CN111039897A - Fluorescent probes and their use in detecting autophagolysosomes - Google Patents

Abstract

The invention discloses a fluorescent probe and a new application thereof in detection of live cell autophagosomes. The fluorescent probe contains a structure shown in the following formula or a compound of a stereoisomer thereof and counter ions, can form a supermolecule aggregate form through self-assembly in a solution system containing metal salt ions or a physiological environment in cells, has two excitation wavelengths of 530-570 nm and 600-650 nm, can realize real-time observation on the autophagy process of a living cell sample, has the advantages of simple specific dyeing, low cytotoxicity and small damage to the biological sample, and is not influenced by the pH value in the cells.

Description

Fluorescent probes and their use in detecting autophagolysosomes

technical field

The present invention relates to the field of analytical chemistry, in particular, to a fluorescent probe and its use in detecting autophagolysosomes, more particularly, to a fluorescent probe and its use in detecting autophagylysosomes and determining cells method for the presence or absence of autophagy lysosomes.

Background technique

Lysosome is a very important organelle with a diameter of about 200-500 nm, which exists in almost all eukaryotic cells. There are many enzymes and proteins in lysosomes, including acid hydrolase, trypsin and cathepsin, which are digestive organs in cells, which can decompose macromolecular substances such as various proteins, and have the function of dissolving or digesting. Autophagolysosomes, also known as lysosomes, are formed by the fusion of primary lysosomes with endogenous vesicles from autophagy, namely autophagosomes, or lysosomes engulfing the cytoplasm. Acts as a "scavenger" as a normal pathway for the natural attrition and renewal of intracellular organelles and other structures. Autophagic lysosomes in normal cells play an important role in digesting, breaking down, and naturally replacing some intracellular structures. When cells are subjected to drug action, radiation exposure and mechanical damage, their number increases significantly. Autophagolysosomes are also frequently seen in diseased cells. Therefore, detection of autophagolysosomes can provide useful information for the diagnosis and treatment tracking of many diseases.

Fluorescent probes have the advantages of simple operation, high sensitivity, and low cytotoxicity, and have attracted extensive attention and research in the field of biological detection. At present, there are several fluorescent probes for labeling lysosomes, such as LysoTracker Red, LysoTracker Green, etc., on the market, but there is still a lack of commercial probes for labeling autophagolysosomes.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to provide a fluorescent probe, which forms supramolecular aggregates in a solution system containing metal salt ions or in an intracellular physiological environment, and has two types of 530-570 nm and 600-650 nm. The excitation wavelength can identify autophagolysosomes, thereby effectively distinguishing whether autophagy occurs with less damage to cells. In addition, the fluorescent probe has good biocompatibility, low cytotoxicity, and good anti-photobleaching performance, which can realize effective observation of cell samples for a long time and is not affected by intracellular pH value.

According to one aspect of the present invention, the present invention provides a fluorescent probe. According to an embodiment of the present invention, the fluorescent probe contains a compound of the structure shown in the following formula or a stereoisomer thereof and a counter ion,

in,

R ₁ is hydrogen, C _1-6 alkyl, aryl or C _1-4 alkyl substituted phenyl;

R ₂ -R ₉ and R ₁₂ -R ₁₅ are independently hydrogen, fluorine, chlorine, bromine, iodine, C _1-6 alkyl, C _1-6 haloalkyl or C _1-6 alkoxy;

R ₁₀ and R ₁₁ are independently a sulfonic acid group or a sulfonic acid group-substituted C _1-6 alkyl group;

X ₁ and X ₂ are independently carbon, oxygen, sulfur, selenium or tellurium.

