Derivatives of triphenylamine and thiazole salt, fluorescent probe as well as preparation method and application of fluorescent probe

Abstract

The invention relates to the technical field of fluorescence imaging, in particular to a derivative of triphenylamine and thiazole salt, a fluorescent probe as well as a preparation method and application of the fluorescent probe. The fluorescent probe disclosed by the invention is a derivative of triphenylamine and thiazole salt. The structure of the fluorescent probe contains a positive charge, and the internal environment of the mitochondria is a negative charge. When the fluorescent probe provided by the invention is used in cell imaging, the fluorescent probe can penetrate through a cell membrane to enter living cells and is adsorbed on mitochondria under the attraction of negative charges in the mitochondria. In the state, after the fluorescent probe is excited to generate fluorescence reaction based on a specific light field, fluorescence imaging of the cells can be realized, so that the internal change process of mitochondria can be observed through real-time fluorescence imaging. The fluorescent probe disclosed by the invention has relatively small cytotoxicity and relatively good photobleaching resistance, can specifically target mitochondria and can realize long-time fluorescence imaging, so that the fluorescent probe disclosed by the invention has relatively good advantages in cell imaging.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence imaging, and in particular to a derivative of triphenylamine and thiazole salt, a fluorescent probe, and a preparation method and application thereof. Background Art

[0002] Mitochondria are membrane-bound organelles present in most cells. They are the main place for cells to carry out aerobic respiration and supply energy. Its basic structure can be divided into four functional areas: the outer membrane of mitochondria, the mitochondrial membrane space, the inner membrane of mitochondria, and the mitochondrial matrix. The inner membrane of mitochondria is coated with its own genetic material, namely mitochondrial DNA, so it is a semi-autonomous organelle. In addition to supplying energy to cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission, and cell apoptosis, and have the ability to regulate cell growth and cell cycle. Therefore, it is of great significance to accurately understand the metabolic process of mitochondria.

[0003] Fluorescence imaging technology has the advantages of simple operation, fast response and high sensitivity. It is widely used in the field of cell imaging. In recent years, with the continuous development of fluorescence imaging technology, small molecule fluorescent probes have gradually attracted the attention of scientific researchers in biological imaging, medical diagnosis and drug screening. As a result, a large number of mitochondrial fluorescent localization probes have emerged. However, the existing mitochondrial fluorescent probes have the disadvantages of high cytotoxicity, poor resistance to photobleaching and weak co-localization ability. Summary of the invention

[0004] In order to solve the technical problems that fluorescent probes in the prior art have high cytotoxicity, poor resistance to photobleaching and weak co-localization ability, the present invention provides a derivative of triphenylamine and thiazole salt, a fluorescent probe and a preparation method and application thereof.

[0005] The present invention is implemented by the following technical scheme: a derivative of triphenylamine and thiazole salt, whose chemical formula is: .

[0006] The present invention also includes a fluorescent probe, which is a derivative of the above triphenylamine and thiazole salt. The fluorescent probe is used for targeting and locating mitochondria in cell imaging.

[0007] As a further improvement of the present invention, the maximum absorption peak of the fluorescent probe in the ultraviolet-visible absorption spectrum is 510 nm.

[0008] As a further improvement of the present invention, the fluorescent probe generates a fluorescence emission spectrum in the range of 540nm-800nm ​​under the excitation condition of 510nm light.

[0009] The present invention also includes a method for preparing a fluorescent probe, which comprises the following steps: Triphenylamine and N,N-dimethylformamide are subjected to Vilsmeier-Haack reaction under catalytic conditions to obtain 4-formyltriphenylamine; the chemical equation of the reaction is as follows: ; 2,3-Dimethylbenzene thiazole iodide and 4-formyl triphenylamine are subjected to Knoevenagel condensation reaction in proportion to obtain an intermediate product 1, and the intermediate product 1 is placed under acidic conditions for protonation to obtain a derivative of triphenylamine and thiazole salt as described in Formula I, i.e., the fluorescent probe; the reaction equation is as follows: .

[0010] As a further improvement of the present invention, the 2,3-dimethylbenzothiazole iodide and the 4-formyltriphenylamine undergo a Knoevenagel condensation reaction as follows: 2,3-Dimethylbenzothiazole iodide and 4-formyltriphenylamine are dissolved in ethanol in proportion and fully mixed. After fully mixing, they are filtered and separated by column chromatography to obtain an intermediate product 1. The intermediate product 1 is protonated under acidic conditions for half an hour to obtain a fluorescent probe.

