CN108794425A - Fluorescence probe and its application - Google Patents
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Abstract
The invention discloses fluorescence probe and its applications.Wherein, fluorescence probe has structure or its stereoisomer shown in Formulas I, wherein R1For hydrogen, C1‑6Alkyl, phenyl or alkyl-substituted phenyl；R2~R9It independently is hydrogen atom, C1‑6Alkyl, C1‑6Halogenated alkyl, C1‑6Alkoxy or halogen atom；R10And R11It independently is sulfonic group or the C of sulfonic group substitution1‑6Alkyl；X1, X2It independently is carbon, oxygen, sulphur, selenium or tellurium.The specific good biocompatibility of the fluorescence probe, cytotoxicity are low, small advantage is damaged to biological sample, and have good anti-light bleachability, may be implemented effectively to observe the cell sample long period, and do not influenced by intracellular ph value.
Description
Technical field
The present invention relates to analytical chemistry fields, and in particular, to fluorescence probe and its application, more particularly, to fluorescence
The fluorescence imaging of the purposes and cell autophagy lysosome of probe, fluorescence probe in targeting intracellular autophagy lysosome fluorescence imaging
Method.
Background technology
Lysosome is a kind of very important organelle, and diameter about 200-500nm is almost present in all eukaryocytes
In.There are many enzymes and protein, including acid hydrolase, membrane proteolytic enzyme and cathepsin in lysosome, disappears for intracellular
Change organ, the macromolecular substances such as various protein of decomposable asymmetric choice net have the function of dissolving or digest.Autophagy lysosome, also known as cell lysis
Enzyme body, it is that primary lysosome is merged with the vesica containing the endogenous material i.e. autophagosome from autophagocytosis or lysosome gulps down
Phagocyte matter and formed, serve in the cell " street cleaner ", as intracellular organelle and the other structures natueal reduction of staffs and more
New usual channel.Autophagy lysosome in normal cell in digestion, decomposition, substitute naturally and risen in some intracellular structures
Important function.When cell is by drug effect, radiation exposure and mechanical damage, quantity significantly increases.In lesion
In cell also often visible autophagy lysosome.Therefore, by detect autophagy lysosome, can be many diseases diagnosis and treat with
Track provides useful information.
Fluorescence probe has the advantages such as easy to operate, high sensitivity, cytotoxicity be small, is caused extensively in field of biological detection
General concern and research.Sold currently on the market it is several label lysosomes fluorescence probe such as LysoTracker Red,
LysoTracker Green etc., but still lack the probe of commercialized label autophagy lysosome.
Invention content
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose a kind of fluorescence probe, the good biocompatibility of the fluorescence probe, cytotoxicity are low, it is small to be damaged to biological sample, and
With good anti-light bleachability, may be implemented effectively to observe the cell sample long period, and not by intracellular ph value
It influences.
According to an aspect of the present invention, the present invention provides a kind of fluorescence probes.According to an embodiment of the invention, this is glimmering
Light probe has structure or its stereoisomer shown in Formulas I,
Wherein,
R1For hydrogen, C1-6Alkyl, phenyl or alkyl-substituted phenyl；
R2-R9It independently is hydrogen atom, C1-6Alkyl, C1-6Halogenated alkyl, C1-6Alkoxy or halogen atom；
R10And R11It independently is sulfonic group or the C of sulfonic group substitution1-6Alkyl；
X1, X2It independently is carbon, oxygen, sulphur, selenium or tellurium.
Fluorescence probe according to the ... of the embodiment of the present invention, membrane permeability is good, cell need not be fixed, is penetrating etc.
Reason carries out specific marker under conditions of keeping cell activity to intracellular autophagy lysosome；There is biocompatibility simultaneously
Well, cytotoxicity is low, small advantage is damaged to biological sample, and has good anti-light bleachability, may be implemented to cell
The sample long period is effectively observed, and is not influenced by intracellular ph value.In addition, the probe ingredient is simple, detection is easy to operate,
Fast, it is expected to become the Universal Dye of detection living cells autophagy lysosome.
In addition, fluorescence probe according to the above embodiment of the present invention can also have following additional technical characteristic：
According to an embodiment of the invention, the alkyl is methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, uncle
Butyl, amyl, isopentyl, n-hexyl or isohesyl.
According to an embodiment of the invention, the alkyl-substituted phenyl is aminomethyl phenyl or 3,5-dimethylphenyl.
