620 nm. As the volume of glycerol increases, the fluorescence at 720 nm gradually increases.

[0037] Fig.25 The probe TXET (3×10 -5 mol / L) in the viscosity range of 7.9-880 centipoise. 720 ) and the logarithm of the system viscosity (logη). It shows a good linear relationship.

[0038] Fig.26 The probe TXET (3×10 -5 The fluorescence intensity of the probe in different ratios of PBS and glycerol mixed solvents varies with pH value. The probe is not affected by pH when detecting viscosity.

[0039] Fig. 27 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules.

[0040] Fig.28 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0041] Fig.29 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0042] Fig.30 The prepared probe TXET (3×10 -6 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , maximum emission wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0043] Fig.31 (A) is the probe TXET (1×10 -5 mol / L) after staining of normal human liver cells (L02) and liver cells with different degrees of damage. (B) is the relative fluorescence intensity diagram in Figure (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nanometers. At the cellular level, the probe TXET has a good ability to monitor the degree of damage to L02 cells.

[0044] Fig.32 (A) is the probe TXET (1×10 -5 mol / L) in vivo and in vitro diagnostic liver injury ability evaluation. (B) is the relative fluorescence intensity diagram in Figure (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nm; ****P<0.0001. The probe TXET can track the process of liver injury and treatment in mice in vivo and in vitro.

[0045] Fig.33 (A) is the probe TXET (1×10 -5 mol / L) fluorescence imaging to monitor liver injury process at the tissue level. (B) is the relative fluorescence intensity diagram in (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nm; ****P<0.0001The probe TXET can successfully monitor the liver injury process at the tissue level.

[0046] In summary, using a simple organic synthesis method, we obtained a method for detecting HSO3 - / ClO -The probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) was used to study the viscosity of the sample. - The solution color (from dark blue to colorless) changed significantly before and after. This indicates that the probe - After that, different optical properties are shown. And with the HSO3 - With the increase of concentration, the fluorescence intensity of the probe at 400 nm wavelength gradually increased, which was due to the addition of HSO3 - After HSO3 - The Michael addition reaction with the probe molecule causes the photoinduced electron transfer effect of the fluorescent molecule to disappear, and emits strong green fluorescence. - The solution color changed significantly (from dark blue to light yellow) before and after, and showed light yellow light under a 365-nanometer hand lamp. This shows that the probe - After that, they show different optical properties. And with the ClO - With the increase of concentration, the fluorescence intensity of the probe at 530 nm gradually increased. - The color of the solvent changed significantly (from dark blue to colorless) before and after. This indicates that the probe - After that, they show different optical properties. And with the ClO - As the concentration increases, the fluorescence intensity of the probe at 720 nm gradually decreases. This is because the probe molecules react with ClO - The response of the probe caused the photoinduced electron transfer effect of the luminescent molecule to disappear, and a strong yellow fluorescence was emitted, while the fluorescence at 720 nm gradually decreased. Finally, with the increase of viscosity, the fluorescence intensity of the probe at a wavelength of 720 nm gradually increased. - / ClO - The fluorescence emission spectrum shows obvious regular changes under different viscosities, which is a good way to detect HSO3 - / ClO - The preparation of this fluorescent probe fills the gap in the field of fluorescent probes and can simultaneously quantify the detection of HSO3 - / ClO - At the same time, during the biological experiment, it can be found that with the occurrence and development of liver damage, the green, yellow and red fluorescence signals of the probe TXET all increased significantly. It is worth noting that during the treatment of liver damage in mice, the three fluorescence signals decreased significantly. This shows that the probe TXET can be used as a sensitive tool to identify liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Preparation and design route of the probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET).

[0048] Figure 2 Detection of HSO3 by TXET probe - , ClO - and the mechanism of response to viscosity.

[0049] Figure 3 The probe TXET (3×10 -6 mol / L) in different solvents.

[0050] Figure 4 The probe TXET (3×10 -6 mol / L) in different solvents at an excitation wavelength of 620 nm.

[0051] Figure 5 The probe TXET (3×10 -5 mol / L) was added to the DMF-PBS system with different DMF contents at an excitation wavelength of 400 nm. - Fluorescence intensity changes before and after.