[0011] As a further improvement of the present invention, in the Knoevenagel condensation reaction, the molar ratio of the 2,3-dimethylbenzothiazole iodide to 4-formyltriphenylamine is 1:1, and the solid-liquid ratio of the 2,3-dimethylbenzothiazole iodide to the ethanol is 1:10.

[0012] As a further improvement of the present invention, the preparation method of 4-formyltriphenylamine is as follows: under a protective atmosphere, N,N-dimethylformamide and phosphorus oxychloride are added to 1,2-dichloroethane to obtain an intermediate product 2, and then triphenylamine is added and reacted at 60° C. to obtain an intermediate product 3; the intermediate product 3 cooled to room temperature is placed in a saturated sodium acetate solution and stirred to obtain a mixed solution 1; dichloromethane is added to the mixed solution 1 to extract and then an organic phase is retained; the solvent in the organic phase is removed and 4-formyltriphenylamine is obtained by column chromatography separation.

[0013] As a further improvement of the present invention, in the Vilsmeier-Haack reaction, the molar ratio of the triphenylamine, the N,N-dimethylformamide and the phosphorus oxychloride is 1:1.2:1.2, and the volume ratio of the N,N-dimethylformamide to 1,2-dichloroethane is 2:7.

[0014] An application of the fluorescent probe as described above in cell imaging comprises: co-incubating living cells with the fluorescent probe to achieve targeting of mitochondria in the living cells.

[0015] The technical solution provided by the present invention has the following beneficial effects: (1) The fluorescent probe of the present invention has a positive charge in its structure, while the internal environment of the mitochondria is negatively charged. Therefore, when the fluorescent probe of the present invention is used in cell imaging, the fluorescent probe can penetrate the cell membrane into the living cell, and then adsorb on the mitochondria in the cell under the attraction of the negative charge inside the mitochondria. In this state, after the fluorescent probe is excited by a specific light field to produce a fluorescent reaction, fluorescent imaging of the cell can be achieved, and the mitochondria in the cell can be located, so as to observe the internal changes of the mitochondria through real-time fluorescence imaging.

[0016] (2) During the synthesis process of the fluorescent probe of the present invention, the conjugated structure is enlarged, so that the emission wavelength of the synthesized fluorescent probe is red-shifted. Therefore, the fluorescent probe of the present invention can emit red light after being excited during the cell imaging process. During the cell imaging process, red light is more conducive to observation during the cell imaging process than blue light or purple light.

[0017] (3) The fluorescent probe of the present invention has less cytotoxicity, better resistance to photobleaching, can specifically target mitochondria and can perform long-term fluorescence imaging, which proves that the fluorescent probe of the present invention has better advantages in cell imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the nuclear magnetic hydrogen spectrum of the fluorescent probe provided by the present invention.

[0019] Figure 2 This is the nuclear magnetic carbon spectrum of the fluorescent probe provided by the present invention.

[0020] Figure 3 This is the mass spectrum of the fluorescent probe provided by the present invention.

[0021] Figure 4 This is the ultraviolet-visible absorption spectrum of the fluorescent probe provided by the present invention.

[0022] Figure 5 This is the fluorescence spectrum of the fluorescent probe provided by the present invention.

[0023] Figure 6 This is a diagram of the cytotoxicity analysis of the fluorescent probe provided by the present invention on HeLa cells.

[0024] Figure 7 This is a diagram of the cytotoxicity analysis of the fluorescent probe provided by the present invention on HepG2 cells.

[0025] Figure 8 This is a co-localization imaging analysis diagram of the fluorescent probe provided by the present invention and mitochondrial commercial dyes in living cells.

[0026] Fig. 9This is a graph analyzing the anti-photobleaching performance of the fluorescent probe and mitochondrial commercial dye provided by the present invention in living cells.

[0027] Fig.10 The diagram is a graph showing changes in relative fluorescence intensity and number of photographs of the fluorescent probe and mitochondrial commercial dye provided by the present invention in living cells.

[0028] Fig.11 This is a fluorescence imaging diagram of the fluorescent probe provided by the present invention during the mitochondrial autophagy process of HeLa cells. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1 This embodiment provides a derivative of triphenylamine and thiazole salt, and its chemical formula is: .

[0031] The derivatives of triphenylamine and thiazole salt can be used as fluorescent probes in the field of cell imaging during practical application. Therefore, the present embodiment also provides a fluorescent probe, which is a derivative of triphenylamine and thiazole salt. Its chemical formula is as described above. According to the above chemical formula, the structure of the fluorescent probe of the present embodiment contains a positive charge, and the internal environment of the mitochondria in the cell is negatively charged. Therefore, when the fluorescent probe and living cells are co-incubated, the fluorescent probe will penetrate the cell membrane into the cell, and under the mutual attraction of positive and negative charges, the fluorescent probe can be targeted to the mitochondria, thereby achieving the purpose of accurately locating the mitochondria in the cell. In the present embodiment, the mitochondria are accurately located by the fluorescent probe, and then the metabolic process of the mitochondria in the cell can be observed by fluorescent imaging, thereby understanding the processes such as cell differentiation and apoptosis.

