阅读说明：本技术 一种基于苯并噁嗪可视有机分子比率型荧光探针及其制备方法和细胞成像应用 (Benzoxazine-based visible organic molecule ratio type fluorescent probe, preparation method thereof and cell imaging application ) 是由 王素华 彭俊翔 陈宏霞 余龙 孙明泰 余欢 于 2020-12-11 设计创作，主要内容包括：本发明公开了一种基于苯并噁嗪可视有机分子比率型荧光探针及其制备方法和应用,本发明提供的荧光探针能够用来检测溶液pH值的变化,在pH变化范围为1.8-5.8内,加入本发明的荧光探针的溶液在自然光下或紫外灯下都能看出明显的比率颜色变化；本发明通过建立荧光强度和溶液pH值的拟合曲线关系,可以快速实现对溶液pH值的实时检测；本发明的荧光探针选择性好,灵敏度高,斯托克斯位移大且细胞毒性低,检测方法简便且效果明显。(The invention discloses a benzoxazine-based visible organic molecule ratio type fluorescent probe and a preparation method and application thereof, the fluorescent probe provided by the invention can be used for detecting the change of the pH value of a solution, and the solution added with the fluorescent probe can show obvious ratio color change under natural light or an ultraviolet lamp within the pH change range of 1.8-5.8; according to the invention, the real-time detection of the pH value of the solution can be rapidly realized by establishing the fitting curve relationship between the fluorescence intensity and the pH value of the solution; the fluorescent probe has the advantages of good selectivity, high sensitivity, large Stokes shift, low cytotoxicity, simple and convenient detection method and obvious effect.)

1. The benzoxazine-based visible organic molecule ratio type fluorescent probe is characterized by comprising a 6-methoxy-2-naphthaldehyde fluorescent group and a benzoxazine group.

2. The method of claim 1, wherein the fluorescent probe comprises a compound having a structure represented by formula II:

3. a method for preparing a benzoxazine-based visible organic molecular ratio-type fluorescent probe, preferably the fluorescent probe of claim 1 or 2, the method comprising:

step 1, preparing a compound having a structure shown in formula I:

and 2, preparing the benzoxazine-based visible organic molecule ratio type fluorescent probe.

4. A method according to claim 3, characterized in that step 1 comprises the steps of:

step 1-1, feeding materials into a reactor for reaction;

and step 1-2, treating the reaction liquid obtained in the step 1-1.

5. The method according to claim 3 or 4, wherein in step 1-1, the charged material comprises an indole compound, a phenol compound, or a nitrile compound.

6. The method of claim 5,

the indole compound comprises any one or more of 2-methylindole, 3-methylindole, 2,3, 3-trimethylindole and 1-butyl-2-methylindole, and preferably 2,3, 3-trimethylindole;

the phenolic compound comprises any one or more of 4-propylphenol, 4-vinyl-2-methoxyphenol, 2-hydroxy-5-nitrobenzyl bromide and 2-methyl-5-isopropylphenol, and preferably 2-hydroxy-5-nitrobenzyl bromide;

the nitrile compound comprises any one or more of methacrylonitrile, adiponitrile, acetonitrile and malononitrile, and is preferably acetonitrile.

7. A method according to claim 3, characterized in that step 2 comprises the steps of:

step 2-1, dissolving the compound I obtained in the step 1 and an aldehyde compound in an organic solvent, and adding acid;

and 2-2, carrying out post-treatment on the reaction in the step 2-1.

8. The method according to claim 7, wherein the aldehyde compound comprises any one or more of hexanal, malonic acetal, methyl glyoxal, and 6-methoxy-2-naphthaldehyde, preferably 6-methoxy-2-naphthaldehyde.

9. Use of a fluorescent probe according to claim 1 or 2 or obtained according to the method of any one of claims 3 to 8 for the visual real-time detection of the pH of a solution.

10. Use of a fluorescent probe according to claim 1 or 2 or obtained according to the method of any one of claims 3 to 8 for imaging cells.

