CN111039897B - Fluorescent probes and their use in detection of autophagy lysosomes - Google Patents
Fluorescent probes and their use in detection of autophagy lysosomes Download PDFInfo
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Abstract
The invention discloses a fluorescent probe and a new application thereof in detecting living cell autophagy lysosomes. The fluorescent probe contains a compound with a structure shown in the following formula or a stereoisomer thereof and a counter ion, can form a supermolecule aggregate form in a solution system containing metal salt ions or in a physiological environment in cells through self-assembly, has two excitation wavelengths of 530-570 nm and 600-650 nm, can realize real-time observation of the autophagy process of living cell samples, has the advantages of simple specific staining, low cytotoxicity and small damage to biological samples, and is not influenced by the pH value in the cells.
Description
Technical Field
The present invention relates to the field of analytical chemistry, in particular to fluorescent probes and their use in the detection of autophagosomes, more particularly to fluorescent probes and their use in the detection of autophagosomes and a method of determining the presence or absence of autophagosomes within a cell.
Background
Lysosomes are a very important organelle, with diameters of about 200-500 nm, found in almost all eukaryotic cells. The lysosome contains a plurality of enzymes and proteins, including acid hydrolase, membrane protease and cathepsin, which are digestive organs in cells, can decompose macromolecular substances such as various proteins, and has the function of dissolving or digesting. Autophagosomes, also known as lysosomes, are formed by fusion of a primary lysosome with an endogenous substance-containing vesicle from autophagy, i.e., an autophagosome, or lysosomes phagocytic cytoplasm, and function as "scavenger" within cells as a normal pathway for natural attenuation and renewal of intracellular organelles and other structures. Autophagic lysosomes in normal cells play an important role in digestion, breakdown, and natural replacement of some intracellular structures. When cells are subjected to drug action, irradiation and mechanical injury, their number increases significantly. Autophagosomes are also often seen in diseased cells. Thus, by detecting autophagosomes, useful information can be provided for diagnosis and treatment follow-up of many diseases.
The fluorescent probe has the advantages of simple operation, high sensitivity, low cytotoxicity and the like, and is widely focused and researched in the field of biological detection. Several lysosome-labeled fluorescent probes such as LysoTracker Red, lysoTracker Green, etc. are currently commercially available, but there is still a lack of commercial autophagy lysosome-labeled probes.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present invention is to provide a fluorescent probe which forms a supramolecular aggregate in a solution system containing metal salt ions or in an intracellular physiological environment, has two excitation wavelengths of 530 to 570nm and 600 to 650nm, and can recognize autophagosomes, thereby effectively distinguishing whether autophagy occurs and having less damage to cells. In addition, the fluorescent probe has good biocompatibility, low cytotoxicity and good photobleaching resistance, can realize long-time effective observation of a cell sample, and is not influenced by the pH value in cells.
According to one aspect of the present invention, there is provided a fluorescent probe. According to an embodiment of the present invention, the fluorescent probe contains a compound having a structure represented by the following formula or a stereoisomer thereof and a counter ion,
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 is hydrogen, C 1-6 Alkyl, aryl or C 1-4 An alkyl-substituted phenyl group;
R 2 -R 9 and R is 12 -R 15 Independently hydrogen, fluorine, chlorine, bromine, iodine, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 An alkoxy group;
R 10 and R is 11 Independently sulfonic or sulfonic-substitutedC 1-6 An alkyl group;
X 1 and X 2 Independently carbon, oxygen, sulfur, selenium or tellurium.