[0032] The maximum absorption peak of the fluorescent probe in the ultraviolet-visible absorption spectrum is 510 nm. Under the excitation condition of 510 nm light, the fluorescence emission spectrum generated by the fluorescent probe ranges from 540 nm to 800 nm.

[0033] In the prior art, triphenylamine fluorescent dyes have excellent physical properties and present blue-violet light in cell imaging. If triphenylamine fluorescent dyes are used as fluorescent probes, they have the following disadvantages: 1. Existing triphenylamine fluorescent dyes cannot locate mitochondria, so when observing the situation of mitochondria in cells through fluorescent probes, it is impossible to accurately observe the changes in mitochondria in cells through cell imaging technology. 2. Existing triphenylamine fluorescent probes have complex synthesis methods, high cytotoxicity, and weak resistance to photobleaching.

[0034] In this embodiment, triphenylamine is formylated by Vilsmeier-Haack reaction to obtain 4-formyl triphenylamine, and then the obtained 4-formyl triphenylamine and 2,3-dimethylbenzothiazole iodide are reacted by Knoevenagel condensation reaction to synthesize a fluorescent probe. The fluorescent probe synthesized by the above-mentioned synthesis method has a large conjugated structure, which can cause the fluorescence emission wavelength of the synthesized fluorescent probe to red-shift, so that red light can be presented in cell imaging. In the process of fluorescence imaging, emitting blue light or purple light is not conducive to the observation of cell imaging, and emitting red light is conducive to the observation of cell imaging. This proves that the fluorescent probe prepared in this embodiment is more conducive to the observation of cells in the process of cell imaging than the existing triphenylamine fluorescent dyes.

[0035] The fluorescent probe of this embodiment is synthesized by modifying 4-formyltriphenylamine on 2,3-dimethylbenzothiazole iodide as a matrix. Therefore, this embodiment also provides a method for preparing a fluorescent probe, and the overall chemical equation for its synthesis is as follows: .

[0036] The preparation method of the fluorescent probe specifically comprises the following steps: (1) Triphenylamine and N,N-dimethylformamide are subjected to a Vilsmeier-Haack reaction under catalytic conditions to obtain 4-formyltriphenylamine. The chemical equation of the reaction is as follows: .

[0037] Among them, the chemical structural formula of triphenylamine is as follows: .

[0038] The chemical structure of N,N-dimethylformamide is as follows: .

[0039] Among them, the specific operation of the Vilsmeier-Haack reaction is as follows: under protective atmosphere conditions, N,N-dimethylformamide and phosphorus oxychloride are added to 1,2-dichloroethane to obtain intermediate product 2. Then, intermediate product 2 is stirred at room temperature and triphenylamine is added, and then heated to 60°C for reaction for 16 hours to obtain intermediate product 3. After intermediate product 3 is cooled to room temperature, intermediate product 3 is placed in a saturated sodium acetate solution and stirred to obtain a mixed solution 1. Then, dichloromethane is added to the mixed solution 1 for multiple extractions, and the organic phase after extraction is retained. The organic phase is dried with anhydrous sodium sulfate, and then the solvent is removed by reduced pressure distillation, and finally the target 4-formyltriphenylamine is obtained by column chromatography.

[0040] The chemical structural formula of the obtained 4-formyltriphenylamine is as follows: .

[0041] When performing the Vilsmeier-Haack reaction, the molar ratio of the triphenylamine, the N,N-dimethylformamide and the phosphorus oxychloride is 1:1.2:1.2, and the volume ratio of the N,N-dimethylformamide to 1,2-dichloroethane is 2:7.

[0042] (2) 2,3-Dimethylbenzene thiazole iodide and 4-formyl triphenylamine are subjected to Knoevenagel condensation reaction in proportion to obtain an intermediate product 1, and the intermediate product 1 is placed under acidic conditions for protonation to obtain a derivative of triphenylamine and thiazole salt as described in Formula I, i.e., the fluorescent probe; the reaction equation is as follows: .

[0043] Among them, the chemical structure of 2,3-dimethylbenzene thiazole iodide is as follows: .