Technical Field

The invention belongs to the technical field of fluorescence spectrum detection and analysis, and particularly relates to a benzoxazine-based visible organic molecule ratio type fluorescent probe, a preparation method thereof and a cell imaging application thereof.

Background

Intracellular pH homeostasis plays a key role in various biological processes, such as ion transport, phagocytosis, cell proliferation and apoptosis, and drug resistance. Cellular organelles have different pH distributions, for example, the cellular environment of lysosomes and endosomes is acidic with a pH of 4.5-6.8, while the environment of cytoplasm and mitochondria is weakly basic with a pH of 6.8-8.0. That is, abnormal cellular pH also reflects abnormal changes in physiological function. Therefore, real-time detection of intracellular pH fluctuations is of crucial importance to investigate the role and understanding of various cellular pH changes in biology and pathology.

Fluorescent probe technology is used as an important means of biological imaging by virtue of its ease of visualization, high sensitivity and good selectivity. In recent years, more and more fluorescent probes based on organic molecules have been designed, which are used for detection of intracellular and extracellular environments with excellent selectivity and biocompatibility and each of which shows unique advantages. However, both the open-type and the quenching-type fluorescent probes are generally susceptible to environmental factors, such as changes in optical path length and temperature, and changes in excitation intensity, when the pH is detected qualitatively. Therefore, designing a fluorescent probe with good selectivity and high sensitivity to detect the pH change in real time is a good method for replacing a single-type fluorescent probe.

The documents Nikolai I.Georgiev, Vladimir B.Bojinov, Peter S.Nikolov.the design, synthesis and photophysical properties of two novel 1,8-naphthalimide fluorescent pH sensors based on PET and ICT.dyes and Pigments 2011,88, 350-fluorescence 357, propose the design of two pH fluorescent probes with 1,8-naphthalimide dyes and condensed heterocyclic derivatives thereof for the real-time detection of pH fluctuations in solution. However, the Stokes shift of the two ratiometric probes is small, and one probe belongs to a single type fluorescent probe and is easily interfered by environmental factors.

In view of the above, there is a need to research a fluorescent probe with good selectivity, high sensitivity and good biocompatibility for real-time detection of pH change.

Disclosure of Invention

In order to overcome the problems, the inventor of the invention carries out intensive research and researches a benzoxazine-based visible organic molecule ratio type fluorescent probe as well as a preparation method and application thereof, the fluorescent probe provided by the invention can be used for detecting the change of the pH value of a solution, and the solution added with the fluorescent probe can show obvious ratio color change under natural light or ultraviolet lamp within the pH change range of 1.8-5.8; according to the invention, the real-time detection of the pH value of the solution can be rapidly realized by establishing the fitting curve relationship between the fluorescence intensity and the pH value of the solution; the fluorescent probe of the invention has good selectivity, high sensitivity, large Stokes shift, low cytotoxicity, simple and convenient detection method and obvious effect, thereby completing the invention.

Specifically, the present invention aims to provide the following:

in a first aspect, a benzoxazine-based visible organic molecule ratio type fluorescent probe is provided, which comprises a 6-methoxy-2-naphthaldehyde fluorescent group and a benzoxazine group.

Wherein the fluorescent probe comprises a compound having a structure represented by formula II:

in a second aspect, there is provided a method for preparing a benzoxazine-based visible organic molecule ratio type fluorescent probe, preferably the method for preparing a fluorescent probe according to the first aspect, the method comprising:

step 1, preparing a compound having a structure shown in formula I:

and 2, preparing the benzoxazine-based visible organic molecule ratio type fluorescent probe.

Wherein, step 1 includes the following steps:

step 1-1, feeding materials into a reactor for reaction;

and step 1-2, treating the reaction liquid obtained in the step 1-1.

Wherein, in the step 1-1, the input materials comprise indole compounds, phenolic compounds and nitrile compounds.