The fluorescent probe according to the embodiment of the invention can enter the lysosome of the cell and does not emit a fluorescent signal. When autophagy occurs in a cell, phagocytosed material (e.g., self cytoplasmic proteins or organelles) will become entrapped into the vesicle and fuse with the lysosome to form an autophagic lysosome. Certain substances in autophagy lysosomes react with the fluorescent probe to generate a detectable fluorescent signal, and whether the cells have autophagy can be effectively judged by detecting the fluorescent signal of the cells. In addition, the fluorescent probe has double excitation wavelengths of 530-570 nm and 600-650 nm, can detect whether fluorescent detection signals appear under the two excitation wavelengths at the same time, and has higher detection accuracy compared with single excitation wavelength. Moreover, the excitation wavelength is longer, and the damage to the cells can be better avoided than a short excitation wavelength. In addition, the fluorescent probe membrane has good permeability, does not need to fix and permeate cells and the like, and specifically marks the autophagy lysosomes in the cells under the condition of keeping the activity of the cells; meanwhile, the method has the advantages of good biocompatibility and low cytotoxicity, has good photobleaching resistance, can realize long-time effective observation of a cell sample, and is not influenced by the pH value in cells. In addition, the probe has simple components, simple and quick detection operation and is hopeful to become a universal dye for detecting the autophagy lysosome of living cells.
In addition, the fluorescent probe according to the above embodiment of the present invention may have the following additional technical features:
according to an embodiment of the 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 invention, the alkyl-substituted phenyl is methylphenyl or dimethylphenyl.
According to an embodiment of the invention, the haloalkyl is monofluoromethane, difluoromethane, trifluoromethane, monobromomethane, dibromomethane or tribromomethane.
According to an embodiment of the invention, the alkoxy group is methoxy, ethoxy or propoxy.
According to an embodiment of the invention, R 1 Is hydrogen, C 1-3 Alkyl or aryl; r is R 2 -R 9 And R is 12 -R 15 Each independently is hydrogen, fluorine, chlorine, bromine, iodine, C 1-3 Alkyl, C 1-3 Haloalkyl or C 1-3 An alkoxy group; r is R 10 And R is 11 Each independently is a sulfonic acid group, a sulfomethyl group, a sulfoethyl group, or a sulfopropyl group.
According to an embodiment of the invention, the counter ion is selected from N + H(C 2 H 5 ) 3 Fluoride, chloride, bromide or iodide.
According to an embodiment of the invention, comprises
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According to a further aspect of the invention, the use of a fluorescent probe as described above for the detection of autophagosomes is proposed. As previously described, fluorescent probes according to embodiments of the present invention may enter the lysosomes of cells, which do not themselves emit fluorescent signals. When autophagy occurs in a cell, phagocytosed material (e.g., self cytoplasmic proteins or organelles) will become entrapped into the vesicle and fuse with the lysosome to form an autophagic lysosome. Certain substances in the autophagy lysosome can react with the fluorescent probe to generate a detectable fluorescent signal, and the presence of the autophagy lysosome in the cell can be effectively judged by detecting the fluorescent signal of the cell. Specifically, as the number of autophagy lysosomes increases, the fluorescent signal at 570-620nm increases and the fluorescent signal at 650-700nm decreases. In addition, it should be noted that the fluorescent probe has all the technical features and advantages of the foregoing fluorescent probe, and will not be described in detail herein.
According to a further aspect of the invention, the use of a fluorescent probe as described above for determining whether autophagy has occurred is presented. As previously described, fluorescent probes according to embodiments of the present invention may enter the lysosomes of cells, which do not themselves emit fluorescent signals. When autophagy occurs in a cell, phagocytosed material (e.g., self cytoplasmic proteins or organelles) will become entrapped into the vesicle and fuse with the lysosome to form an autophagic lysosome. Certain substances in autophagy lysosomes react with the fluorescent probe to generate a detectable fluorescent signal, and the cell autophagy can be effectively judged by detecting the fluorescent signal of the cell. In particular, in addition, it should be noted that the fluorescent probe has all the technical features and advantages of the foregoing fluorescent probe, and will not be described in detail herein.