[0044] The specific operation of the Knoevenagel condensation reaction is as follows: 2,3-dimethylbenzothiazole iodide and 4-formyltriphenylamine obtained in step (1) are dissolved in ethanol. Stir at room temperature for more than 12 hours to obtain a mixed solution 2. Filter the mixed solution 2 to obtain a solid crude product, and then purify it by column chromatography to obtain an intermediate product 1. Then, the intermediate product 1 is protonated under acidic conditions for half an hour to obtain the fluorescent probe. The entire preparation process is simple and easy to perform, so that the fluorescent probe can be prepared by the above preparation method in practical applications.

[0045] The chemical structure of the fluorescent probe is as follows: .

[0046] Specifically, the yield of the intermediate product 1 after purification by column chromatography is between 60% and 85%, which indicates that the above operation can effectively prepare the desired intermediate product 1. The intermediate product 1 after purification by column chromatography is a neutral product.

[0047] It is understandable that, in the present embodiment, the acidic conditions during the operation of placing the intermediate product 1 under acidic conditions for protonation for half an hour can be a concentrated hydrochloric acid environment. And the purpose of protonation is to obtain a positive charge on the intermediate product 1, that is, to obtain the fluorescent probe we need. Through this operation, the prepared fluorescent probe is made to carry a positive charge. When the fluorescent probe prepared by the present embodiment is used for cell imaging operation, the fluorescent probe will penetrate the cell membrane into the living cell, and under the attraction of the negative charge carried by the mitochondria, the fluorescent probe entering the cell will bind to the mitochondria. Thus, the purpose of locating the mitochondria in the cell is achieved. By locating the mitochondria in the cell with the fluorescent probe, it is possible to facilitate researchers to quickly find the mitochondria in the cell when observing the inside of the cell through fluorescent imaging, thereby realizing the monitoring of the internal changes of the mitochondria by real-time fluorescence imaging, and the imaging effect of the above-mentioned probe in observing the internal changes of the mitochondria is not affected by the changes of the intracellular biologically active small molecules and the fluorescent probe itself.

[0048] In the Knoevenagel condensation reaction, the molar ratio of the 2,3-dimethylbenzothiazole iodide to 4-formyltriphenylamine is 1:1, and the solid-liquid ratio of the 2,3-dimethylbenzothiazole iodide to the ethanol is 1:10.

[0049] The Vilsmeier-Haack reaction and the Knoevenagel condensation reaction of this embodiment need to be carried out under a protective atmosphere. In practical applications, the type of gas used in the protective atmosphere is not limited, and various types of protective gases well known to those skilled in the art, such as nitrogen and inert gases, can be used.

[0050] The typical application scenario of the fluorescent probe provided in this embodiment is to realize cell imaging. Because the fluorescent probe carries a positive charge, it can specifically bind to the mitochondria in the cell. For example, after the living cells are co-incubated with the fluorescent probe, the fluorescent probe can penetrate the cell membrane into the living cells, and then adsorb on the mitochondria in the cell under the attraction of the negative charge inside the mitochondria. In this state, after the fluorescent probe is excited to produce a fluorescent reaction based on a specific light field, fluorescent imaging of the cell can be realized, and the mitochondria in the cell can be located, so as to observe the internal changes of the mitochondria through real-time fluorescence imaging.

[0051] In order to verify the properties and performance of the materials provided in this embodiment, technicians verified the scheme of Example 1 in subsequent test examples and experiments, experimentally synthesized relevant samples, analyzed the physical and chemical properties of the synthesized substances in subsequent experiments, and tested the various properties of the material when used as a fluorescent probe.

[0052] Test Example 1 (1) Preparation of 4-formyltriphenylamine 2.8 mL of N, N-dimethylformamide with a concentration of 36.6 mmol and 3.4 mL of phosphorus oxychloride with a concentration of 36.6 mmol were measured and dissolved in 10 mL of 1,2-dichloroethane under inert gas conditions to obtain intermediate product 2. After stirring intermediate product 2 at room temperature for 5 min, 5.98 g of triphenylamine with a concentration of 24.4 mmol was added, and then heated to 60 ° C for 16 h to obtain intermediate product 3. After the reaction, intermediate product 3 was cooled to room temperature. After cooling to room temperature, intermediate product 3 was poured into 200 mL of saturated sodium acetate solution and stirred for 30 min to obtain mixed solution 1. Dichloromethane was added to mixed solution 1 for multiple extractions, and the organic phase after extraction was retained. The organic phase after extraction was dried over anhydrous sodium sulfate and the solvent in the organic phase was removed by reduced pressure distillation, and finally the target 4-formyl triphenylamine was obtained by column chromatography.