Wherein the content of the first and second substances,

the indole compound comprises any one or more of 2-methylindole, 3-methylindole, 2,3, 3-trimethylindole and 1-butyl-2-methylindole, and preferably 2,3, 3-trimethylindole;

the phenolic compound comprises any one or more of 4-propylphenol, 4-vinyl-2-methoxyphenol, 2-hydroxy-5-nitrobenzyl bromide and 2-methyl-5-isopropylphenol, and preferably 2-hydroxy-5-nitrobenzyl bromide;

the nitrile compound comprises any one or more of methacrylonitrile, adiponitrile, acetonitrile and malononitrile, and is preferably acetonitrile.

Wherein, step 2 includes the following steps:

step 2-1, dissolving the compound I obtained in the step 1 and an aldehyde compound in an organic solvent, and adding acid;

and 2-2, carrying out post-treatment on the reaction in the step 2-1.

Wherein the aldehyde compound comprises any one or more of hexanal propyl diacetal, methyl glyoxal and 6-methoxy-2-naphthaldehyde, and 6-methoxy-2-naphthaldehyde is preferred.

In a third aspect, there is provided a use of the fluorescent probe according to the first aspect or the fluorescent probe obtained by the method of the second aspect for visualizing and detecting the pH of a solution in real time.

In a fourth aspect, there is provided a use of the fluorescent probe according to the first aspect or the fluorescent probe obtained by the method of the second aspect for imaging cells.

The invention has the advantages that:

(1) the organic molecular ratio type fluorescent probe provided by the invention detects the change of the pH value of the solution in real time, and the solution added with the probe can see the obvious change of the fluorescent color under ultraviolet light, thereby realizing the qualitative detection of the pH value of the solution; and (3) quantitatively detecting the real-time pH of the solution by establishing a fitting curve relation between the fluorescence intensity ratio value and the pH of the solution.

(2) The organic molecular ratio type fluorescent probe provided by the invention has good biocompatibility, is quick to operate, does not need complex instruments and is low in cost when being used for detecting the pH value in living cells.

(3) The organic molecular ratio type fluorescent probe provided by the invention can avoid good selectivity and strong anti-interference performance, and can effectively avoid the interference of a plurality of influence factors in the environment inside and outside the cell; the detection steps are simple and convenient, the response speed is high, and the detection effect is good.

Drawings

FIG. 1 shows fluorescence spectra and visualized photographs of fluorescent probes in different pH buffers of Experimental example 2;

FIG. 2 shows fluorescence intensity ratio values (F) of fluorescent probes in different pH buffers in Experimental example 2₅₇₅/F₄₃₀) And pH;

FIG. 3 shows experimental example 3 with fluorescent probes added at different concentrations H⁺The subsequent fluorescence spectrogram;

FIG. 4 shows the fluorescent probes of Experimental example 4 with different OH concentrations^-The subsequent fluorescence spectrogram;

FIG. 5 shows cell imaging photographs of the fluorescent probe of Experimental example 5 in HeLa cells under different pH environments.

Detailed Description

The present invention will be described in further detail below with reference to the drawings, examples and experimental examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. While the various aspects of the embodiments are presented in drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

In a first aspect of the invention, the invention aims to provide a benzoxazine-based visible organic molecular ratio type fluorescent probe, which comprises a 6-methoxy-2-naphthaldehyde fluorescent group and a benzoxazine group.

In a preferred embodiment, the fluorescent probe comprises a compound having the structure shown in formula II below:

in a second aspect of the present invention, it is an object to provide a method for preparing a benzoxazine-based visible organic molecular ratio type fluorescent probe, preferably the method for preparing a fluorescent probe according to the first aspect, the method comprising:

step 1, preparing a compound having a structure shown in formula I:

in a preferred embodiment, the step 1 comprises the steps of:

step 1-1, feeding materials into a reactor for reaction.

In step 1-1, the material to be charged includes indole compounds, phenol compounds, nitrile compounds.

In the step 1-1, the indole compound comprises any one or more of 2-methylindole, 3-methylindole, 2,3, 3-trimethylindole and 1-butyl-2-methylindole, and preferably 2,3, 3-trimethylindole.