[0052] Figure 6 The probe TXET (3×10 -5 mol / L) were added to the mixed solvent of PBS and DMF in equal proportions. - The fluorescence intensity before and after the experiment changes with time.

[0053] Figure 7 The probe TXET (3×10 -5 mol / L) were added with different concentrations of HSO3 in a mixed solvent of PBS and DMF in equal proportions. - UV-visible absorption spectrum.

[0054] Figure 8 The probe TXET (3×10 -5 mol / L) were added to the mixed solvent of PBS and DMF at an excitation wavelength of 400 nm with different concentrations of HSO3 - Fluorescence emission spectrum of .

[0055] Fig. 9 The probe TXET (3×10 -5mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The concentration range is 0mmol / L-0.13mmol / L 500 Linear relationship diagram.

[0056] Fig.10 The probe TXET (3×10 -5 mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The fluorescence intensity before and after changes with pH value.

[0057] Fig.11 The probe TXET (3×10 -5 mol / L) for the anti-interference of biological small molecules. From left to right: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0058] Fig.12 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ .

[0059] Fig.13 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2-、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - .

[0060] Fig.14 The probe TXET (3×10 -5 mol / L) were added to THF-PBS systems with different THF contents at an excitation wavelength of 400 nm. - Fluorescence intensity changes before and after.

[0061] Fig.15 The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF at different concentrations of ClO - The fluorescence intensity before and after the experiment changes with time.

[0062] Fig.16 The probe TXET (3×10 -5 mol / L) were added into THF-PBS mixed solvent containing 80% THF with different concentrations of ClO - UV-visible absorption spectrum.

[0063] Fig.17 The probe TXET (3×10 -5 mol / L) were added to different concentrations of ClO in a THF-PBS mixed solvent containing 80% THF at an excitation wavelength of 400 nm. - Fluorescence emission spectrum of .

[0064] Fig.18 The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF. - The concentration range is 40μmol / L-80μmol / L 530 Linear relationship diagram.

[0065] Fig.19 The probe TXET (3×10 -5 mol / L) was added to a THF-PBS mixed solvent containing 80% THF. - The fluorescence intensity before and after changes with pH value.

[0066] Fig. 20 The probe TXET (3×10 -5mol / L) for the anti-interference of biological small molecules. From left to right: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0067] Fig.21 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ .

[0068] Fig. 22 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - .

[0069] Fig.23 The probe TXET (3×10 -5 mol / L) in mixed solvents of PBS and glycerol at different ratios.

[0070] Fig.24The probe TXET (3×10 -5 mol / L) in different proportions of PBS and glycerol mixed solvents at an excitation wavelength of 620 nm.

[0071] Fig.25 The probe TXET (3×10 -5 mol / L) in the viscosity range of 7.9-880 centipoise. 720 ) and the linear relationship between the logarithm of the system viscosity (logη).

[0072] Fig.26 The probe TXET (3×10 -5 mol / L) in mixed solvents of PBS and glycerol with different pH values.

[0073] Fig. 27 The probe TXET (3×10 -5 mol / L) for the anti-interference of biological small molecules. From left to right: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0074] Fig.28 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ .

[0075] Fig.29 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS- 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - .

[0076] Fig.30 The prepared probe TXET (3×10 -6 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , Maximum emission peak wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0077] Fig.31 (A) is the probe TXET (1×10 -5 mol / L) after staining of normal human liver cells (L02) and liver cells with different degrees of damage. (B) is the relative fluorescence intensity diagram in Figure (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nanometers.

[0078] Fig.32 (A) is the probe TXET (1×10 -5 mol / L) in vivo and in vitro diagnostic liver injury ability evaluation. (B) is the relative fluorescence intensity diagram in Figure (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nm; ****P<0.0001.