[0053] 10 mmol of 2,3-dimethylbenzothiazole iodide and 10 mmol of 4-formyltriphenylamine were dissolved in 30 mL of ethanol. The reaction was stirred at room temperature overnight, and then filtered to obtain a solid crude product, which was purified by column chromatography to obtain an intermediate product 1, and then the intermediate product 1 was placed in a concentrated hydrochloric acid environment for protonation for half an hour to obtain the fluorescent probe.

[0054] Performance Testing 1. Product composition analysis 1.1 Fluorescent probes This experiment firstly performs nuclear magnetic resonance hydrogen spectrum analysis on the fluorescent probe prepared in Test Example 1, and obtains the following Figure 1 shown 1 H NMR spectrum, through Figure 1 of 1 The H NMR graph was analyzed and the analysis results were as follows: 1H NMR(400 MHz, DMSO-d6) δ 8.33 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 8.4Hz, 1H), 8.08 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.79 (t, J =7.8 Hz, 1H), 7.77 – 7.67 (m, 2H), 7.39 (t, J = 7.7 Hz, 4H), 7.18 (dd, J =21.1, 7.6 Hz, 6H), 6.87 (d, J = 8.5 Hz, 2H), 5.71 (s, 1H), 4.25 (s, 3H).

[0055] This experiment also performed nuclear magnetic resonance carbon spectrum analysis on the fluorescent probe prepared in Test Example 1, and obtained the following Figure 2 shown 13 C NMR spectrum, through Figure 2 The analysis results are as follows: 13 C NMR (101 MHz, DMSO-d6) δ 172.25, 151.90, 149.13, 146.05, 142.54,132.39, 130.58, 129.72, 128.56, 127.92, 126.72, 126.02, 124.61, 119.53,117.03, 110.65, 55.46.

[0056] This experiment also analyzed the mass spectrometry characteristics of the fluorescent probe prepared in Test Example 1, and obtained the following Figure 3 The mass spectrum shown. Figure 3 The analysis showed that the relative molecular mass of the fluorescent probe in Test Example 1 was 419.

[0057] pass Figure 1 , Figure 2 and Figure 3 From the data, it can be seen that the chemical structure of the fluorescent probe synthesized in Test Example 1 is as follows: .

[0058] 2. Fluorescence characteristics test 2.1 UV absorption performance The fluorescent probe prepared in Test Example 1 was placed in a PBS buffer solution, and the UV-visible absorption spectrum of the fluorescent probe prepared in Test Example 1 in the PBS buffer solution was measured. The parameters in this operation are as follows: a double-pass cuvette with a size of 1 cm was selected, 2 mL of PBS buffer solution containing the fluorescent probe with a working concentration of 10 μM was added to the double-pass cuvette, and then the UV-visible absorption spectrum was measured by a UV-visible spectrophotometer. Figure 4 The UV-visible absorption spectrum of the fluorescent probe. In order to be consistent with the subsequent cell experiments, when conducting the UV absorption performance test, the UV-visible spectrophotometer can use a 510nm wavelength laser to excite the fluorescent probe, set the emission wavelength range to 450nm-900nm, set the slit width to 5nm, and set the excitation voltage to 400V.

[0059] Through Figure 4 Analysis shows that the absorption peak of the fluorescent probe synthesized in Test Example 1 is around 510 nm.

[0060] 2.2 Fluorescence spectrum test The fluorescent probe prepared in Test Example 1 was placed in a PBS buffer solution, and the fluorescence spectrum of the fluorescent probe prepared in Test Example 1 in the PBS buffer solution was measured. The parameters in this operation are as follows: a four-way cuvette with a size of 1 cm was selected, 2 mL of PBS buffer solution containing the fluorescent probe with a working concentration of 10 μM was added to the four-way cuvette, and then the fluorescence spectrum was measured by a fluorescence spectrophotometer as shown below: Figure 5 Fluorescence spectrum of the fluorescent probe. In order to be consistent with the subsequent cell experiments, when conducting the fluorescence spectrum test, the fluorescence spectrophotometer can use a 510nm wavelength laser to excite the fluorescent probe, set the emission wavelength range to 450nm-900nm, set the slit width to 5nm, and set the excitation voltage to 400V.

[0061] Through Figure 5 Analysis shows that the fluorescence emission wavelength of the fluorescent probe synthesized in Test Example 1 is between 540nm and 800nm, with the maximum emission wavelength being around 651nm.

