阅读说明：本技术 基于混合组装的混合聚集体fret探针及其在检测线粒体自噬中的用途 (Mixed assembly based mixed aggregate FRET probes and their use in detecting mitochondrial autophagy ) 是由 孙红霞 郭晓朦 唐亚林 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种基于混合组装的混合聚集体FRET探针及其在检测线粒体自噬中的用途。该探针体系含有A和B两种分子,具有如下式所示的结构特征。A和B分子可以在水溶液环境中通过混合组装,构建出同时含有A和B的混合聚集体。A的荧光发射波长与B的吸收波长有部分交叠,能够产生荧光共振能量转移(FRET)效应。在活细胞内,该探针体系可以被用来检测自噬。(The invention discloses a mixed aggregate FRET probe based on mixed assembly and application thereof in detecting mitophagy. The probe system contains two molecules of A and B and has the structural characteristics shown as the following formula. The A and B molecules can be assembled by mixing in an aqueous solution environment to construct a mixed aggregate containing both A and B. The fluorescence emission wavelength of A is partially overlapped with the absorption wavelength of B, and the Fluorescence Resonance Energy Transfer (FRET) effect can be generated. In living cells, the probe system can be used to detect autophagy.)

1. A FRET fluorescent molecular pair is composed of a compound A or a stereoisomer thereof and a compound B or a stereoisomer thereof, wherein the structural formula of the compound A is shown as the following,

the structural formula of the compound B is shown as follows,

wherein R is₁Is hydrogen, C_1-6Alkyl, aryl or C_1-4Alkyl-substituted phenyl;

R₂-R₉independently hydrogen, fluorine, chlorine, bromine, iodine, C_1-6Alkyl radical, C_1-6Haloalkyl or C_1-6An alkoxy group;

R₁₀and R₁₁Each independently is C_1-6Alkylsulfonic acid group, C_1-6Alkyl hydroxy, C_1-6An alkyl group;

X₁and X₂Independently is-CH₂-, oxygen, sulfur, selenium or tellurium.

2. A FRET fluorescent molecule pair according to claim 1, characterized in that: said C is_1-6Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl;

said C is_1-4The alkyl-substituted phenyl is methylphenyl or dimethylphenyl;

said C is_1-6The halogenated alkyl is monofluoromethane, difluoromethane, trifluoromethane, monobromomethane, dibromomethane or tribromomethane;

said C is_1-6Alkoxy is methoxy, ethoxy or propoxy;

said C is_1-6The alkyl sulfonic group is n-propyl sulfonic group or ethyl sulfonic group；

Said C is_1-6The alkylhydroxy group being-CH₂CH₂OH。

3. A FRET fluorescent molecule pair according to claim 1 or 2, characterized in that: the compound A is any one of the following or a stereoisomer thereof:

the compound B is any one of the following or a stereoisomer thereof:

4. a FRET fluorescent molecule pair according to any one of claims 1-3, characterized in that: in the FRET fluorescent molecule pair, the molar ratio of the compound A or the stereoisomer thereof to the compound B or the stereoisomer thereof is any ratio between 0:1 and 20:1, and the endpoint value of 0 is not taken.

5. A mixed aggregate formed by spontaneous aggregation of compound A or a stereoisomer thereof and compound B or a stereoisomer thereof constituting the FRET fluorescent molecule pair according to any one of claims 1 to 4 in an aqueous solution system.

6. Use of a FRET fluorescent molecule pair of any one of claims 1-4 or a mixed aggregate of claim 5 as a fluorescent probe to detect mitophagy.

7. A method of determining the presence or absence of mitophagy in a cell comprising: contacting a FRET fluorescent molecule of any one of claims 1-4 with a cell; and detecting the fluorescence signal of the contacted cells.

8. The method of claim 7, wherein: the presence of a change in FRET fluorescent signal in the contacted cells is indicative of mitophagy within the cells.