According to an embodiment of the invention, the halogenated alkyl be a fluoromethane, difluoromethane, fluoroform, a bromomethane,
Methylene bromide or bromoform.
According to an embodiment of the invention, the alkoxy is methoxyl group, ethyoxyl or propoxyl group.
According to an embodiment of the invention, the halogen atom is fluorine, chlorine, bromine or iodine.
According to an embodiment of the invention, R1For hydrogen or C1-3Alkyl；R2-R9It independently is hydrogen atom, C1-3Alkyl, C1-3
Halogenated alkyl, methoxyl group or halogen atom；R10And R11It independently is sulfonic group, sulfonomethyl or sulfonic group ethyl.
According to an embodiment of the invention, include the structure of one of：
According to another aspect of the invention, the present invention provides fluorescence probes above-mentioned to target intracellular autophagy lysosome
Purposes in fluorescence imaging.Since the fluorescence probe membrane permeability and good biocompatibility, cytotoxicity are low, are damaged to biological sample
Hinder small advantage, and there is good anti-light bleachability, intracellular autophagy lysosome fluorescence is being targeted using the fluorescence probe
The processing such as cell need not be fixed, be penetrating, to intracellular autophagy lysosome under conditions of keeping cell activity in imaging
Specific marker is carried out, and may be implemented effectively to observe the cell sample long period, and is not influenced by intracellular ph value.
Furthermore, it is necessary to explanation, the fluorescence probe have the advantages that all technical features of aforementioned fluorescent probe and, herein no longer one by one
It repeats.
In accordance with a further aspect of the present invention, the present invention provides a kind of methods of the fluorescence imaging of cell autophagy lysosome.
According to an embodiment of the invention, this method includes：Fluorescence probe above-mentioned is mixed with solvent, it is molten to obtain fluorescence probe
Liquid；The fluorescence probe solution and cell are subjected to contact culture, to obtain the cell of fluorescent marker；And to the fluorescence
The cell of label carries out fluorescence imaging.Since the fluorescence probe has membrane permeability and good biocompatibility, cytotoxicity low, right
Biological sample damages small advantage, and has good anti-light bleachability, and cell autophagy lyase is carried out using the fluorescence probe
The processing such as cell need not be fixed, be penetrating in the fluorescence imaging of body, under conditions of keeping cell activity to it is intracellular from
It bites lysosome and carries out specific marker, and may be implemented effectively to observe the cell sample long period, and not by internal pH
The influence of value.Furthermore, it is necessary to explanation, the fluorescence probe have the advantages that all technical features of aforementioned fluorescent probe and,
This is no longer repeated one by one.
According to an embodiment of the invention, the solvent is molten selected from physiological saline, trishydroxymethylaminomethane-hydrochloride buffer
Liquid, phosphatic buffer solution, methanol solution, ethanol solution, acetonitrile solution, dimethyl sulfoxide solution and diformamide solution are extremely
Few one kind.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obviously, or practice through the invention is recognized.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination following accompanying drawings to embodiment
Obviously and it is readily appreciated that, wherein：
Fig. 1 shows fluorescence imaging schematic diagram according to an embodiment of the invention；
Fig. 2 shows fluorescence imaging schematic diagram according to an embodiment of the invention；
Fig. 3 shows fluorescence imaging schematic diagram according to an embodiment of the invention；
Fig. 4 shows fluorescence imaging schematic diagram according to an embodiment of the invention；
Fig. 5 shows fluorescence imaging schematic diagram according to an embodiment of the invention；
Fig. 6 shows fluorescence imaging schematic diagram according to an embodiment of the invention.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and is only used for explaining the present invention, and is not considered as limiting the invention.
Fluorescence probe
According to an aspect of the present invention, the present invention provides a kind of fluorescence probes.According to an embodiment of the invention, this is glimmering
Light probe has structure or its stereoisomer shown in Formulas I,
Wherein,
R1For hydrogen, C1-6Alkyl, phenyl or alkyl-substituted phenyl；
R2-R9It independently is hydrogen atom, C1-6Alkyl, C1-6Halogenated alkyl, C1-6Alkoxy or halogen atom；
R10And R11It independently is sulfonic group or the C of sulfonic group substitution1-6Alkyl；
X1, X2It independently is carbon, oxygen, sulphur, selenium or tellurium.
Fluorescence probe according to the ... of the embodiment of the present invention, membrane permeability is good, cell need not be fixed, is penetrating etc.