[0062] By testing the fluorescence characteristics of the fluorescent probe of the present invention, it can be known that the maximum absorption wavelength of the fluorescent probe of the present invention in PBS buffer is about 510nm, the maximum emission wavelength is about 651nm, and its fluorescence quantum efficiency is calculated to be 16%. The above data are much higher than those of traditional triphenylamine dyes, and the fluorescent probe of the present invention has a large Stokes shift (Stokes shift is the difference between the maximum emission wavelength and the maximum excitation wavelength). Due to its large Stokes shift, the synthesized fluorescent probe will be red-shifted, so that the fluorescent probe of the present invention emits red light after excitation.

[0063] 3. Biological Toxicity This experiment uses the MTT method to test the biological toxicity of the fluorescent probe prepared in Test Example 1 to living cells. Among them, in the biological toxicity of living cells, HeLa cells and HepG2 cells are generally used for the biological toxicity test of living cells. Therefore, in this experiment, HeLa cells and HepG2 cells are respectively selected for the biological toxicity test of living cells.

[0064] 2.1 Biological toxicity of fluorescent probes to HeLa cells The specific experimental operations are as follows: (1) The fluorescent probe solution prepared in Test Example 1 was prepared into the following concentrations: 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 15 μM, and 20 μM.

[0065] (2) HeLa cells were seeded into 96-well plates at a density of 1×104 cells per well. The 96-well plates were placed in a cell culture incubator with 10 culture plates. The culture conditions were: 37°C, 5% CO 2 , saturated humidity; culture for 24 hours to allow it to completely adhere to the wall. After that, the above 10 culture media were replaced with fresh culture media, and the 10 fluorescent probe solutions of different concentrations prepared in step (1) were added to 10 fresh culture media respectively. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well, and incubated for another 4 hours. Then, 100 μL DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance of the solution in each well of the 96-well plate was measured, and the cell survival rate was calculated according to the following formula: Cell survival rate = (absorbance of the test group - absorbance of the blank group) / (absorbance of the cell group - absorbance of the blank group) × 100%. The result is as follows: Figure 6 of experimental data.

[0066] 2.2 Biological toxicity of fluorescent probes to HepG2 cells The specific experimental operations are as follows: (1) The fluorescent probe solution prepared in Test Example 1 was prepared into the following concentrations: 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 15 μM, and 20 μM.

[0067] (2) HepG2 cells were seeded into 96-well plates at a density of 1×104 cells per well. The 96-well plates were placed in a cell culture incubator with 10 culture plates. The culture conditions were: 37°C, 5% CO 2, saturated humidity; culture for 24 hours to allow it to completely adhere to the wall. After that, the above 10 culture media were replaced with fresh culture media, and the 10 fluorescent probe solutions of different concentrations prepared in step (1) were added to 10 fresh culture media respectively. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well, and incubated for another 4 hours. Then, 100 μL DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance of the solution in each well of the 96-well plate was measured, and the cell survival rate was calculated according to the following formula: Cell survival rate = (absorbance of the test group - absorbance of the blank group) / (absorbance of the cell group - absorbance of the blank group) × 100%. The result is as follows: Figure 7 of experimental data.

[0068] Through Figure 6 and Figure 7 Analysis shows that when the concentration of the fluorescent probe synthesized in Test Example 1 is within the range of 0-20 μM, the survival rates of HeLa cells and HepG2 cells are both above 90%. This indicates that the fluorescent probe synthesized in Test Example 1 has almost no cytotoxicity within the range of 0-20 μM.

[0069] IV. Cellular Imaging Effects of Fluorescent Probes In order to verify the fluorescence imaging effect of the fluorescent probe provided by the present invention in cells, this experiment adopted a co-localization experimental method. In the experiment, Mito-Tracker Green, a mitochondrial commercial dye with a significant difference in excitation light from the fluorescent probe of the present invention, was selected as a control material to achieve performance calibration. Among them, the model of the mitochondrial commercial dye is Mito-Tracker Green, purchased from Sigma. The specific operation steps are as follows: First, remove the original culture medium in the cell culture dish and wash it several times with PBS buffer. Then add 1 mL of serum-free culture medium and the fluorescent probe synthesized by the present invention, and ensure that the working concentration of the fluorescent probe is 10 μM, and incubate for 30 minutes. After the incubation is completed, remove the culture medium containing the fluorescent probe, and then wash it three times with PBS buffer to ensure that the fluorescent probe that has not entered the cell is completely removed. Then add the mitochondrial commercial dye Mito-Tracker Green to the serum-free culture medium, adjust its working concentration to 1 μM, and incubate for 20 minutes, and then perform confocal imaging observation.

[0070] During the imaging process, we used a 510nm laser to excite the fluorescent probe and a 488nm laser to excite the mitochondrial commercial dye Mito-Tracker Green. Figure 8 .