9. The method according to claim 7 or 8, characterized in that: the FRET fluorescent molecule pair respectively enters mitochondria and lysosomes, has no FRET fluorescent signal in a non-autophagy state, and generates the FRET signal when autophagy occurs; or the FRET fluorescent molecule pair simultaneously enters mitochondria or lysosomes, a FRET signal appears in a non-autophagy state, and the FRET signal is weakened or even disappears when autophagy occurs;

and (3) judging whether the mitochondrial autophagy phenomenon occurs in the cell or not by detecting the FRET fluorescent signal in the cell.

10. The method according to any one of claims 7-9, wherein: the FRET fluorescent molecule is provided in the form of a solution in which a solvent is selected from at least one of physiological saline, potassium salt solution, tris-hcl buffer solution, phosphate buffer solution, methanol solution, ethanol solution, acetonitrile solution, dimethyl sulfoxide solution, and dimethylformamide solution.

Technical Field

The present invention relates to the field of chemical biology, in particular to a fluorescent probe and its use in detecting mitophagy, and more particularly, to a fluorescent probe having Fluorescence Resonance Energy Transfer (FRET) properties, its use in detecting mitophagy, and a method of determining the presence or absence of mitophagy in a cell.

Background

Fluorescence Resonance Energy Transfer (FRET) refers to the fact that if the emission spectrum of one fluorophore (donor) overlaps the absorption spectrum of the other fluorophore (acceptor) in two different fluorophores, the distance between the two fluorophores is appropriate (generally smaller than the distance between the two fluorophores)) The phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed, i.e., fluorescence emitted from the former group is observed when excited at the excitation wavelength of the latter group. In short, it is the process of energy transfer from donor to acceptor mediated by a pair of dipoles in the excited state of the donor group, which is non-radiative without the participation of photons. When the donor molecule is excited, the acceptor molecule is spaced from the donor molecule, and the energy difference between the vibrational energy levels of the ground states of the donor and acceptor and the first excited electronic state are adapted to each other, the donor in the excited state will transfer some or all of the energy to the acceptor, causing the acceptor to be excited, without involving emission and reabsorption of photons during the entire energy transfer process.

The conditions under which efficient energy transfer between energy donor-acceptor pairs occurs are harsh and include, among other things: (1) the emission spectrum of the energy donor must overlap with the absorption spectrum of the energy acceptor; (2) the fluorescent chromophores of the energy donor and the energy acceptor must be arranged in a suitable manner; (3) the energy donor and the energy acceptor must be close enough to each other so that the chance of energy transfer is high. In addition, there are many requirements for suitable donor and acceptor molecules in terms of quantum yield, extinction coefficient, water solubility, interference resistance, etc. A suitable donor-acceptor pair is not readily available.

FRET has wide application in many fields such as nanotechnology, analytical chemistry, biosensors, biological research, and the like. The common method is to connect two fluorophores at different sites of the same molecule through chemical bonds, and when the conformation of the molecule changes, the FRET signal changes; alternatively, two fluorophores can be attached to two different molecules, and the FRET signal can be changed simultaneously when the two molecules interact with each other. The FRET fluorescence pair constructed by the chemical bond has the disadvantages of complicated preparation method and high cost.

Disclosure of Invention

An object of the present invention is to provide a FRET fluorescent molecule pair, which is composed of two cyanine dye molecules with different structures, and can spontaneously aggregate to form a mixed aggregate containing both a and B in an aqueous solution system or an intracellular physiological environment. The mixed aggregate has the capability of generating FRET fluorescence, has good biocompatibility and low cytotoxicity, and can be used for monitoring the physiological process of mitophagy.