Reason carries out specific marker under conditions of keeping cell activity to intracellular autophagy lysosome；There is biocompatibility simultaneously
Well, cytotoxicity is low, small advantage is damaged to biological sample, and has good anti-light bleachability, may be implemented to cell
The sample long period is effectively observed, and is not influenced by intracellular ph value.In addition, the probe ingredient is simple, detection is easy to operate,
Fast, it is expected to become the Universal Dye of detection living cells autophagy lysosome.
Furthermore, it is necessary to which explanation, the fluorescence probe are also used as laser dye, nonlinear optical material, Yi Jisheng
Object sensor etc..
Term " alkyl " indicates that 1-20 carbon atom or 1-10 carbon atom or 1-8 carbon atom or 1-6 carbon are former
The univalence hydrocarbyl of the saturated straight chain or branch of son or 1-4 carbon atom or 1-3 carbon atom, wherein alkyl can be independent and be appointed
Selection of land is replaced by one or more substituent groups described in the invention.The example of alkyl includes, but is not limited to, methyl
(Me ,-CH3), ethyl (Et ,-CH2CH3), n-propyl (n-Pr ,-CH2CH2CH3), isopropyl (i-Pr ,-CH (CH3)2), normal-butyl
(n-Bu ,-CH2CH2CH2CH3), isobutyl group (i-Bu ,-CH2CH(CH3)2), sec-butyl (s-Bu ,-CH (CH3)CH2CH3), tertiary fourth
Base (t-Bu ,-C (CH3)3), n-pentyl (- CH2CH2CH2CH2CH3), 2- amyls (- CH (CH3)CH2CH2CH3), 3- amyls (- CH
(CH2CH3)2), 2- methyl -2- butyl (- C (CH3)2CH2CH3), 3- methyl -2- butyl (- CH (CH3)CH(CH3)2), 3- methyl-
1- butyl (- CH2CH2CH(CH3)2), 2-methyl-1-butene base (- CH2CH(CH3)CH2CH3), n-hexyl (-
CH2CH2CH2CH2CH2CH3), 2- hexyls (- CH (CH3)CH2CH2CH2CH3), 3- hexyls (- CH (CH2CH3)(CH2CH2CH3))、2-
Methyl -2- amyls (- C (CH3)2CH2CH2CH3), 3- methyl -2- amyls (- CH (CH3)CH(CH3)CH2CH3), 4- methyl -2- penta
Base (- CH (CH3)CH2CH(CH3)2), 3- methyl -3- amyls (- C (CH3)(CH2CH3)2), 2- methyl -3- amyls (- CH
(CH2CH3)CH(CH3)2), 2,3- dimethyl -2- butyl (- C (CH3)2CH(CH3)2), 3,3- dimethyl -2- butyl (- CH (CH3)
C(CH3)3), n-heptyl, n-octyl etc..Term " alkyl " and its prefix " alkane " use here, all include straight chain and branch
Saturated carbon chains.Term " alkylene " uses here, indicates that eliminating two hydrogen atoms from linear chain or branched chain saturation hydrocarbons obtains
Saturated divalent hydrocarbon radical, such example includes, but is not limited to, methylene, ethylidine, secondary isopropyl etc..
Term " alkoxy " used in the present invention, is related to alkyl, as defined herein, is connected by oxygen atom
Onto main carbochain, such example includes, but is not limited to methoxyl group, ethyoxyl, propoxyl group, butoxy etc..And institute
It can be substituted or unsubstituted to state alkoxy, and wherein substituent group can be, but be not limited to, deuterium, hydroxyl, amino, halogen, cyanogen
Base, alkoxy, alkyl, alkenyl, alkynyl, sulfydryl, nitro etc..
Term " halogenated alkyl " indicates the case where alkyl can be replaced by one or more halogen atoms, such example
Include, but is not limited to trifluoromethyl, difluoro-methoxy etc..
In addition, it is necessary to explanation, unless otherwise explicitly pointing out, describing mode as used throughout this document
" each ... independently be ", " ... independently be " and " ... be each independently " can be interchanged, and shall be understood in a broad sense, both can be with
Refer among the different groups, not influencing mutually, can also indicating identical between expressed specific option between the same symbol
Group in, do not influenced mutually between expressed specific option between the same symbol, with R9For, " C1-6Alkyl " and " halogen
Substituted alkyl " R between the two9Specific option it is unaffected from each other.