According to the invention, the lone pair electrons on the nitrogen of the indole compound provide a driving force for the ring-opening reaction, and preferably, 2,3, 3-trimethylindole is the indole compound, so that the ring-opening reaction has better stability.

In step 1-1, the phenolic compound comprises any one or more of 4-propylphenol, 4-vinyl-2-methoxyphenol, 2-hydroxy-5-nitrobenzyl bromide and 2-methyl-5-isopropylphenol, and preferably 2-hydroxy-5-nitrobenzyl bromide.

According to the present invention, 2-hydroxy-5-nitrobenzyl bromide is preferably selected as a phenolic compound, which can improve the speed of photochromic response and the effect of accelerating thermal conversion rate, and the introduction of bromine can also improve the fatigue resistance of discoloration.

In step 1-1, the nitrile compound includes any one or more of methacrylonitrile, adiponitrile, acetonitrile and malononitrile, and acetonitrile is preferred.

According to the invention, the nitrile compound contains a nitrile group, is a rigid group, has larger polarity, can perform potential cross-linking reaction with certain reactive groups, improves the stability of the fluorescent probe, preferably selects acetonitrile as the nitrile compound, has low viscosity and is easy to react, and the prepared fluorescent probe has higher yield.

In step 1-1, optionally, the reaction is carried out under the protection of inert gas, wherein the inert gas comprises any one or more of helium, argon and nitrogen, and nitrogen is preferred.

In the step 1-1, the mol ratio of the indole compound to the phenol compound is (0.5-6): 1, preferably (1-4): 1, more preferably 2: 1.

according to the invention, the molar ratio of the indole compound to the phenol compound is different, the reaction products are different, namely the purity of the target product fluorescent probe is different, and when the molar ratio of the indole compound to the phenol compound is (0.5-6): 1, in particular 2: 1, the obtained fluorescent probe has the highest purity and the least side reaction.

In step 1-1, the reaction time is 4 to 24 hours, preferably 6 to 18 hours, and more preferably 12 hours.

According to the invention, the conversion rate of the phenolic compound gradually increases with the extension of the reaction time, and the reaction time is too long, so that the method has no significance for the reaction.

In a further preferred embodiment, the reaction occurring in step 1-1 is as shown in formula III:

and step 1-2, treating the reaction liquid obtained in the step 1-1.

In the step 1-2, the treatment process comprises standing, filtering, extracting, drying, separating and purifying.

According to the present invention, it is preferable to stand at a low temperature, for example, the reaction liquid obtained in step 1-1 is placed in a refrigerator.

According to the invention, ultrapure water is preferably used for filtration, so that the production cost is reduced, and the environmental pollution caused by using a solvent can be reduced.

According to the invention, the extractant is preferably an ether compound, more preferably diethyl ether.

According to the invention, the drying agent is preferably a sulfate salt, more preferably anhydrous sodium sulfate. As the reaction solution in the step 1-1 is alkalescent, the anhydrous sodium sulfate has large water absorption capacity, low price, large particles, convenient treatment and stable property.

According to the invention, the eluent is preferably any one or combination of alkane and ester compounds, and is more preferably a mixture of dichloromethane and ethyl acetate.

In a preferred embodiment, the molar ratio of dichloromethane to ethyl acetate is (6-15): 1, preferably (8-13): 1, more preferably 10: 1.

And 2, preparing the benzoxazine-based visible organic molecule ratio type fluorescent probe.

In a preferred embodiment, the step 2 comprises the steps of:

and 2-1, dissolving the compound I obtained in the step 1 and an aldehyde compound in an organic solvent, and adding acid.

In step 2-1, the aldehyde compound comprises any one or more of hexanal, malonal, methyl glyoxal and 6-methoxy-2-naphthaldehyde, and 6-methoxy-2-naphthaldehyde is preferred.

According to the invention, preferably, the indole compound and the phenol compound are firstly reacted, and then the aldehyde compound is added, so that the reaction is more thorough; the aldehyde group has a catalytic effect on benzoxazine ring-opening polymerization, and promotes the synthesis of the fluorescent probe, preferably 6-methoxy-2-naphthaldehyde is used as the aldehyde compound, and the synthesized fluorescent probe has excellent characteristics.