The FRET fluorescent molecular pair provided by the invention is composed of a compound A or a stereoisomer thereof and a compound B or a stereoisomer thereof, wherein the structural formula of the compound A is shown as follows,

the structural formula of the compound B is shown as follows,

wherein R is₁Can be hydrogen or C_1-6Alkyl (specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, n-hexyl, or isohexyl), aryl, or C_1-4Alkyl-substituted phenyl (methylphenyl or dimethylphenyl); specifically, R₁Can be H, ethyl;

R₂-R₉each independently of the others is hydrogen, fluorine, chlorine, bromine, iodine, C_1-6Alkyl radical, C_1-6Haloalkyl (specifically, monofluoromethyl, difluoromethyl, trifluoromethyl, monobromomethyl, dibromomethyl or tribromomethyl) or C_1-6Alkoxy (specifically, methoxy, ethoxy or propoxy); utensil for cleaning buttockThe body can be: trifluoromethyl, methoxy, fluoro;

R₁₀and R₁₁Each independently is C_1-6Alkylsulfonic acid group (specifically, n-propylsulfonic acid group, ethylsulfonic acid group), and C_1-6An alkylhydroxy group (which may be specifically-CH)₂CH₂OH)、C_1-6An alkyl group (which may be specifically an ethyl group);

X₁and X₂Independently is carbon (-CH)₂-), oxygen, sulfur, selenium or tellurium.

Specifically, R₁Can be hydrogen or C_1-3An alkyl or aryl group;

R₂-R₉each independently of the others is hydrogen, fluorine, chlorine, bromine, iodine, C_1-3Alkyl radical, C_1-3Haloalkyl or C_1-3An alkoxy group; r₁₀And R₁₁Each independently is C_1-6Alkyl radical, C_1-6Alkylsulfonic acid group, C_1-6Alkyl hydroxy, C_1-6An alkoxy group.

According to an embodiment of the present invention, the compound a may specifically be any one of the following or stereoisomers thereof:

the compound B is any one of the following or a stereoisomer thereof:

in the FRET fluorescent molecule pair, the molar ratio of the compound A or the stereoisomer thereof to the compound B or the stereoisomer thereof can be 0:1 to 20:1, and the endpoint value of 0 is not selected and can be 1:1 to 8:1 specifically;

the mixed aggregate formed by spontaneous aggregation of the compound A or the stereoisomer thereof and the compound B or the stereoisomer thereof in an aqueous solution system also belongs to the protection scope of the invention.

The invention also aims to provide an application of the FRET fluorescent molecule pair or a mixed aggregate formed by spontaneous aggregation of the compound A or the stereoisomer thereof and the compound B or the stereoisomer thereof in an aqueous solution system as a fluorescent probe in detecting mitochondrial autophagy.

The FRET fluorescent molecule pair can respectively enter mitochondria and lysosomes, has no FRET fluorescent signal in a non-autophagy state, and generates the FRET signal when autophagy occurs; or the FRET fluorescent molecule pair can enter mitochondria or lysosomes at the same time, the FRET signal appears under the non-autophagy state, and the FRET signal is weakened or even disappears when autophagy occurs. Whether the mitophagy phenomenon occurs in the cells can be effectively judged by detecting the FRET fluorescent signal in the cells. The membrane permeability of the fluorescent molecules is good, the cells do not need to be fixed, permeated and the like, and the intracellular autophagy is detected under the condition of keeping the activity of the cells; meanwhile, the method has the advantages of good biocompatibility and low cytotoxicity, and is not easily influenced by the pH value in cells. In addition, the FRET fluorescent molecule pair is simple to prepare, simple and convenient to detect and operate, and is expected to be developed into a universal dye for detecting living cell mitophagy.

It is yet another object of the present invention to provide a method for determining the presence or absence of mitophagy in a cell.

The method for determining whether mitophagy exists in cells provided by the invention comprises the following steps: contacting the pair of FRET fluorescent molecules as a fluorescent probe with a cell; and detecting fluorescence signals of the contacted cells;

wherein a change in FRET fluorescence signal in the contacted cell is indicative of mitophagy within the cell.