The fluorescent probes according to the embodiments of the present invention can enter the lysosomes of cells, and do not emit fluorescent signals themselves. When cells undergo autophagy, phagocytosed substances (such as self-cytoplasmic proteins or organelles) are encapsulated into vesicles and fused with lysosomes to form autophagolysosomes. Certain substances in the autophagolysosome will react with the fluorescent probe to generate a detectable fluorescent signal. By detecting the fluorescent signal of the cell, it can effectively determine whether the cell has autophagy. Moreover, the fluorescent probe has dual excitation wavelengths, 530-570 nm and 600-650 nm, which can simultaneously detect whether a fluorescence detection signal occurs at these two excitation wavelengths, and the detection accuracy is higher than that of a single excitation wavelength. Moreover, the excitation wavelength is longer, which can better avoid cell damage compared with the short excitation wavelength. In addition, the fluorescent probe has good membrane permeability, and does not require cell fixation, permeabilization, etc., and can specifically label intracellular autophagy-lysosomes under the condition of maintaining cell activity; at the same time, it has biocompatibility. It has the advantages of good quality, low cytotoxicity, and good anti-photobleaching performance, which can realize effective observation of cell samples for a long time, and is not affected by intracellular pH value. In addition, the probe has simple components, simple and fast detection operation, and is expected to become a universal dye for detecting autophagy-lysosomes in living cells.

In addition, the fluorescent probe according to the above embodiments of the present invention may also have the following additional technical features:

According to an embodiment of the present invention, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl .

According to an embodiment of the present invention, the alkyl-substituted phenyl group is methylphenyl group or dimethylphenyl group.

According to an embodiment of the present invention, the haloalkyl group is monofluoromethane, difluoromethane, trifluoromethane, monobromomethane, dibromomethane or tribromomethane.

According to an embodiment of the present invention, the alkoxy group is a methoxy group, an ethoxy group or a propoxy group.

According to an embodiment of the present invention, R ₁ is hydrogen, C _1-3 alkyl or aryl; R ₂ -R ₉ and R ₁₂ -R ₁₅ are each independently hydrogen, fluorine, chlorine, bromine, iodine, C _{1- 3} alkyl, C _1-3 haloalkyl or C _1-3 alkoxy; R ₁₀ and R ₁₁ are each independently sulfonic acid, sulfomethyl, sulfoethyl or sulfopropyl.

According to an embodiment of the present invention, the counter ion is selected from N ⁺ H(C ₂ H ₅ ) ₃ , fluoride ion, chloride ion, bromide ion or iodide ion.

According to an embodiment of the present invention, containing

According to another aspect of the present invention, the present invention proposes the use of the aforementioned fluorescent probe in detecting autophagolysosomes. As mentioned above, the fluorescent probe according to the embodiment of the present invention can enter the lysosome of the cell, and does not emit a fluorescent signal by itself. When cells undergo autophagy, phagocytosed substances (such as self-cytoplasmic proteins or organelles) are encapsulated into vesicles and fused with lysosomes to form autophagolysosomes. Certain substances in the autophagolysosome will react with the fluorescent probe to generate a detectable fluorescent signal, and the presence of autophagolysosome in the cell can be effectively determined by detecting the fluorescent signal of the cell. Specifically, with the increase in the number of autophagolysosomes, the fluorescence signal at 570-620 nm was enhanced, and the fluorescence signal at 650-700 nm was weakened. In addition, it should be noted that this fluorescent probe has all the technical features and advantages of the aforementioned fluorescent probes, which will not be repeated here.

According to another aspect of the present invention, the present invention proposes the use of the aforementioned fluorescent probe in determining whether autophagy occurs. As mentioned above, the fluorescent probe according to the embodiment of the present invention can enter the lysosome of the cell, and does not emit a fluorescent signal by itself. When cells undergo autophagy, phagocytosed substances (such as self-cytoplasmic proteins or organelles) are encapsulated into vesicles and fused with lysosomes to form autophagolysosomes. Certain substances in the autophagolysosome will react with the fluorescent probe to generate a detectable fluorescent signal, and the detection of the fluorescent signal in the cell can effectively determine whether autophagy occurs. Specifically, in addition, it should be noted that this fluorescent probe has all the technical features and advantages of the aforementioned fluorescent probes, which will not be repeated here.

According to yet another aspect of the present invention, the present invention provides a method for determining whether autophagolysosome exists in a cell. According to an embodiment of the present invention, the method includes: contacting the aforementioned fluorescent probes with cells; and performing fluorescence signal detection on the contacted cells; wherein, the fluorescent signals appearing in the contacted cells indicate that the cells are present in the cells. Indication of autophagy to lysosomes. As mentioned above, the fluorescent probe according to the embodiment of the present invention can enter the lysosome of the cell, and does not emit a fluorescent signal by itself. When cells undergo autophagy, phagocytosed substances (such as self-cytoplasmic proteins or organelles) are encapsulated into vesicles and fused with lysosomes to form autophagolysosomes. Certain substances in the autophagolysosome will react with the fluorescent probe to generate a detectable fluorescent signal. By detecting the fluorescent signal of the cell, it can be effectively judged whether there is an autophagolysosome in the cell. In addition, it should be noted that this fluorescent probe has all the technical features and advantages of the aforementioned fluorescent probes, which will not be repeated here.