[0071] Through Figure 8Analysis shows that Figure 8 (a) is a cell imaging diagram of the fluorescent probe of the present invention; Figure 8 Middle (b) is a cell imaging image of the commercial mitochondrial dye Mito-Tracker Green; Figure 8 (c) is a superimposed image of the cell imaging image of the fluorescent probe of the present invention and the cell imaging image of the mitochondrial commercial dye Mito-Tracker Green. By analyzing the above three images, it can be seen that the fluorescence signal overlap degree of the fluorescent probe of the present invention and the mitochondrial commercial dye Mito-Tracker Green is extremely high, and further analysis by confocal software shows that the colocalization coefficient of the cell imaging image of the fluorescent probe of the present invention and the cell imaging image of the mitochondrial commercial dye Mito-Tracker Green is as high as 0.87. This proves that the fluorescent probe of the present invention can specifically target mitochondria and achieve the positioning of mitochondria.

[0072] In addition, it was observed in this experiment that when a 510nm laser was used to excite the fluorescent probe, the emission wavelength of the fluorescent probe was in the range of 620nm-650nm. When a 488nm laser was used to excite the mitochondrial commercial dye Mito-Tracker Green, the emission wavelength of the mitochondrial commercial dye Mito-Tracker Green was in the range of 500-530nm.

[0073] Furthermore, it can be seen from the above description that the fluorescent probe is used for fluorescent imaging, which is simple to operate and has good imaging effect. Therefore, the fluorescent probe of the present invention has broad application prospects in the medical field.

[0074] 5. Anti-photobleaching performance of fluorescent probes In order to verify the anti-photobleaching performance of the fluorescent probe provided by the present invention in cells, this experiment took 100 photos continuously during the co-incubation process of the fluorescent probe and cells, and obtained the fluorescence intensity of the fluorescent probe in this process. The mitochondrial commercial dye Mito-Tracker Green was co-incubated with cells, and 100 photos were taken continuously at the same time point to obtain the fluorescence intensity of the mitochondrial commercial dye Mito-Tracker Green in this process. Fig. 9 and Fig.10 .

[0075] in, Fig. 9 The row of red pictures in the upper middle part are the 1st, 20th, 40th, 60th, 80th and 100th pictures among 100 pictures taken continuously during the co-incubation of the fluorescent probe of the present invention with cells. Fig. 9The green images in the lower middle part are the 100 images taken continuously during the co-incubation of the mitochondrial commercial dye Mito-Tracker Green with cells. The first, 20th, 40th, 60th, 80th and 100th images are shown in Figure 1. Fig. 9 Comparative analysis of the graph in the upper and lower parts shows that for the mitochondrial commercial dye Mito-Tracker Green, as the number of photos increases, the fluorescence intensity emitted by the mitochondrial commercial dye Mito-Tracker Green is gradually weakened. It can be seen that as time goes by, the fluorescence intensity emitted by the mitochondrial commercial dye Mito-Tracker Green is gradually weakened. For the fluorescent probe of the present invention, as the number of photos increases, the fluorescence intensity of the fluorescent probe of the present invention does not change significantly. It can be seen that the fluorescent probe of the present invention has better resistance to photobleaching than the mitochondrial commercial dye Mito-Tracker Green, and can minimize the light damage to biological samples.

[0076] Fig.10 This is a graph showing the relationship between the relative fluorescence intensity of the fluorescent probe and the number of photos taken in 100 consecutive photos. Fig.10 It is known from the analysis that when 100 pictures are taken continuously in the same time period, as the number of pictures taken increases, the relative fluorescence intensity of the fluorescent probe of the present invention only decreases by less than 10%, while the relative fluorescence intensity of the mitochondrial commercial dye Mito-Tracker Green decreases by about 30%. This proves that the fluorescent probe of the present invention has better resistance to photobleaching than the mitochondrial commercial dye Mito-Tracker Green, so that it has excellent photostability, and can be used to track mitochondria for a long time in practical applications.

[0077] 6. Fluorescent probes to detect mitochondrial autophagy Autophagy is the process by which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy-related genes. In short, the lysosomes of cells can clear mitochondria.

[0078] The specific operation is as follows: the fluorescent probe of the present invention is used to mark the mitochondria of HeLa cells, and the commercial mitochondrial dye Mito-Tracker Green is used to mark the lysosomes of HeLa cells, and the autophagy process of mitochondria is observed. Pictures are taken at 0min, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min and 90min, and the following are obtained: Fig.11 .