As described above, the FRET fluorescent molecule pair according to the embodiment of the present invention may enter mitochondria and lysosomes, respectively, and has no FRET fluorescent signal in a non-autophagy state, and a FRET signal occurs when autophagy occurs; or the FRET fluorescent molecule pair can enter mitochondria or lysosomes at the same time, the FRET signal appears under the non-autophagy state, and the FRET signal is weakened or even disappears when autophagy occurs. Whether the mitophagy phenomenon occurs in the cells can be effectively judged by detecting the FRET fluorescent signal in the cells. It should be noted that the mitophagy detection method has all the technical features and advantages of the fluorescent probe, and is not described herein again.

According to an embodiment of the present invention, the fluorescent probe is provided in the form of a solution in which a solvent is selected from at least one of physiological saline, a potassium salt solution, a tris-hcl buffer solution, a phosphate buffer solution, a methanol solution, an ethanol solution, an acetonitrile solution, a dimethylsulfoxide solution, and a dimethylformamide solution.

The "methanol solution" may be pure methanol or a mixture of methanol and water at an arbitrary ratio. Similarly, the same applies to the "ethanol solution", the "acetonitrile solution", the "dimethyl sulfoxide solution" and the "dimethylformamide solution", and the description thereof is omitted.

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 to 8.2, and the concentrations thereof are both 0.1 to 50 mmol/L.

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.

The FRET fluorescent probe film has good permeability, does not need to carry out treatments such as fixation, permeability and the like on cells, and carries out specific marking on mitochondrion autophagy in the cells under the condition of keeping the activity of the cells; meanwhile, the probe has the advantages of good photostability and low cytotoxicity, and can realize effective observation of cell samples for a long time. In addition, the probe has simple components, is simple and convenient to detect and operate, and is expected to become a universal dye for detecting living cell mitophagy.

Drawings

FIG. 1 shows the preparation of Compound (1) according to example 1 of the present invention¹H-NMR chart;

FIG. 2 shows a mass spectrum of the compound (1) obtained in example 1 of the present invention;

FIG. 3 shows a cytotoxicity assay chart of Compound (2) in example 2 of the present invention;

FIG. 4 is a graph showing absorption spectra of a mixed aggregate of the compound (2) and the compound (4) in example 3 of the present invention;

FIG. 5 shows a FRET fluorescence spectrum of a mixed aggregate of compound (2) and compound (4) in example 4 of the present invention;

FIG. 6 shows a FRET fluorescence spectrum of a mixed aggregate of compound (1) and compound (5) in example 5 of the present invention;

FIG. 7 shows a cell location map of a mixed aggregate of Compound (7) and Compound (8) in example 6 of the present invention;

FIG. 8 shows a cell location map of a mixed aggregate of Compound (3) and Compound (4) in example 7 of the present invention;

FIG. 9 shows a cell location map of a mixed aggregate of Compound (6) and Compound (9) in example 8 of the present invention;

FIG. 10 shows imaging graphs of mitophagy of FRET fluorescence pairs composed of compound (3) and compound (4) in example 9 of the present invention;

FIG. 11 shows imaging graphs of mitophagy of FRET fluorescence pairs composed of compound (2) and compound (5) in example 10 of the present invention;

FIG. 12 shows imaging graphs of mitophagy of FRET fluorescence pairs composed of compound (6) and compound (4) in example 11 of the present invention.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

The fluorescent probe can also be used as a composition of a laser dye, a nonlinear optical material, a biosensor, and the like.

Unless otherwise expressly indicated, the descriptions "… independently for each 8932 independently", "… independently for" and "… independently for each …" used throughout this document are interchangeable and should be construed broadly to mean that particular items expressed between the same symbols do not interfere with each other in different groups or that particular items expressed between the same symbols do not interfere with each other in the same groups.