According to an embodiment of the present invention, the fluorescent probe is provided in the form of a solution, and the solvent in the solution is selected from physiological saline, potassium salt solution, tris-hydrochloric acid buffer solution, and phosphate buffer at least one of a solution, a methanol solution, an ethanol solution, an acetonitrile solution, a dimethyl sulfoxide solution, and a dimethyl amide solution. Among them, it should be noted that the "methanol solution" may be pure methanol, or may be a solution obtained by mixing methanol and water in any ratio. Similarly, "ethanol solution", "acetonitrile solution", "dimethyl sulfoxide solution" and "dimethyl amide solution" are also the same, and will not be repeated here.

According to the embodiment of the present invention, the pH values of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution and the phosphate buffer solution are both 6.2-8.2, and the concentrations are both 0.1-50 mmol/L. Therefore, the pH value of the buffer solution is close to the pH value in the cells, and the biocompatibility with the cells is good.

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows a cytotoxicity analysis chart according to an embodiment of the present invention;

Figure 2 shows an absorption spectrogram according to an embodiment of the present invention;

3-7 show schematic diagrams of fluorescence imaging according to an embodiment of the present invention;

Figure 8 shows a mass spectrum according to one embodiment of the present invention;

FIG. 9 shows a ¹ H-NMR spectrum according to another embodiment of the present invention.

Detailed ways

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, only used to explain the present invention, and should not be construed as a limitation of the present invention.

It should be noted that the fluorescent probe can also be used as a composition of laser dyes, nonlinear optical materials, and biosensors.

Unless otherwise clearly stated, the description methods "each independently", "...independently" and "...independently" used throughout this document are interchangeable, and should be understood in a broad sense. It can mean that in different groups, the specific options expressed between the same symbols do not affect each other, or it can mean that in the same group, the specific options expressed between the same symbols do not affect each other.

Definitions and conventions of stereochemistry in the present invention are generally used by reference to the following references: S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S. , "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including, but not limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention . Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D, L or R, S are used to denote the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to designate the sign of the plane-polarized light rotation of the compound, (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory. The chemical structures of these stereoisomers are the same, but their steric structures are not the same. A particular stereoisomer can be an enantiomer, a mixture of isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, devoid of optical activity.

It should be noted here that, for the compound of formula (I), its preparation method can refer to Hamer, F.M. The Chemistry of Heterocyclic Compounds. The Cyanine Dyes and Related Compounds. Interscience Publishers, New York-London, 1964 and Ficken, G.E. The Chemistry of Synthetic The synthetic routes described in Dyes. Cyanine Dyes. Academic Press, 1971 can also be prepared using other methods well known in the art.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If the specific technique or condition is not indicated in the embodiment, according to the technique or condition described in the literature in this area (for example, with reference to J. Sambrook etc., "Molecular Cloning Experiment Guide" translated by Huang Peitang etc., 3rd edition, Science Press) or follow the product instructions. The reagents or instruments used without specifying the manufacturer are conventional products that can be obtained commercially, for example, can be purchased from Sigma.

The instrument used to observe cell fluorescence in the following examples is a laser confocal microscope (OLYMPUS FV1000-IX81 (Olympus, Japan)).

Example 1

1. The synthesis methods of the compounds (1) to (5) are as follows, wherein the reaction ratio and the purification method can adopt the conventional ratio or conventional purification method in the field. In addition, the inventors confirmed that the structure of the above-mentioned compounds is correct by analyzing the hydrogen spectrum, carbon spectrum and/or mass spectrum data of each compound, wherein the mass spectrum and ¹ H-NMR spectrum of compound (2) refer to FIG. 8 and FIG. 9 , respectively. shown.

Example 2

The compounds of the formulae (1) to (5) have similar properties, and the properties of the compounds of the formulae (1) to (3) as the fluorescent probes (1) to (3) are described below as examples.