[0079] Fig.33 (A) is the probe TXET (1×10 -5mol / L) fluorescence imaging to monitor liver injury process at the tissue level. (B) is the relative fluorescence intensity diagram in (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600)nm; red channel: λ ex / λ em =620 / (700-750) nm; ****P<0.0001. Specific implementation examples

[0080] Example 1: Synthesis of Compound A1 Chloroform (25 mL) and phosphorus tribromide (4.5 mL) were added to anhydrous N,N-dimethylformamide (6 mL), and the mixture was stirred at 0°C for 1 hour. Then, cyclohexanone (2 mL) was added to the chloroform to mix well, and the mixture was added to the reaction system, vacuumed, and reacted at room temperature for 18 hours. After the reaction was completed, the pH was adjusted to neutral with saturated NaHCO3, and then the organic phase was extracted with dichloromethane. The organic phase was collected and the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column (eluent: dichloromethane: ethanol = 30:1), and 1.83 g of light yellow oily product 2-chloro-1-cyclooctene-1-carbaldehyde (A1, yield 50.4%) was obtained after vacuum drying.

[0081] Example 2: Synthesis of Compound A2 4-(Diphenylamino)phenylboronic acid (2.4mmol, 693.94mg) and 4-bromo-2-hydroxybenzaldehyde (2mmol, 402.11mg) were ultrasonically dissolved in tetrahydrofuran (THF) solution, and potassium carbonate aqueous solution (2mol / L) and tetrabutylammonium bromide (1mmol, 322mg) were added and ultrasonically dissolved, and stirred at room temperature for 30 minutes. Tetrakis(triphenylphosphine)palladium (0.002mmol, 2.4mg) was then added to the reaction system and ultrasonically dissolved and vacuumed, and heated to reflux for about 6 hours; after the reaction was completed, it was cooled to room temperature and extracted with dichloromethane (DCM), the organic phase was collected and the solvent was removed by rotary evaporation, and purified on a silica gel column (eluent was petroleum ether: dichloromethane = 4:1) to obtain 641.70mg fluorescent green solid product 4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carboxaldehyde (A2, with a yield of 87.8%).

[0082] Example 3: Synthesis of Compound A3 A1 (2 mmol, 375.96 mg) and A2 (2 mmol, 730.28 mg) and cesium carbonate (1.2 mmol, 166 mg) were dissolved in anhydrous DMF, vacuumized, and then reacted at room temperature for 20 hours. After the reaction, the reaction solution was spin-dried to obtain a crude product, which was separated and purified by column chromatography. The eluent was petroleum ether: ethyl acetate = 15: 1. After vacuum drying, 633.63 mg of a yellow solid product 6-(4-(diphenylamine)phenyl)-2,3-dihydro-1H-xanthene-4-carboxaldehyde (A3, yield 69.6%) was obtained.

[0083] Example 4: Synthesis of Compound A4 2-Methylbenzothiazole (10 mmol, 1.49 g) and iodoethane (10 mmol, 1.56 g) were added to ethanol and heated under reflux for about 12 hours. At the end of the reaction, the reaction solution was cooled to room temperature, a large amount of precipitate was precipitated and filtered, washed with ethanol three times, and dried in a vacuum drying oven to obtain 2.64 g of off-white solid 3-ethyl-2-methylbenzothiazol-3-ium iodide (A4, yield 86.4%).

[0084] Example 5: Synthesis of probe TXET A3 (0.5 mmol, 227.60 mg) and A4 (0.5 mmol, 152.49 mg) were dissolved in anhydrous ethanol, and the mixture was fully dissolved by ultrasonic treatment and heated under reflux for 8 hours. When the reaction was over, the reaction solution was cooled to room temperature, and a precipitate was generated. The precipitate was filtered and washed with ethanol, and dried to obtain 238.40 mg of a dark blue product (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-oxanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET, yield 64.2%).

[0085] Example 6: Detection of HSO3 in solvent by probe TXET - and ClO - And viscosity application.