The definition and convention of stereochemistry in the present invention is generally used with reference to 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 in different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including, but in no way 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 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 indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to designate the sign of the rotation of plane polarized light of the compound, with (-) or l indicating that the compound is left-handed and the prefix (+) or d indicating that the compound is right-handed. The chemical structures of these stereoisomers are identical, but their stereo structures are different. A particular stereoisomer may be an enantiomer, a mixture of isomers commonly referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during the chemical reaction. 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 compounds of A and B, reference may be made to the Synthetic routes described in Hamer, F.M. the Chemistry of Heterocyclic compounds, the cyano Dyes and Related compounds, Interscience Publishers, New York-London,1964 and Ficken, G.E. the Chemistry of Synthetic Dyes, cyano Dyes, academic Press,1971, and that other methods known in the art may also be used.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

The instrument used to observe the fluorescence of the cells in the examples described below was a confocal laser microscope (OLYMPUS FV1000-IX81(Olympus, Japan)).

Example 1

1. The synthesis methods of the compounds (1) to (3) are shown below, and the reaction ratio and the purification method may be those conventionally used in the art or those conventionally used in the art. Further, the inventors confirmed that the structure of the compound (1) is correct by analyzing the hydrogen spectrum, carbon spectrum and/or mass spectrum data of each compound¹The H-NMR and mass spectra are shown in FIGS. 1 and 2, respectively.

Example 2

The compound of the patent has similar characteristics, and the cytotoxicity test is carried out by using the compound (2) of the embodiment of the invention, which comprises the following steps:

(1) dissolving the compound (2) with a small amount of methanol;

(2) adding compound (2) solutions with different concentrations into the cultured HeLa and MCF-7 cells, and continuously culturing for 48 h;

(3) after the culture medium is sucked dry, adding 10% MTT solution, and continuously culturing for 4 h;

(4) after the medium is blotted dry, DMSO is added for dissolution, and the absorbance at 559nm is measured by using an enzyme-labeling instrument. The absorbance value at 559nm of the cells without the compound was taken as a control, the absorbance at 559nm of the cells with the compound added was divided by the absorbance at 559nm of the cells without the compound added as an ordinate, and the concentration value of the compound (2) was taken as an abscissa, and plotted. As shown in FIG. 3, the absorbance value at 559nm after the addition of the compound (2) was not significantly different from that of the control group, indicating that the compound (2) had low cytotoxicity.

Example 3

The compound (2) and the compound (4) of the embodiment of the present invention are assembled in an aqueous solution containing potassium ions to form a mixed aggregate having FRET properties, specifically as follows:

(1) dissolving the compound (2) and the compound (4) with a small amount of methanol, respectively;

(2) mixing the compound (2) and compound (4) solutions obtained in the step (1) according to different concentration ratios, diluting the mixture with water containing potassium ions, and preparing a mixed aggregate with FRET performance, wherein the molar concentration ratio of the compound (2) to the compound (4) is 0:1, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1 and 8: 1; the molar concentration of compound 4 was 4 μ M;

(3) and (3) detecting the absorption spectrum of the sample solution in the step (2) by using an ultraviolet absorption spectrometer, wherein the result is shown in fig. 4, and the absorbance of the compound (4) is gradually increased along with the increase of the concentration of the compound (2), which indicates that the compound (2) and the compound (4) are self-assembled to form a mixed aggregate. (the arrow in FIG. 4 indicates an increase in the concentration of Compound (2))

Example 4

The compound (2) and the compound (4) of the embodiment of the present invention are assembled in an aqueous solution containing potassium ions to form a mixed aggregate having FRET properties, specifically as follows:

(1) dissolving the compound (2) and the compound (4) with a small amount of methanol, respectively;

(2) mixing the compound (2) and compound (4) solutions obtained in the step (1) according to different concentration ratios, diluting the mixture with water containing potassium ions, and preparing a mixed aggregate with FRET performance, wherein the molar concentration ratio of the compound (2) to the compound (4) is 0:1, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1 and 8: 1; the molar concentration of compound 4 was 4 μ M;