Using the fluorescent probe (1) of the embodiment of the present invention to carry out a cytotoxicity experiment, the details are as follows:

(1) Dissolve the fluorescent probe (1) with a small amount of methanol;

(2) The cultured HeLa and MCF-7 cells were added with different concentrations of probe (1) solution, and the culture was continued for 24 hours;

(3) Add 10% MTT solution after drying the medium, and continue to cultivate for 4h;

(4) Add DMSO to dissolve the medium after blotting dry, and measure the absorbance at 559 nm with a microplate reader. Take the absorbance value at 559 nm as the ordinate and the concentration value of the probe (1) as the abscissa to draw a graph. The results are shown in FIG. 1 , the absorbance value at 559 nm has no significant difference at different concentrations of probe (1), indicating that probe (1) has no inhibitory effect on cell growth.

Example 3

Utilize the fluorescent probe (1) of the embodiment of the present invention to assemble in an aqueous solution containing potassium ions to form supramolecular J-aggregates, as follows:

(1) Dissolve the fluorescent probe (1) with a small amount of methanol;

(2) diluting the fluorescent probe solution in step (1) with water containing different concentrations of potassium ions, and configuring respectively into an aqueous solution containing the fluorescent probe (1) at a concentration of 4 μM;

(3) Using an ultraviolet absorption spectrometer to detect the absorption spectrum of the aqueous solution of the fluorescent probe (1) in step (2), the results are shown in Figure 2, with the increase of potassium ion concentration, a new absorption peak is generated at 650 nm, indicating that the probe Needle (1) forms supramolecular J-aggregates.

Example 4

Fluorescent imaging of cells is performed by using the fluorescent probe (1) and the LysoTracker probe in the embodiment of the present invention, as follows:

(1) Dissolve the fluorescent probe (1) and the LysoTracker probe with a small amount of dimethyl sulfoxide respectively;

(2) adding the two fluorescent probe solutions of step (1) into the culture medium, respectively, and configuring them into a culture solution containing the fluorescent probe (1) at a concentration of 4 μM and a culture solution of LysoTracker at a concentration of 1.0 μM;

(3) Pipette 1 mL of the culture solution prepared in step (2) with a pipette, respectively, add it to a culture dish culturing HEK293 cells, and place it in a 37° C., 5% CO ₂ incubator for 30 min;

(4) The cultured cells were washed three times with PBS, and then 1 mL of blank mixed medium was added for confocal fluorescence imaging. The excitation wavelength was 405 nm, and the collection wavelength was 420-500 nm; the excitation wavelength was 559 nm, and the collection wavelength was 570-620 nm. And the excitation wavelength is 633nm, and the collection band is 650-700nm. The results are shown in Figure 3, where a is the schematic diagram of the fluorescence confocal imaging of LysoTracker, Figure b is the schematic diagram of the fluorescence confocal imaging of the fluorescent probe (1) at 570-620 nm, and Figure c is the fluorescent probe (1) at 650- Schematic diagram of fluorescence confocal imaging at 700 nm. The fluorescence signal in Figure a is highly coincident with the fluorescence signals in Figures b and c, indicating that the fluorescent probe (1) has good targeting to intracellular lysosomes.

Example 5

The fluorescence imaging of the process of cell starvation-induced lysosomal autophagy is respectively monitored by the fluorescent probe (1) of the embodiment of the present invention, and the details are as follows:

(1) dissolve the fluorescent probe (1) with a small amount of dimethyl sulfoxide;

(2) adding the fluorescent probe solution of step (1) into a blank medium without embryonic bovine serum, and configuring it into a blank culture medium containing the fluorescent probe (1) at a concentration of 4 μM;

(3) Pipette 1 mL of the blank culture solution prepared in step (2) with a pipette, and add them to the culture dishes of cervical cancer HeLa cells that have been starved for 0, 0.5 and 1 hour respectively, and put them in Incubate in a 37°C, 5% _CO2 incubator for 20min;