According to a further aspect of the invention, the invention provides a method of determining the presence or absence of autophagosomes in a cell. According to an embodiment of the invention, the method comprises: contacting the fluorescent probe with a cell; detecting fluorescent signals of the contacted cells; wherein the presence of a fluorescent signal in the cell after contact is indicative of the presence of autophagosomes within said cell. As previously described, fluorescent probes according to embodiments of the present invention may enter the lysosomes of cells, which do not themselves emit fluorescent signals. When autophagy occurs in a cell, phagocytosed material (e.g., self cytoplasmic proteins or organelles) will become entrapped into the vesicle and fuse with the lysosome to form an autophagic lysosome. Certain substances in the autophagy lysosome can react with the fluorescent probe to generate a detectable fluorescent signal, and the presence of the autophagy lysosome in the cell can be effectively judged by detecting the fluorescent signal of the cell. In addition, it should be noted that the fluorescent probe has all the technical features and advantages of the foregoing fluorescent probe, and will not be described in detail herein.
According to an embodiment of the present invention, the fluorescent probe is provided in the form of a solution, wherein the solvent is at least one selected from the group consisting of physiological saline, a potassium salt solution, a tris-hydrochloric acid buffer solution, a phosphate buffer solution, a methanol solution, an ethanol solution, an acetonitrile solution, a dimethyl sulfoxide solution, and a dicarboxamide solution. The "methanol solution" may be pure methanol, or a solution obtained by mixing methanol and water at an arbitrary ratio. Similarly, the same is true of "ethanol solution", "acetonitrile solution", "dimethyl sulfoxide solution" and "dicarboxamide solution", and will not be described in detail herein.
According to the embodiment of the invention, the pH value of the buffer solution of the tris-hydrochloric acid and the buffer solution of the phosphate are respectively 6.2-8.2, and the concentration is respectively 0.1-50mmol/L. Thus, the pH value of the buffer solution is close to the pH value in the cells, and the buffer solution has good biocompatibility with the cells.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a cytotoxicity assay according to one embodiment of the invention;
FIG. 2 shows an absorption spectrum according to an embodiment of the present invention;
FIGS. 3-7 show a schematic representation of fluorescence imaging according to one embodiment of the invention;
FIG. 8 shows a mass spectrum according to one embodiment of the invention;
FIG. 9 shows another embodiment of the invention 1 H-NMR spectrum.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
The fluorescent probe may be used as a component of a laser dye, a nonlinear optical material, a biosensor, or the like.
Unless explicitly indicated otherwise, the descriptions used throughout this document, "… each independently," "… each independently," and "… each independently" are interchangeable, and are to be construed broadly as meaning that particular items expressed between the same symbols in different groups do not affect each other, or that particular items expressed between the same symbols in the same groups do not affect each other.
The definition and use of stereochemistry in the present invention is generally referred to in the following documents: 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 invention may contain asymmetric or chiral centers and thus exist as different stereoisomers. All stereoisomeric forms of the compounds of the invention, including, but in no way limited to, diastereomers, enantiomers, atropisomers and mixtures thereof, such as racemic mixtures, form part of the invention. Many organic compounds exist in optically active form, i.e. they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefix D, L or R, S is used to denote the absolute configuration of the chiral center of the molecule. The prefix d, l or (+), (-) is used to name the sign of the compound plane polarization rotation, where (-) or l means that the compound is left-handed and the prefix (+) or d means that the compound is right-handed. The chemical structures of these stereoisomers are identical, but their stereoisomers are different. The particular stereoisomers may be enantiomers, mixtures of isomers commonly referred to as enantiomeric mixtures. The 50:50 enantiomeric mixture is known as a racemic mixture or racemate, which may result in the absence of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.
It is to be noted here that, for the compound of the formula (I), the preparation method can be referred to the synthetic route described in 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 dyes.cyanine dyes.academic Press,1971, and can also be prepared by other methods well known in the art.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are conventional products commercially available, for example, from Sigma company.
The instrument used for observing cell fluorescence in the following examples was a confocal laser microscope (OLYMPUS FV1000-IX81 (OLYMPUS, japan)).