(3) the fluorescence spectrum of the sample solution in step (2) was measured by a fluorescence spectrometer with 430nm as the excitation wavelength, and the result is shown in fig. 5, in which the fluorescence intensity at 600nm gradually increased with the increase of the concentration of compound (2), indicating that FRET occurred in the mixed aggregate formed by compound (2) and compound (4). (the arrow in FIG. 5 indicates an increase in the concentration of Compound (2))

Example 5

The compound (1) and the compound (5) of the embodiment of the present invention are assembled in an aqueous solution containing potassium ions to form a mixed aggregate having FRET properties, specifically as follows:

(1) dissolving the compound (1) and the compound (5) with a small amount of methanol, respectively;

(2) mixing the compound (1) and compound (5) solutions obtained in the step (1) according to different concentration ratios, diluting the mixture with water containing potassium ions, and preparing a mixed aggregate with FRET performance, wherein the molar concentration ratio of the compound (1) to the compound (5) is 0:1, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 15:1 and 20: 1; the concentration of compound 5 was 10 μ M;

(3) the fluorescence spectrum of the sample solution in step (2) was measured by a fluorescence spectrometer with 420nm as the excitation wavelength, and as a result, as shown in fig. 6, the fluorescence intensity of compound (5) at 600nm gradually increased with the increase in the concentration of compound (1), indicating that FRET occurred in the mixed aggregates formed by compound (1) and compound (5). (the arrow in FIG. 6 indicates an increase in the concentration of Compound (1))

Example 6

The cell localization was performed using the compound (7) and the compound (8) of the present example, respectively, as follows:

(1) dissolving the compound (7), the compound (8) and the Lyso Brite NIR probe with a small amount of dimethyl sulfoxide, respectively;

(2) adding the two compound solutions of step (1) to culture media, respectively, to prepare a culture solution containing 10. mu.M of compound (7) and 10. mu.M of compound (8) and a culture solution of 50nM concentration of Lyso Brite NIR;

(3) using a pipette to pipette 1mL of the culture solution prepared in step (2), adding the culture solution to a culture dish in which MCF-7 cells are cultured, and placing the culture dish at 37 ℃ and 5% CO₂Culturing for 4h in an incubator;

(4) washing the cultured cells with PBS for three times respectively, adding 1mL of blank mixed culture medium for fluorescence confocal imaging, wherein the excitation wavelength is 405nm, and the collection waveband is 420-500 nm; the excitation wavelength is 559nm, and the collection wavelength band is 570-620 nm; the excitation wavelength is 633nm, and the collection band is 650-700 nm. The results are shown in FIG. 7, in which FIG. a is a schematic diagram of fluorescence confocal imaging of compound (7) at 420-500 nm; FIG. b is a schematic diagram of fluorescence confocal imaging of compound (8) at 570-620 nm; panel c is a schematic representation of fluorescence confocal imaging of Lyso Brite NIR at 650-700 nm. The fluorescence signals of panels a and b are highly coincident with the fluorescence signal of panel c, indicating that compound (7) and compound (8) have good targeting to intracellular lysosomes.

Example 7

The cell localization was performed using the compound (3) and the compound (4) of the present example, respectively, as follows:

(1) dissolving the compound (3), the compound (4), the Lyso Brite NIR and the Mito Tracker Deep Red probe with a small amount of dimethyl sulfoxide, respectively;

(2) adding the two compound solutions of step (1) into culture medium respectively to prepare culture solution containing 20 μ M compound (3), 10 μ M compound (4), 50 nLyso Brite NIR and 50nMMito Tracker Deep Red;

(3) using a pipette to pipette 1mL of the culture solution prepared in step (2), adding the culture solution to a culture dish in which MCF-7 cells are cultured, and placing the culture dish at 37 ℃ and 5% CO₂Culturing for 4h in an incubator;