(4) The cultured cells were washed three times with PBS, and then 1 mL of blank mixed medium was added for confocal fluorescence imaging. The excitation wavelength was 559 nm, the collection wavelength was 570-620 nm, the excitation wavelength was 633 nm, and the collection wavelength was 650-700 nm. The results are shown in Figure 4, where a is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0 hours, b is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0.5 hours, and c is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0.5 hours. Schematic diagram of confocal fluorescence imaging of cervical cancer HeLa starved for 1 hour. With the increase of starvation time, the fluorescence signal of fluorescent probe (1) at 570-620 nm in the cell gradually increased, and the fluorescence signal at 650-700 nm gradually weakened. It was shown that with the increase of starvation time, the intracellular autophagy-lysosomes increased, and the experimental results showed that the fluorescent probe (1) could detect the changes of autophagy-lysosomes in living cells induced by starvation.

Example 6

The fluorescence imaging of the rapamycin-induced lysosomal autophagy process is monitored by using the fluorescent probe (1) of the embodiment of the present invention, and the details are as follows:

(1) dissolve the fluorescent probe (1) with a small amount of dimethyl sulfoxide;

(2) adding the fluorescent probe solution of step (1) into a blank medium without embryonic bovine serum, and configuring it into a blank culture medium containing the fluorescent probe (1) at a concentration of 4 μM;

(3) Pipette 1 mL of the culture solution prepared in step (2) with a pipette, add it to the culture dish of human breast cancer cell MCF-7 treated with 0, 0.5 and 1 μM rapamycin, respectively, and add it Incubate in a 37°C, 5% _CO2 incubator for 10min;

(4) The cultured cells were washed three times with PBS respectively, and then 1 mL of blank mixed medium was added for confocal fluorescence imaging. The results are shown in Figure 5, where a is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0 μM rapamycin, and b is a human breast cancer cell MCF-7 treated with 0.5 μM rapamycin 7 is a schematic diagram of fluorescence confocal imaging, c is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 1 μM rapamycin. With the increase of the concentration of rapamycin, the fluorescence signal of the fluorescent probe (1) at 570-620 nm in the cells gradually increased, and the fluorescence signal at 650-700 nm gradually weakened. Thus, it is indicated that fluorescent probe 1 can monitor rapamycin-induced intracellular autophagy lysosomes.

Example 7

The fluorescence imaging of the rapamycin-induced lysosomal autophagy process is monitored by using the fluorescent probe (2) of the embodiment of the present invention, and the details are as follows:

(1) dissolve the fluorescent probe (2) with a small amount of dimethyl sulfoxide;

(2) adding the fluorescent probe solution of step (1) into a blank culture medium without embryonic bovine serum, and respectively configuring it into a blank culture solution containing the fluorescent probe (2) at a concentration of 4 μM;

(3) Pipette 1 mL of the culture solution prepared in step (2) with a pipette, add it to the culture dish of human breast cancer cell MCF-7 treated with 0, 0.5 and 1 μM rapamycin, respectively, and add it Incubate in a 37°C, 5% _CO2 incubator for 10min;

(4) The cultured cells were washed three times with PBS respectively, and then 1 mL of blank mixed medium was added for confocal fluorescence imaging. The results are shown in Figure 6, where a is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0 μM rapamycin, and b is a human breast cancer cell MCF-7 treated with 0.5 μM rapamycin 7 is a schematic diagram of fluorescence confocal imaging, c is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 1 μM rapamycin. With the increase of the concentration of rapamycin, the fluorescence signal of the fluorescent probe (2) at 570-620 nm in cells gradually increased, and the fluorescence signal at 650-700 nm gradually weakened. Thus, it is indicated that the fluorescent probe (2) can monitor rapamycin-induced intracellular autophagy lysosomes.

Example 8

The fluorescence imaging of the cell starvation-induced lysosomal autophagy process is respectively monitored by using the fluorescent probe (3) in the embodiment of the present invention, and the details are as follows:

(1) dissolve the fluorescent probe (3) with a small amount of dimethyl sulfoxide;

(2) adding the fluorescent probe solution of step (1) into a blank medium without embryonic bovine serum, and configuring it into a blank culture medium containing the fluorescent probe (3) at a concentration of 3 μM;

(3) Pipette 1 mL of the blank culture solution prepared in step (2) with a pipette, and add them to the culture dishes of cervical cancer HeLa cells that have been starved for 0, 0.5 and 1 hour respectively, and put them in Incubate in a 37°C, 5% _CO2 incubator for 20min;