[0086] HSO3 in solution - Detection: Figure 5 The probe TXET (3×10 -5 mol / L) was added to the DMF-PBS system with different DMF contents at an excitation wavelength of 400 nm. - Fluorescence intensity change diagram before and after. When the volume percentage of N,N-dimethylformamide is 50%, 0.13mmol / LHSO3 is added - There is an obvious difference in fluorescence intensity before and after. Figure 6 The probe TXET (3×10 -5mol / L) were added to the mixed solvent of PBS and DMF in equal proportions. - The fluorescence intensity changes with time before and after adding HSO3 - After 15 minutes, the fluorescence intensity did not change significantly. Figure 7 The probe TXET (3×10 -5 mol / L) were added with different concentrations of HSO3 in a mixed solvent of PBS and DMF in equal proportions. - UV-visible absorption spectrum of the probe. - There is a significant difference in the UV absorption spectrum. - With the increase of concentration, the absorption peak at 400 nm gradually increases, and the absorption peak at 600 nm gradually decreases. Figure 8 The probe TXET (3×10 -5 mol / L) were added to the mixed solvent of PBS and DMF at an excitation wavelength of 400 nm with different concentrations of HSO3 - Fluorescence emission spectrum of. With the HSO3 - With the increase of concentration, the fluorescence intensity (I 500 ) is also gradually increasing. Fig. 9 The probe TXET (3×10 - 5 mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The concentration range is 0mmol / L-0.13mmol / L 500 The linear relationship diagram of - The concentration range is 0mmol / L-0.13mmol / L, showing a good linear relationship. Fig.10 The probe TXET (3×10 -5 mol / L) in a mixed solvent of PBS and DMF in equal proportions and HSO3 - The fluorescence intensity changes with pH before and after. The probe detects HSO3 under neutral and alkaline conditions - The effect is better. Fig.11 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules. Fig.12 The probe TXET (3×10 -5mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Fig.13 The probe TXET (5×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0087] ClO in solution - Detection: ClO under low polarity conditions - Detection: Fig.14 The probe TXET (3×10 -5 mol / L) were added to THF-PBS systems with different THF contents at an excitation wavelength of 400 nm. - When the volume percentage of tetrahydrofuran is 80%, 80 μmol / L ClO - There is an obvious difference in fluorescence intensity before and after. Fig.15The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF at different concentrations of ClO - The fluorescence intensity changes with time before and after addition of ClO - After 3 minutes, the fluorescence intensity did not change significantly with time. Fig.16 The probe TXET (3×10 -5 mol / L) were added into a THF-PBS mixed solvent containing 80% THF with different concentrations of ClO - The probe was subjected to the addition of different concentrations of ClO - There is a significant difference in the UV absorption spectrum. - With the increase of concentration, the absorption peak at 400 nm gradually decreases, and the absorption peak at 600 nm also gradually decreases. Fig.17 The probe TXET (3×10 -5 mol / L) were added to different concentrations of ClO in a THF-PBS mixed solvent containing 80% THF at an excitation wavelength of 400 nm. - Fluorescence emission spectrum of. With the increase of ClO - With the increase of concentration, the fluorescence intensity (I 530 ) is gradually increasing. Fig.18 The probe TXET (3×10 -5 mol / L) was added to a THF-PBS mixed solvent containing 80% THF. - The concentration range is 40μmol / L-80μmol / L 530 The linear relationship diagram of - The concentration range is 40μmol / L-80μmol / L, showing a good linear relationship. Fig.19 The probe TXET (3×10 -5 mol / L) was added to a THF-PBS mixed solvent containing 80% THF. - The fluorescence intensity changes with pH before and after. The probe detects ClO under neutral and alkaline conditions - The effect is better. Fig. 20 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules. Fig.21The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2 + Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Fig. 22 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0088] ClO in solution - Detection: ClO under high viscosity conditions - Detection: Fig.23 The probe TXET (3×10 -5 80 μmol / L ClO was added to the glycerol-PBS system with different glycerol contents at an excitation wavelength of 600 nm. - When the volume percentage of glycerol is 99%, 80 μmol / L ClO- There is an obvious difference in fluorescence intensity before and after. Fig.24 The probe TXET (3×10 -5 mol / L) were added to a mixed solvent containing 99% glycerol with different concentrations of ClO - The fluorescence intensity changes with time before and after addition of ClO - After 1 minute, the fluorescence intensity did not change significantly with time. Fig.25 The probe TXET (3×10 -5 mol / L) were added to a mixed solvent containing 99% glycerol with different concentrations of ClO - The probe was subjected to the addition of different concentrations of ClO - There is a significant difference in the UV absorption spectrum. - With the increase of concentration, the absorption peak at 400 nm gradually decreases, and the absorption peak at 600 nm also gradually decreases. Fig.26 The probe TXET (3×10 - 5 mol / L) were added to a mixed solvent containing 99% glycerol at an excitation wavelength of 600 nm. - Fluorescence emission spectrum of. With the increase of ClO - With the increase of concentration, the fluorescence intensity (I 720 ) is gradually decreasing. Fig. 27 The probe TXET (3×10 - 5 mol / L) was added to a mixed solvent containing 99% glycerol. - The concentration range is 0μmol / L-80μmol / L 720 The linear relationship diagram of - The concentration range is 0μmol / L-80μmol / L, showing a good linear relationship. Fig.28 The probe TXET (3×10 -5 mol / L) was added to a mixed solvent containing 99% glycerol. - The fluorescence intensity changes with pH before and after. The probe detects ClO at high viscosity - Not affected by pH. Fig.29 The probe TXET (3×10 -5mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules. Fig.30 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Fig.31 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl - Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0089] Viscosity detection in solvents: Fig.23 The probe TXET (3×10 -5mol / L) in different proportions of PBS and glycerol mixed solvents. The UV absorption spectra of the probe at different viscosities are significantly different. Fig.24 The probe TXET (3×10 -5 mol / L) in different proportions of PBS and glycerol mixed solvents at an excitation wavelength of 620 nm. As the solvent viscosity increases, the fluorescence intensity gradually increases. Fig.25 The probe TXET (3×10 -5 mol / L) in the viscosity range of 7.9-880 centipoise (logI 720 ) and the logarithm of the system viscosity (logη). As can be seen from the figure, a good linear relationship is presented. Fig.26 The probe TXET (3×10 -5 mol / L) in different ratios of PBS and glycerol mixed solvents. It can be seen from the figure that the effect of pH on the probe is almost negligible. Fig. 27 The probe TXET (3×10 -5 mol / L) to small biological molecules. From left to right are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. The interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties to small biological molecules. Fig.28 The probe TXET (3×10 -5 mol / L) to metal ions. From left to right are Blank, K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Ag + Sn 2+ 、Co 2+ 、Ni 2+ , Mn 2+ 、Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Fig.29 The probe TXET (3×10 -5 mol / L) to anion anti-interference study. From left to right are Blank, F - , Cl- Br - ,I - 、SO4 2- , S 2- , HS - 、SCN - PO4 3- 、H2PO4 - 、P2O7 4- 、C2O4 2- 、CO3 2- 、HCO3 - 、Cr2O7 2- 、CrO4 2- 、NO3 - 、NO2 - 、BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Fig.30 The prepared probe TXET (3×10 -6 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , maximum emission wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0090] In biological experiments, Fig.31 The probe TXET (1×10 -5 mol / L) after human normal liver cells (L02) and liver cells with different degrees of damage. As the degree of damage of L02 cells increases, the green, yellow and red fluorescence signals of the probe TXET are significantly enhanced, indicating that HSO3 - / ClO - The concentration increases, and with it the viscosity level. Fig.32 The probe TXET (1×10 -5 mol / L) in vivo and in vitro diagnostic ability of liver injury. Compared with the control group, the three fluorescence intensities of liver injury mice and mouse organs were significantly increased. After the treatment of liver injury, the three fluorescence intensities of liver injury mice and mouse organs were significantly reduced. Fig.33 The probe TXET (1×10 -5 mol / L) can be used to monitor the liver injury process at the tissue level by fluorescence imaging, which can obtain Fig.32 The consistent experimental results further demonstrated that the probe TXET can be used as a sensitive tool to identify liver damage.