Example 1
1. The synthesis methods of the compounds (1) to (5) are shown below, wherein the reaction ratio and the purification method are those conventional in the art or conventional purification methods. In addition, the inventors confirmed that the structure of the above-mentioned compound was correct by analyzing hydrogen spectrum, carbon spectrum and/or mass spectrum data of each compound, wherein the mass spectrum of the compound (2) and 1 the H-NMR spectra are shown with reference to FIGS. 8 and 9, respectively.
Example 2
The compounds of the formulae (1) to (5) have similar characteristics, and the characteristics of the compounds of the formulae (1) to (3) will be mainly described below as examples of the fluorescent probes (1) to (3), respectively.
Cytotoxicity experiments were performed using the fluorescent probe (1) of the embodiment of the present invention, specifically as follows:
(1) Dissolving the fluorescent probes (1) with a small amount of methanol, respectively;
(2) Adding probe (1) solutions with different concentrations into the cultured HeLa and MCF-7 cells, and continuously culturing for 24 hours;
(3) After the culture medium is sucked up, 10% MTT solution is added for continuous culture for 4 hours;
(4) After the medium was blotted, DMSO was added for dissolution, and absorbance at 559nm was measured using an enzyme-labeled instrument. Plotting is performed with the absorbance at 559nm as ordinate and the concentration of the probe (1) as abscissa. As shown in FIG. 1, there was no significant difference in absorbance at 559nm between the concentrations of probe (1), indicating that probe (1) had no inhibitory effect on cell growth.
Example 3
The fluorescent probe (1) of the embodiment of the invention is assembled in an aqueous solution containing potassium ions to form a supermolecule J-aggregate, and the method comprises the following steps:
(1) Dissolving the fluorescent probe (1) with a small amount of methanol;
(2) Diluting the fluorescent probe solution in the step (1) with water containing potassium ions with different concentrations, and preparing aqueous solutions containing the fluorescent probes (1) with the concentration of 4 mu M respectively;
(3) The absorption spectrum of the aqueous solution of the fluorescent probe (1) in the step (2) was detected by an ultraviolet absorption spectrometer, and as shown in FIG. 2, a new absorption peak was generated at 650nm with the increase of the potassium ion concentration, indicating that the probe (1) formed a supramolecular J-aggregate.
Example 4
The fluorescence probe (1) and the LysoTracker probe of the embodiment of the invention are respectively utilized for carrying out fluorescence imaging on cells, and the specific steps are as follows:
(1) Respectively dissolving the fluorescent probe (1) and the LysoTracker probe by using a small amount of dimethyl sulfoxide;
(2) Adding the two fluorescent probe solutions in the step (1) into a culture medium respectively to prepare a culture solution containing the fluorescent probe (1) with the concentration of 4 mu M and a culture solution containing the LysoTracker with the concentration of 1.0 mu M;
(3) 1mL of the culture solution prepared in the step (2) is respectively removed by a pipette, respectively added into a culture dish in which HEK293 cells are cultured, and placed in a temperature of 37 ℃ and 5% CO 2 Culturing in an incubator for 30min;
(4) Washing the cultured cells with PBS for three times, adding 1mL of blank mixed culture medium for fluorescence confocal imaging, wherein the excitation wavelength is 405nm, and the collection wave band is 420-500nm; the excitation wavelength is 559nm, the collection wave band is 570-620nm and the excitation wavelength is 633nm, and the collection wave band is 650-700nm. The results are shown in FIG. 3, wherein a is a fluorescent confocal imaging schematic diagram of the Lysotracker, b is a fluorescent confocal imaging schematic diagram of the fluorescent probe (1) at 570-620nm, and c is a fluorescent confocal imaging schematic diagram of the fluorescent probe (1) at 650-700nm. The fluorescence signal of FIG. a is highly coincident with the fluorescence signal of FIGS. b and c, indicating that the fluorescent probe (1) has good targeting to intracellular lysosomes.