(4) The cultured cells were washed three times with PBS respectively, and then 1 mL of blank mixed medium was added to perform fluorescence confocal imaging. The results are shown in Figure 7, where a is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0 hours, b is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0.5 hours, and c is a schematic diagram of confocal fluorescence imaging of cervical cancer HeLa that has been starved for 0.5 hours Schematic diagram of confocal fluorescence imaging of cervical cancer HeLa starved for 1 hour. With the increase of starvation time, the fluorescence signal of fluorescent probe (1) at 570-620 nm in the cell gradually increased, and the fluorescence signal at 650-700 nm gradually weakened. It was shown that with the increase of starvation time, the intracellular autophagy-lysosomes increased. The experimental results showed that the fluorescent probe (3) could detect the changes of autophagy-lysosomes in living cells induced by starvation.

Summarize

From the comprehensive examples, it can be concluded that the fluorescent probe membrane of the embodiment of the present invention has good permeability, does not require fixation, permeabilization, etc. of cells, and can specifically target autophagy-lysosomes in cells under the condition of maintaining cell activity. At the same time, the probe has the advantages of good photostability and low cytotoxicity, and can effectively observe cell samples for a long time. In addition, the probe has simple components, simple and fast detection operation, and is expected to become a universal dye for detecting autophagy-lysosomes in living cells.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, The scope of the invention is defined by the claims and their equivalents.

Claims (9)

1. A fluorescent probe, characterized in that, a compound and a counter ion containing a structure shown in the following formula or its stereoisomer,

in, R ₁ is hydrogen, C _1-6 alkyl, aryl or C _1-4 alkyl substituted phenyl; R ₂ -R ₉ and R ₁₂ -R ₁₅ are independently hydrogen, fluorine, chlorine, bromine, iodine, C _1-6 alkyl, C _1-6 haloalkyl or C _1-6 alkoxy; R ₁₀ and R ₁₁ are independently a sulfonic acid group or a sulfonic acid group-substituted C _1-6 alkyl group; X ₁ and X ₂ are independently carbon, oxygen, sulfur, selenium or tellurium. 2. The fluorescent probe according to claim 1, wherein the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl , isopentyl, n-hexyl or isohexyl; Optionally, the alkyl-substituted phenyl is methylphenyl or dimethylphenyl; Optionally, the haloalkyl is monofluoromethane, difluoromethane, trifluoromethane, monobromomethane, dibromomethane, or tribromomethane; Optionally, the alkoxy group is methoxy, ethoxy or propoxy. 3. The fluorescent probe according to claim 1, wherein R ₁ is hydrogen, C _1-3 alkyl or aryl; R ₂ -R ₉ and R ₁₂ -R ₁₅ are each independently hydrogen, fluorine, chlorine, bromine, iodine, C _1-3 alkyl, C _1-3 haloalkyl or C _1-3 alkoxy; R ₁₀ and R ₁₁ are each independently sulfonic acid, sulfomethyl, sulfoethyl, or sulfopropyl. 4 . The fluorescent probe according to claim 1 , wherein the counter ion is selected from N ⁺ H(C ₂ H ₅ ) ₃ , fluoride ion, chloride ion, bromide ion or iodide ion. 5 . 5. The fluorescent probe according to claim 1, characterized in that, containing 6. Use of the fluorescent probe according to any one of claims 1 to 5 in detecting autophagolysosomes. 7. Use of the fluorescent probe according to any one of claims 1 to 5 in determining whether autophagy occurs. 8. A method for determining whether there is an autophagolysosome in a cell, comprising: contacting the fluorescent probe of any one of claims 1 to 5 with a cell; and Detecting the fluorescent signal of the contacted cells; The presence of a fluorescent signal in the contacted cells is an indication of the presence of autophagolysosomes in the cells. 9. The method according to claim 8, wherein the fluorescent probe is provided in the form of a solution, and the solvent in the solution is selected from physiological saline, potassium salt solution, tris-hydroxymethylaminomethane- At least one of hydrochloric acid buffer solution, phosphate buffer solution, methanol solution, ethanol solution, acetonitrile solution, dimethyl sulfoxide solution and dimethyl amide solution.

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