[0091] In summary, using a simple organic synthesis method, we obtained a method for detecting HSO3 - / ClO- The probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) was used to study the viscosity of the sample. - The color of the solution changed significantly (from blue to colorless). This indicates that the probe - After that, different optical properties are shown. And with the HSO3 - With the increase of concentration, the fluorescence intensity of the probe at 500 nm wavelength gradually increased, which was due to the addition of HSO3 - After HSO3 - The Michael addition reaction with the probe molecule causes the photoinduced electron transfer effect of the fluorescent molecule to disappear, and emits strong green fluorescence. - The color of the solution changed significantly (from blue to light yellow). This indicates that the probe - After that, they show different optical properties. And with the ClO - With the increase of concentration, the fluorescence intensity of the probe at a wavelength of 530 nm gradually increased; the probe was added with ClO at high viscosity. - The color of the solution changed significantly (from blue to light colorless). This indicates that the probe - After that, they show different optical properties. And with the ClO - With the increase of concentration, the fluorescence intensity of the probe at 720 nm gradually decreases. This is because the probe molecules react with ClO - The response of the probe causes the photoinduced electron transfer effect of the luminescent molecule to disappear, and a strong yellow fluorescence is emitted, and the red light gradually decreases. Finally, with the increase of viscosity, the fluorescence intensity of the probe at a wavelength of 720 nm gradually increases. - / ClO - The fluorescence emission spectrum shows obvious regular changes under different viscosities, which is a good way to detect HSO3 - / ClO - The preparation of this fluorescent probe fills the gap in the field of fluorescent probes and can simultaneously quantify the detection of HSO3 - / ClO - At the same time, during the biological experiment, it can be found that with the occurrence and development of liver damage, the green, yellow and red fluorescence signals of the probe TXET all increased significantly. It is worth noting that during the treatment of liver damage in mice, the three fluorescence signals decreased significantly, indicating that the probe TXET can be used as a sensitive tool to identify liver damage.