(4) washing the cultured cells with PBS for three times respectively, adding 1mL of blank mixed culture medium for fluorescence confocal imaging, wherein the excitation wavelength is 405nm, and the collection waveband is 420-500 nm; the excitation wavelength is 559nm, and the collection wavelength band is 570-620 nm; the excitation wavelength is 633nm, and the collection band is 650-700 nm. The results are shown in FIG. 8, in which a is a schematic diagram of fluorescence confocal imaging of compound (3) at 420-500 nm; FIG. b is a schematic diagram of fluorescence confocal imaging of compound (4) at 570-620 nm; FIG. c is a schematic diagram of fluorescence confocal imaging of Lyso Brite NIR at 650-700 nm; panel d is a schematic representation of fluorescence confocal imaging of Mito Tracker Deep Red at 650-700 nm. The fluorescence signals of panels a and c are highly coincident, indicating that compound (1) has good targeting to intracellular lysosomes. The fluorescence signals in panels b and d are highly coincident, indicating that compound (4) has good targeting to mitochondria within cells.

Example 8

The cell localization was performed using the compound (6) and the compound (9) of the present example, respectively, as follows:

(1) dissolving the compound (6), the compound (9) and the Mito Tracker Deep Red probe respectively by using a small amount of dimethyl sulfoxide;

(2) adding the two compound solutions obtained in the step (1) into culture media respectively to prepare culture solutions containing 4 mu M of compound (6), 4 mu M of compound (9) and 50nM Mito Tracker Deep Red respectively;

(3) using a pipette to pipette 1mL of the culture solution prepared in step (2), adding the culture solution into a culture dish with HT1080 cells, and placing the culture dish at 37 ℃ and 5% CO₂Culturing in an incubator for 30 min;

(4) washing the cultured cells with PBS for three times respectively, adding 1mL of blank mixed culture medium for fluorescence confocal imaging, wherein the excitation wavelength is 405nm, and the collection waveband is 420-500 nm; the excitation wavelength is 559nm, and the collection wavelength band is 570-620 nm; the excitation wavelength is 633nm, and the collection band is 650-700 nm. The results are shown in FIG. 9, in which a is a schematic diagram of fluorescence confocal imaging of compound (3) at 420-500 nm; FIG. b is a schematic diagram of fluorescence confocal imaging of compound (4) at 570-620 nm; panel c is a schematic representation of fluorescence confocal imaging at 650-700nm by Mito Tracker Deep Red. The fluorescence signals of the panels a, b and c are highly coincident, indicating that the compounds (6) and (9) have good targeting to mitochondria within cells.

Example 9

Fluorescence imaging for monitoring cell starvation induced mitochondrial autophagy process by using FRET fluorescence pairs consisting of the compound (3) and the compound (4) in the embodiment of the invention is specifically as follows:

(1) dissolving the compound (3) and the compound (4) with a small amount of dimethyl sulfoxide;

(2) adding the two compound solutions obtained in the step (1) into culture media respectively to prepare culture solutions containing 20 mu M of the compound (3) and 10 mu M of the compound (4);

(3) by movingTransferring 1mL of the culture solution prepared in step (2) by using a liquid gun, adding the culture solution into a culture dish for culturing HT1080 cells, and placing the culture dish at 37 ℃ and 5% CO₂Culturing for 8h in an incubator;

(4) replacing the culture solution of step (3) with 1mL of blank culture solution without fetal bovine serum, and placing the blank culture solution at 37 ℃ and 5% CO₂Starving the cells in the incubator for 0, 0.5 and 1 hour, respectively;

(4) the cultured cells are washed three times by PBS respectively, and then 1mL of blank mixed culture medium is added for fluorescence confocal imaging, the excitation wavelength is 405nm, and the FRET fluorescence collection band is 550-620 nm. The results are shown in FIG. 10, in which a is a schematic diagram of fluorescence confocal imaging of HT1080 after 0 hour starvation, b is a schematic diagram of fluorescence confocal imaging of HT1080 after 0.5 hour starvation, c is a schematic diagram of fluorescence confocal imaging of HT1080 after 1 hour starvation, and as the starvation time increases, the FRET fluorescence composed of compound (3) and compound (4) gradually increases with the fluorescence signal at 550-620nm in the cell, indicating that the autophagy in the cell increases as the starvation time increases. The experimental result shows that FRET fluorescence composed of the compound (3) and the compound (4) can detect starvation-induced mitophagy.