[0079] right Fig.11 The analysis shows that Fig.11 The red parts are mitochondria and the green parts are lysosomes. Fig.11 In the 9 images, the red color did not darken significantly within 0-90 minutes, while the commercial mitochondrial dye Mito-Tracker Green darkened significantly after 50 minutes, indicating that the fluorescent probe of the present invention can perform long-term fluorescence imaging in cells. This proves that the dynamic changes of mitochondria can be tracked over a long period of time using the fluorescent probe of the present invention. This conclusion provides certain guidance and reference for subsequent clinical research.

[0080] In summary, by performing the performance tests of the fluorescent probe of the present invention as items 1 to 6 above, the following conclusions can be drawn: the fluorescent probe of the present invention has a large Stokes shift and a high quantum yield, can effectively meet the conditions of fluorescence imaging, can not only image for a long time, but also minimize the light damage to biological samples. And the fluorescent probe of the present invention is applied to long-term fluorescence imaging in cells, and a cell imaging effect with clear cell structure and resistance to photobleaching can be obtained. This shows that the fluorescent probe of the present invention is of great significance for understanding the metabolism and behavior of living cells, so that it has a wide range of application prospects in the medical field.

[0081] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention to be protected. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A derivative of triphenylamine and thiazole salt, characterized in that: Its chemical formula is: 。 2. A fluorescent probe, characterized in that It is a derivative of triphenylamine and thiazole salt as claimed in claim 1, and the fluorescent probe is used for targeting and locating mitochondria in cell imaging.

3. The fluorescent probe according to claim 2, characterized in that The maximum absorption peak of the fluorescent probe in the ultraviolet-visible absorption spectrum is 510 nm.

4. The fluorescent probe according to claim 2, characterized in that Under the excitation condition of 510nm light, the fluorescent probe generates a fluorescence emission spectrum in the range of 540nm-800nm.

5. A method for preparing a fluorescent probe according to any one of claims 2 to 4, characterized in that: It includes the following steps: Triphenylamine and N,N-dimethylformamide are subjected to Vilsmeier-Haack reaction under catalytic conditions to obtain 4-formyltriphenylamine; the chemical equation of the reaction is as follows: ； 2,3-Dimethylbenzene thiazole iodide and 4-formyl triphenylamine are subjected to Knoevenagel condensation reaction in proportion to obtain an intermediate product 1, and the intermediate product 1 is placed under acidic conditions for protonation to obtain a derivative of triphenylamine and thiazole salt as described in Formula I, i.e., the fluorescent probe; the reaction equation is as follows: 。 6. The method for preparing a fluorescent probe according to claim 5, characterized in that: The 2,3-dimethylbenzothiazole iodide and the 4-formyltriphenylamine undergo a Knoevenagel condensation reaction as follows: 2,3-Dimethylbenzothiazole iodide and 4-formyltriphenylamine are dissolved in ethanol in proportion and fully mixed. After fully mixing, they are filtered and separated by column chromatography to obtain an intermediate product 1. The intermediate product 1 is protonated under acidic conditions for half an hour to obtain a fluorescent probe.

7. The method for preparing a fluorescent probe according to claim 6, characterized in that: In the Knoevenagel condensation reaction, the molar ratio of the 2,3-dimethylbenzothiazole iodide to 4-formyltriphenylamine is 1:1, and the solid-liquid ratio of the 2,3-dimethylbenzothiazole iodide to the ethanol is 1:

10.

8. The method for preparing a fluorescent probe according to claim 5, characterized in that: The preparation method of 4-formyltriphenylamine is as follows: under a protective atmosphere, N,N-dimethylformamide and phosphorus oxychloride are added to 1,2-dichloroethane to obtain an intermediate product 2, and then triphenylamine is added and reacted at 60°C to obtain an intermediate product 3; the intermediate product 3 cooled to room temperature is placed in a saturated sodium acetate solution for stirring to obtain a mixed solution 1; dichloromethane is added to the mixed solution 1 for extraction and an organic phase is retained; the solvent in the organic phase is removed and the 4-formyltriphenylamine is obtained by column chromatography separation.

9. The method for preparing a fluorescent probe according to claim 8, characterized in that: In the Vilsmeier-Haack reaction, the molar ratio of the triphenylamine, the N,N-dimethylformamide and the phosphorus oxychloride is 1:1.2:1.2, and the volume ratio of the N,N-dimethylformamide to 1,2-dichloroethane is 2:

7.

10. Use of the fluorescent probe according to any one of claims 2 to 4 in cell imaging, characterized in that: The living cells are co-incubated with the fluorescent probe to achieve targeting of mitochondria in the living cells.

Images