Claims

1. A method for detecting HSO3 - / ClO - The invention discloses a method for preparing a fluorescent probe having a high viscosity and a high viscosity, and its application, (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET), whose structural formula is:

2. A method for simultaneously detecting HSO3 according to claim 1. - / ClO - The preparation method and application of the fluorescent probe of viscosity are as follows: Chloroform and phosphorus tribromide were added to anhydrous N,N-dimethylformamide (DMF), and the mixture was stirred at 0°C for 1 hour. Cyclohexanone was then added to the chloroform to mix the mixture thoroughly. The mixture was added to the reaction system, vacuumed, and reacted at room temperature for 18 hours. After the reaction was completed, the pH was adjusted to neutral with saturated NaHCO3, and the organic phase was extracted with dichloromethane. The organic phase was collected and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column to obtain a light yellow oily product 2-chloro-1-cyclooctene-1-carboxaldehyde (A1). 4-(Diphenylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde are ultrasonically dissolved in tetrahydrofuran (THF) solution, potassium carbonate aqueous solution and tetrabutylammonium bromide are added and ultrasonically dissolved, stirred at room temperature for 30 minutes, and then tetrakis(triphenylphosphine)palladium is added to the reaction system, ultrasonically dissolved, vacuumized, and heated to reflux for about 6 hours; after the reaction is completed, it is cooled to room temperature, extracted with dichloromethane (DCM), the organic phase is collected and the solvent is removed by rotary evaporation, and purified by silica gel column to obtain a fluorescent green solid product 4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carboxaldehyde (A2); A1, A2 and cesium carbonate were dissolved in anhydrous DMF, vacuumized, and then reacted at room temperature for 20 hours; after the reaction, the reaction solution was spin-dried to obtain a crude product, which was separated and purified by column chromatography, and dried to obtain a yellow solid product 6-(4-(diphenylamine)phenyl)-2,3-dihydro-1H-xanthene-4-carboxaldehyde (A3); 2-Methylbenzothiazole and iodoethane were added to ethanol and heated under reflux for about 12 hours. When the reaction was finished, the reaction solution was cooled to room temperature, a large amount of precipitate was separated out and filtered and washed, and then dried in a vacuum drying oven to obtain an off-white solid 3-ethyl-2-methylbenzothiazol-3-ium iodide (A4). A3 and A4 were dissolved in anhydrous ethanol, ultrasonicated to fully dissolve them, and heated under reflux for 8 hours; when the reaction was over, the reaction stock solution was cooled to room temperature, and a precipitate was generated, which was filtered and washed with ethanol, and dried to obtain a dark blue product (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET); The synthesis path is as follows:

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