In the step 2-1, the organic solvent is preferably alcohols, including methanol, ethanol and butanediol, more preferably ethanol, the dielectric constant of the ethanol is small, the influence on the reaction is small, the reaction is favorably carried out, the phase separation is easy after the reaction is finished, the formation of byproducts can be reduced, and the operation of the ethanol is safer.

In step 2-1, the acid is preferably a weak acid, more preferably hydrofluoric acid.

In step 2-1, optionally, the reaction is carried out under the protection of an inert gas, wherein the inert gas comprises any one or more of helium, argon and nitrogen, and nitrogen is preferred.

In the step 2-1, the molar ratio of the compound I obtained in the step 1 to the aldehyde compound is (0.1-3): 1, preferably (0.5-2): 1, more preferably 1: 1.

according to the invention, the molar ratio of the compound I obtained in the step 1 to the aldehyde compound is different, so that the prepared fluorescent probes have different purities, and when the molar ratio of the compound I obtained in the step 1 to the aldehyde compound is (0.1-3): 1, in particular 1: 1, the obtained fluorescent probe has the highest purity and the least side reaction.

In the step 2-1, the reaction time is 6 to 20 hours, preferably 10 to 14 hours, and more preferably 12 hours.

According to the invention, as the reaction time is prolonged, the selectivity and the yield of the fluorescent probe are gradually increased, the reaction time is too long, the selectivity and the yield of the fluorescent probe are basically unchanged, and even the selectivity and the yield of the product fluorescent probe are reduced.

In a further preferred embodiment, the reaction occurring in step 2-1 is represented by formula IV:

and 2-2, carrying out post-treatment on the reaction in the step 2-1.

In step 2-2, the post-treatment comprises filtration, extraction, drying, separation and purification.

According to the invention, filtration using ultrapure water is preferred.

According to the present invention, the extractant is preferably an ether compound, and more preferably the same extractant as used in step 1-2.

According to the invention, the drying agent is preferably a sulphate, more preferably the drying agent used in step 1-2 is the same.

According to the invention, the eluent is preferably any one or combination of alkane and ether compounds, and is more preferably a mixture of dichloromethane and petroleum ether.

In a preferred embodiment, the molar ratio of dichloromethane to petroleum ether is (1-7): (3-10), preferably (2-5): (5-8), more preferably 3: 7.

In a third aspect, the invention aims to provide an application of the fluorescent probe according to the first aspect or the fluorescent probe obtained by the method of the second aspect in visualizing and detecting the pH of the solution in real time.

According to a preferred embodiment, the fluorescent probe according to the first aspect or the fluorescent probe obtained according to the method of the second aspect is added to a buffer solution in which a polar solution and phosphate salts of different pH values are mixed.

The polar solvent comprises a solvent with higher polarity such as methanol, ethanol, dioxane and dimethyl sulfoxide, and a solvent with lower polarity such as chloroform, toluene and xylene, preferably the solvent with higher polarity, more preferably dimethyl sulfoxide, so that the fluorescent probe has higher resolution.

Wherein the volume ratio of the polar solution to the phosphate is 1: (5-12), preferably 1: (7-10), and more preferably 1:9, wherein the fluorescent probe has higher resolution and better selectivity.

The fluorescent probe provided by the invention can be used for detecting the change of the pH value of a solution, and can see obvious ratio color change under natural light or ultraviolet light within the pH range of 1.0-6.5, preferably 1.5-6.2, and more preferably 1.8-5.8.