The definition of neutral body chemistry of the present invention and the use of convention are typically referenced to 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 compound of the present invention can be comprising asymmetric center or chiral centre, therefore
There are different stereoisomers.All stereoisomeric forms in any ratio of the compound of the present invention, including but not limited to, diastereomeric
Body, enantiomter, atropisomer and their mixture, such as racemic mixture constitute the part of the present invention.Very
More organic compounds all exist with optical active forms, i.e. the plane of their capable Plane of rotation polarised lights.In description optics
When reactive compound, prefix D, L or R, S are used for indicating the absolute configuration at molecular chiral center.Prefix d, l or (+), (-) are used for
The symbol of compound linearly polarized light rotation is named, (-) or l refer to that compound is left-handed, and prefix (+) or d refer to compound
It is dextrorotation.The chemical constitution of these stereoisomers is identical, but their stereochemical structure is different.It is specific three-dimensional
Isomers can be enantiomer, isomers mixture be commonly referred to as enantiomeric mixture.50:50 mixture of enantiomers
It is referred to as racemic mixture or racemic modification, this may lead to do not have stereoselectivity or stereotaxis in chemical reaction process
Property.Term " racemic mixture " and " racemic modification " refer to the mixture of equimolar two enantiomters, lack optics
Activity.
It should be noted that for the compound of Formulas I, preparation method can refer to LeslieG.S., Brooker
And Frank L.W., JACS, 1935, the synthetic route described in 547-551 can also use its other party well known in the art
It is prepared by method.
According to an embodiment of the invention, alkyl is methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, tertiary fourth
Base, amyl, isopentyl, n-hexyl or isohesyl.
According to an embodiment of the invention, alkyl-substituted phenyl is aminomethyl phenyl or 3,5-dimethylphenyl.
According to an embodiment of the invention, halogenated alkyl is a fluoromethane, difluoromethane, fluoroform, a bromomethane, dibromo
Methane or bromoform.
According to an embodiment of the invention, alkoxy is methoxyl group, ethyoxyl or propoxyl group.
According to an embodiment of the invention, halogen atom is fluorine, chlorine, bromine or iodine.
According to an embodiment of the invention, R1For hydrogen or C1-3Alkyl；R2-R9It independently is hydrogen atom, C1-3Alkyl, C1-3
Halogenated alkyl, methoxyl group or halogen atom；R10And R11It independently is sulfonic group, sulfonomethyl or sulfonic group ethyl.
According to an embodiment of the invention, which can include the structure of one of：
The application of fluorescence probe
According to another aspect of the invention, the present invention provides fluorescence probes above-mentioned to target intracellular autophagy lysosome
Purposes in fluorescence imaging.Since the fluorescence probe has, membrane permeability is good, good biocompatibility, cytotoxicity are low, to biology
The small advantage of sample damage, and there is good anti-light bleachability, targeting intracellular autophagy lyase using the fluorescence probe
The processing such as cell need not be fixed, be penetrating, to intracellular autophagy under conditions of keeping cell activity in body fluorescence imaging
Lysosome carries out specific marker, and may be implemented effectively to observe the cell sample long period, and not by intracellular ph value
Influence.Furthermore, it is necessary to explanation, the fluorescence probe have the advantages that all technical features of aforementioned fluorescent probe and, herein
No longer repeat one by one.
In accordance with a further aspect of the present invention, the present invention provides a kind of methods of the fluorescence imaging of cell autophagy lysosome.
According to an embodiment of the invention, this method includes：Fluorescence probe above-mentioned is mixed with solvent, it is molten to obtain fluorescence probe
Liquid；The fluorescence probe solution and cell are subjected to contact culture, to obtain the cell of fluorescent marker；And to the fluorescence
The cell of label carries out fluorescence imaging.Due to the fluorescence probe membrane permeability and good biocompatibility, cytotoxicity it is low, to biology
The small advantage of sample damage, and there is good anti-light bleachability, carry out cell autophagy lysosome using the fluorescence probe
The processing such as cell need not be fixed, be penetrating in fluorescence imaging, molten to intracellular autophagy under conditions of keeping cell activity
Enzyme body carries out specific marker, and may be implemented effectively to observe the cell sample long period, and not by intracellular ph value
It influences.Furthermore, it is necessary to explanation, the fluorescence probe have the advantages that all technical features of aforementioned fluorescent probe and, herein not
It repeats one by one again.