Example 5
The fluorescent probe (1) of the embodiment of the invention is used for monitoring the fluorescent imaging of the cell starvation induced lysosome autophagy process, and the specific steps are as follows:
(1) Dissolving the fluorescent probe (1) with a small amount of dimethyl sulfoxide;
(2) Adding the fluorescent probe solution in the step (1) into a blank culture medium without embryo bovine serum, and preparing blank culture solutions containing the fluorescent probes (1) with the concentration of 4 mu M respectively;
(3) 1mL of the empty prepared in the step (2) is respectively removed by a pipetteWhite culture solution is added into culture dishes of cervical cancer HeLa cells starved for 0, 0.5 and 1 hr, and placed at 37deg.C and 5% CO 2 Culturing in an incubator for 20min;
(4) The cultured cells were washed three times with PBS, and then fluorescence confocal imaging was performed by adding 1mL of a blank mixed medium, the excitation wavelength was 559nm, the collection wavelength was 570-620nm, the collection wavelength was 633nm, and the collection wavelength was 650-700nm, as shown in FIG. 4, where a is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa after 0 hour starvation treatment, b is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa after 0.5 hour starvation treatment, c is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa after 1 hour starvation treatment, the fluorescence signal of fluorescent probe (1) in the cell was gradually increased, the fluorescence signal of 650-700nm was gradually decreased, indicating that the intracellular autophagy lysosome was increased with the increase of starvation time, and experimental results indicate that fluorescent probe (1) was able to detect the change of starvation-induced living cell autophagy lysosome.
Example 6
The fluorescent probe (1) of the embodiment of the invention is used for monitoring the fluorescent imaging of the rapamycin induced lysosome autophagy process, and the specific steps are as follows:
(1) Dissolving the fluorescent probe (1) with a small amount of dimethyl sulfoxide;
(2) Adding the fluorescent probe solution in the step (1) into a blank culture medium without embryo bovine serum, and preparing blank culture solutions containing the fluorescent probes (1) with the concentration of 4 mu M respectively;
(3) 1mL of the culture solution prepared in the step (2) was removed by a pipette, and added to a petri dish of human breast cancer cells MCF-7 treated with 0, 0.5 and 1. Mu.M rapamycin, respectively, and placed in a 5% CO solution at 37 ℃ 2 Culturing in an incubator for 10min;
(4) The cultured cells are respectively washed three times by PBS, and then 1mL of blank mixed culture medium is added for fluorescence confocal imaging, the excitation wavelength is 559nm, the collection wave band is 570-620nm and the excitation wavelength is 633nm, and the collection wave band is 650-700nm. The results are shown in FIG. 5, wherein a is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 0. Mu.M rapamycin, b is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 0.5. Mu.M rapamycin, and c is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 1. Mu.M rapamycin. As the rapamycin concentration increases, the fluorescence signal of the fluorescent probe (1) in the cell at 570-620nm is gradually increased, and the fluorescence signal at 650-700nm is gradually reduced. Thus, it was shown that fluorescent probe 1 was able to monitor rapamycin-induced intracellular autophagy lysosomes.
Example 7
The fluorescent probe (2) of the embodiment of the invention is used for monitoring the fluorescent imaging of the rapamycin induced lysosome autophagy process, and the specific steps are as follows:
(1) Dissolving the fluorescent probe (2) with a small amount of dimethyl sulfoxide;
(2) Adding the fluorescent probe solution in the step (1) into a blank culture medium without embryo bovine serum, and preparing blank culture solutions containing fluorescent probes (2) with the concentration of 4 mu M respectively;
(3) 1mL of the culture solution prepared in the step (2) was removed by a pipette, and added to a petri dish of human breast cancer cells MCF-7 treated with 0, 0.5 and 1. Mu.M rapamycin, respectively, and placed in a 5% CO solution at 37 ℃ 2 Culturing in an incubator for 10min;
(4) The cultured cells are respectively washed three times by PBS, and then 1mL of blank mixed culture medium is added for fluorescence confocal imaging, the excitation wavelength is 559nm, the collection wave band is 570-620nm and the excitation wavelength is 633nm, and the collection wave band is 650-700nm. The results are shown in FIG. 6, wherein a is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 0. Mu.M rapamycin, b is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 0.5. Mu.M rapamycin, and c is a schematic representation of fluorescence confocal imaging of human breast cancer cells MCF-7 treated with 1. Mu.M rapamycin. As the rapamycin concentration increases, the fluorescence signal of the fluorescent probe (2) in the cell at 570-620nm is gradually increased, and the fluorescence signal at 650-700nm is gradually reduced. Thus, it was shown that fluorescent probe (2) was able to monitor rapamycin induced intracellular autophagosomes.