In particular, the ratiometric fluorescent probes of the invention take full advantage of the nature of charge transfer within the organic fluorophore molecule. At different pH's, the fluorescent probes of the invention show two fluorescent emissions under UV light, which result from intramolecular charge transfer processes at 430nm and 575nm, respectively. Thus, the ratio of the fluorescence intensities of two different emission bands can be used to reflect the solution pH. When H is in solution⁺When the amount of the active ingredients is increased, the emission peak at 575nm is enhanced, and the solution is orange under the irradiation of an ultraviolet lamp. When OH is in solution^-When the amount of the active component is increased, the emission peak at 430nm is enhanced, and the solution presents blue under the irradiation of an ultraviolet lamp. By establishing a fitted curve relationship between the fluorescence intensity and the pH value of the solution, the dissociation constant (pKa) of the fluorescent probe can be rapidly detected to be 3.49 in real time.

In a fourth aspect, the invention is directed to a fluorescent probe according to the first aspect or the fluorescent probe obtained by the method of the second aspect for use in imaging cells.

According to a preferred embodiment, an appropriate amount of the fluorescent probe of the first aspect or the organic molecular ratio fluorescent probe obtained by the method of the second aspect is added to Hela (HeLa) cells at different pH values, and the color change of the cells is observed under a confocal fluorescence microscope.

Wherein the pH range is 1.0-6.5, preferably 1.5-6.2, and more preferably 1.8-5.8.

Specifically, the probe solution of the present invention was added to the cultured HeLa cells and incubated in buffer solutions of different pH. Then, the blue fluorescence of the HeLa cells gradually decreased and the orange fluorescence gradually increased with the change of the pH value from large to small as observed under a confocal fluorescence microscope. The process of observing the change of the cell fluorescence by the confocal fluorescence microscope can be used as a basis for qualitatively detecting the change of the pH value in the cell.

According to the ratio-type fluorescent probe, the real-time detection of the pH of the solution can be completed, and the biological imaging of the pH change in living cells can be completed.

Examples

Example 1

Synthesis of organic molecular ratio type fluorescent Probe (Ox-L)

(1) 1.8mmol of 2,3, 3-trimethylindole, 0.9mmol of 2-hydroxy-5-nitrobenzyl bromide and 5ml of acetonitrile are mixed and reacted for 12 hours at normal temperature under the nitrogen atmosphere. After the reaction is finished, putting the product in a refrigerator for 12 hours, and then respectively filtering the product by ultrapure water, extracting the product by diethyl ether and drying the product by anhydrous sodium sulfate; separating and purifying by column chromatography through ethyl acetate/dichloromethane eluent with the ratio of 10:1 to obtain a compound I;

(2) 0.5mmol of the compound I, 0.5mmol of 6-methoxy-2-naphthaldehyde and 10ml of ethanol are mixed, 0.3ml of hydrofluoric acid is added, and the mixture is reacted for 12 hours under a nitrogen atmosphere. After the reaction is finished, ultrapure water is used for filtration, ether extraction and anhydrous sodium sulfate drying respectively; and then the mixture is separated and purified by a column chromatography mode through dichloromethane/petroleum ether eluent with the ratio of 3:7 to obtain the chemofluorescence probe Ox-L.

Mass spectrometry and nuclear magnetic analysis were performed on the fluorescent probe Ox-L to obtain the following data:

mass spectrometry analysis: HR-MS: m/z (%): [ M + H ]]⁺478.1893,found478.1920。

Nuclear magnetic analysis: 1H NMR (400MHz, CDCl3) δ 7.91(m,2H),7.65-7.68(m,2H),7.53(d, J ═ 7.2Hz,1H),7.39(d, J ═ 7.3Hz,1H),7.01-6.91(m,5H), 6.55-6.51 (m,2H),6.25(s,2H),4.61(s,1H),3.73(s,3H),1.39(s,6H), 13C NMR (100MHz, DMSO-d6) δ ppm (164.46,158.64,139.79,139.76,134.44,131.95,131.16,128.54,128.24,126.85,126.84,126.76,126.16,122.27,121.93,121.10,120.23,116.77,114.78,112.88,111.84,107.16, 55.98,49.04,47.87, 18.72.