Example 10

Fluorescence imaging for monitoring the rapamycin-induced mitochondrial autophagy process by using FRET fluorescence pairs consisting of the compound (2) and the compound (5) in the embodiment of the invention is specifically as follows:

(1) dissolving the compound (2) and the compound (5) with a small amount of dimethyl sulfoxide;

(2) adding the compound (2) and compound (5) solution obtained in the step (1) into a culture medium containing fetal bovine serum to prepare culture solutions containing 10 mu M of the compound (2) and 10 mu M of the compound (5) respectively;

(3) using a pipette to pipette 1mL of the culture solution prepared in step (2) and adding to human milk treated with 0, 0.5 and 1. mu.M rapamycinAdenocarcinoma cell MCF-7 in a petri dish at 37 deg.C with 5% CO₂Culturing for 2 hours in an incubator;

(4) the cultured cells are washed three times by PBS respectively, and then 1mL of blank medium is added for fluorescence confocal imaging, the excitation wavelength is 405nm, and the FRET fluorescence collection wavelength is 570-620 nm. The results are shown in FIG. 11, in which a is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0. mu.M rapamycin, b is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0.5. mu.M rapamycin, and c is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 1. mu.M rapamycin. As the concentration of rapamycin increases, the FRET fluorescence of the compound (2) and the compound (5) gradually decreases from the fluorescence signal at 570-620nm in the cells. Thus, it was shown that compound (2) and compound (5) FRET fluorescent pairs are capable of monitoring rapamycin-induced mitophagy.

Example 11

Fluorescence imaging for monitoring rapamycin-induced lysosomal autophagy processes using compound (6) and compound (4) of the examples of the invention, respectively, is as follows:

(1) dissolving the compound (6) and the compound (4) with a small amount of dimethyl sulfoxide;

(2) adding the compound (6) and compound (4) solution obtained in the step (1) into a culture medium containing fetal bovine serum to prepare culture solutions containing 10 mu M of the compound (6) and 10 mu M of the compound (4) respectively;

(3) 1mL of each of the culture solutions prepared in step (2) was pipetted using a pipette gun, and added to each of petri dishes of human breast cancer cells MCF-7 treated with 0, 0.5 and 1. mu.M of rapamycin, and placed at 37 ℃ with 5% CO₂Culturing for 2 hours in an incubator;

(4) the cultured cells are washed three times by PBS respectively, and then 1mL of blank medium is added for fluorescence confocal imaging, the excitation wavelength is 405nm, and the FRET fluorescence collection wavelength is 570-620 nm. The results are shown in FIG. 12, in which a is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0. mu.M rapamycin, b is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 0.5. mu.M rapamycin, and c is a schematic diagram of fluorescence confocal imaging of human breast cancer cell MCF-7 treated with 1. mu.M rapamycin. As the concentration of rapamycin increases, the FRET fluorescence of the compound (6) and the compound (4) gradually decreases from the fluorescence signal at 570-620nm in the cells. Thus, it was shown that compound (6) and compound (4) FRET fluorescent pairs are capable of monitoring rapamycin-induced mitophagy.

Summary of the invention

The comprehensive embodiment proves that the FRET fluorescent probe film has good permeability, does not need to carry out treatments such as fixation and permeability on cells, and carries out specific marking on the mitophagy in the cells under the condition of keeping the activity of the cells; meanwhile, the probe has the advantages of good photostability and low cytotoxicity, and can realize effective observation of cell samples for a long time. In addition, the probe has simple components, is simple and convenient to detect and operate, and is expected to become a universal dye for detecting living cell mitophagy.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above 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.

While embodiments of the 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 to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.