Example 8
The fluorescent probe (3) of the embodiment of the invention is used for monitoring the fluorescent imaging of the cell starvation induced lysosome autophagy process, and the specific steps are as follows:
(1) Dissolving the fluorescent probe (3) with a small amount of dimethyl sulfoxide;
(2) Adding the fluorescent probe solution in the step (1) into a blank culture medium without embryo bovine serum, and preparing blank culture solutions containing fluorescent probes (3) with the concentration of 3 mu M respectively;
(3) 1mL of the blank culture solution prepared in the step (2) is respectively removed by a pipette, and the blank culture solution is respectively added into culture dishes of cervical cancer HeLa cells subjected to starvation treatment for 0, 0.5 and 1 hour, and the culture dishes are placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 20min;
(4) The cultured cells were washed three times with PBS, and then subjected to fluorescence confocal imaging by adding 1mL of a blank mixed medium, the excitation wavelength was 559nm, the collection wavelength was 570-620nm and the excitation wavelength was 633nm, the collection wavelength was 650-700nm, and the results are shown in FIG. 7, wherein a is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa subjected to 0-hour starvation treatment, b is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa subjected to 0.5-hour starvation treatment, c is a fluorescence confocal imaging schematic diagram of cervical cancer HeLa subjected to 1-hour starvation treatment, the fluorescence signal of the fluorescent probe (1) in the cell was gradually increased, the fluorescence signal of the fluorescent probe (1) was 650-700nm was gradually decreased, and the results of experiments indicate that the fluorescent probe (3) was capable of detecting the change of starvation-induced autophagosome in the cell.
Summary
The comprehensive embodiment shows that the fluorescent probe membrane of the embodiment of the invention has good permeability, does not need to fix and permeate cells and the like, and carries out specific labeling on the autophagy lysosomes in the cells under the condition of keeping the activity of the cells; meanwhile, the probe has the advantages of good light stability and low cytotoxicity, and can realize long-time effective observation of a cell sample. In addition, the probe has simple components, simple and quick detection operation and is hopeful to become a universal dye for detecting the autophagy lysosome of living cells.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example 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 embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (4)
1. Use of a fluorescent probe for the detection of non-disease diagnostic purposes in autophagy lysosomes, said fluorescent probe having the structure shown below:
2. use of fluorescent probes for non-disease diagnostic purposes in determining whether autophagy has occurred, saidThe structure of the fluorescent probe is as follows:
3. a method for determining the presence or absence of autophagy lysosomes within cells for non-disease diagnostic purposes, comprising:
contacting a fluorescent probe with the cell; and
detecting fluorescent signals of the contacted cells;
wherein, the fluorescence signal appears in the cell after the contact, which is the indication of the autophagosome in the cell, and the structure of the fluorescence probe is as follows:
4. the method according to claim 3, wherein the fluorescent probe is provided in the form of a solution, and the solvent in the solution is at least one selected from the group consisting of physiological saline, a potassium salt solution, a tris-hydrochloric acid buffer solution, a phosphate buffer solution, a methanol solution, an ethanol solution, an acetonitrile solution, a dimethyl sulfoxide solution, and a dicarboxamide solution.
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