Examples of the experiments

Experimental example 1 measurement of solution pH

Detection of solution pH using organic molecular ratio-type fluorescent probe prepared in example 1: adding 3ul of probe solution into DMSO-phosphate buffer solutions with different pH respectively, andrespectively carrying out fluorescence detection, and setting the pH range to be 1.8-5.8. With the gradual increase of the pH value of the solution, the fluorescence intensity of the emission peak at 575nm is gradually reduced, the fluorescence intensity of the emission peak at 430nm is gradually increased, and the ratio value F₅₇₅/F₄₃₀Gradually decreases until it is almost constant. Accordingly, a fitting curve relation between the fluorescence intensity ratio and the pH value of the solution is established, and the real-time detection of the pH value of the solution is realized.

Experimental example 2 measurement of solution pH

Experimental example 2 is identical to the procedure of experimental example 1 except that the pH range is set to 1.8 to 7, and the fluorescence spectra and the visible photographs of the fluorescent probe prepared in example 1 in the buffers with different pH are shown in fig. 1, and it can be seen that the optimal excitation wavelength of the fluorescent probe is 360 to 380nm at pH 2.0 and pH 6.0, the emission wavelength is 520 to 700nm at pH 2.0, and 400 to 520nm at pH 6.0.

The fluorescence intensity ratio values obtained by adding the fluorescent probe prepared in example 1 to buffers of different pH (F) were obtained from FIG. 1₅₇₅/F₄₃₀) And pH, as shown in FIG. 2, it was calculated that the dissociation constant (pKa) of the fluorescent probe was 3.49.

⁺Experimental example 3 imaging at different H concentrations

The organic molecular ratio type fluorescent probe prepared in example 1 was added with H at various concentrations⁺In solution of (2), H⁺The concentrations of the solutions were 1.5. mu. mol/L, 3. mu. mol/L, 4.5. mu. mol/L, 6. mu. mol/L, 7.5. mu. mol/L, 9. mu. mol/L, 10.5. mu. mol/L, 12. mu. mol/L, respectively, and the obtained fluorescence spectra are shown in FIG. 3, and it can be seen that as H is added⁺The fluorescence emission peak at 575nm changes from weak to strong when the concentration is increased.

^-Experimental example 4 imaging at different OH concentrations

The organic molecular ratio type fluorescent probe prepared in example 1 was added to OH of various concentrations^-In solution of (2), OH^-The concentrations of the solutions were 1.5. mu. mol/L, 3. mu. mol/L, 4.5. mu. mol/L, 6. mu. mol/L, 7.5. mu. mol/L, 9. mu. mol/L, 10.5. mu. mol/L, 12. mu. mol/L, respectively,the obtained fluorescence spectrum is shown in FIG. 4, and it can be seen that the fluorescence spectrum varies with OH^-The concentration is increased, and the fluorescence emission peak at 430nm is changed from weak to strong.

Experimental example 5 cellular imaging at different pH

Biological imaging of HeLa cells at different pH using the organic molecular ratio-type fluorescent probe prepared in example 1; 10 μ M of a fluorescent probe was added to the cultured HeLa cells, and cultured for 2 hours. After completion of the culture, the cell culture medium was changed to buffer solutions of pH 3.0, 4.0 and 5.0, respectively, and 10 μ M of nigericin was added thereto, and the mixture was cultured at 37 ℃ for 15 min. And then observing cell images under a confocal fluorescence microscope, wherein the excitation wavelength of the fluorescence microscope is set to be 405nm, so that the fluorescent probe can be used for realizing cell imaging application under different pH values.

Fig. 5 shows photographs of cells in HeLa cells under different pH environments using the organic molecular ratio type fluorescent probe prepared in example 1, and it can be seen that the orange color exhibited by HeLa cells gradually becomes dark and the blue color gradually becomes strong, exhibiting a ratio change, in the course of pH from 3.0 to 5.0.

The invention has been described in detail with reference to the preferred embodiments and illustrative examples. It should be noted, however, that these specific embodiments are only illustrative of the present invention and do not limit the scope of the present invention in any way. Various modifications, equivalent substitutions and alterations can be made to the technical content and embodiments of the present invention without departing from the spirit and scope of the present invention, and these are within the scope of the present invention. The scope of the invention is defined by the appended claims.