According to an embodiment of the invention, solvent be selected from physiological saline, trishydroxymethylaminomethane-hydrochloric acid buffer solution,
Phosphatic buffer solution, methanol solution, ethanol solution, acetonitrile solution, dimethyl sulfoxide solution and diformamide solution are at least
It is a kind of.Wherein, it should be noted that " methanol solution " can be pure methanol, can also be that first alcohol and water is mixed with arbitrary proportion
Close obtained solution.Similarly, " ethanol solution ", " acetonitrile solution ", " dimethyl sulfoxide solution " and " diformamide solution " is also such as
This, this is no longer going to repeat them.
According to an embodiment of the invention, trishydroxymethylaminomethane-hydrochloric acid buffer solution and phosphatic buffer solution
PH value is 6.2-8.2, and concentration is 0-50mmol/L.The pH value of buffer solution and intracellular pH value are close as a result, with cell
Good biocompatibility.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, according to technology described in document in the art or condition, (such as with reference to works such as J. Pehanorm Brookers, Huang Peitang etc. is translated
's《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Production firm person is not specified, being can be with conventional products that are commercially available, such as can purchase from Sigma companies.
Instrument in following embodiments for observing cell fluorescence is laser confocal microscope (OLYMPUS FV1000-
IX81(Olympus,Japan))。
Embodiment 1
The fluorescence probe (1) and LysoTracker Red probes for being utilized respectively the embodiment of the present invention carry out fluorescence to cell
Imaging, it is specific as follows：
(1) fluorescence probe (1) and LysoTracker Red probes are dissolved respectively with a small amount of dimethyl sulfoxide (DMSO)；
(2) two kinds of fluorescence probe solution of step (1) are added separately in culture medium, are each configured to dense containing 10 μM
The culture solution of the culture solution of the fluorescent dye 1 of degree and the LysoTracker Red of 1.0 μM of concentration；
(3) culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun, being separately added into culture has HEK293 cells
In culture dish, and put it into 37 DEG C, 5%CO230min is cultivated in incubator；
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 1, wherein a is the fluorescence co-focusing imaging schematic diagram of fluorescence probe (1), and b is
The fluorescence co-focusing imaging schematic diagram of LysoTracker Red, the fluorescence signal for scheming a are overlapped with the fluorescence signal height of figure b, table
Mark substance is lysosome to bright fluorescence probe (1) in the cell.The experimental results showed that fluorescence probe 1 has intracellular lysosome
Good targeting.
Embodiment 2
It is utilized respectively the fluorescence probe (2) of the embodiment of the present invention and induces the glimmering of lysosome autophagy process to monitor cell starvation
Light is imaged, specific as follows：
(1) fluorescence probe (2) is dissolved respectively with a small amount of ethyl alcohol；
(2) the fluorescence probe solution of step (1) is added in the culture medium containing 0,5% and 10% fetal bovine serum, is matched
It is set to the culture solution of the fluorescent dye 2 containing 4 μM of concentration；
(3) culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun is added separately to containing 0,5% and 10% embryo
In the culture dish of the human lung cancer cell A549 of fetal calf serum medium culture, and put it into 37 DEG C, 5%CO2In incubator
Cultivate 1h；
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 2, wherein a is using culture medium (containing 0 fetal bovine serum) training without fetal bovine serum
The fluorescence co-focusing imaging schematic diagram of foster cell, b are the fluorescence using the cell of the medium culture containing 5% fetal bovine serum
Co-focusing imaging schematic diagram, c are to be shown using the fluorescence co-focusing imaging of the cell of the medium culture containing 10% fetal bovine serum
It is intended to, with the increase of fetal bovine serum concentration, the fluorescence signal of fluorescence probe (2) in the cell gradually weakens, and shows embryo
Bovine serum concentration increases, and cell hunger intensity weakens, and intracellular autophagy lysosome is reduced, the experimental results showed that 2 energy of fluorescence probe
The variation of enough hungry induction living cells lysosome autophagy of detection.
Embodiment 3
It is utilized respectively the fluorescence probe (3) of the embodiment of the present invention and induces the glimmering of lysosome autophagy process to monitor cell starvation
Light is imaged, specific as follows：
(1) fluorescence probe (3) is dissolved respectively with a small amount of methanol；
(2) the fluorescence probe solution of step (1) is added in the blank cultures without fetal bovine serum, is respectively configured
At the blank culture solution of the fluorescent dye 3 containing 5 μM of concentration；
(3) the blank culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun, is added separately to carry out 0,0.5 and
In the culture dish for the Cervical Cancer HeLa Cells that 1 hour Nature enemy is crossed, and put it into 37 DEG C, 5%CO2It is cultivated in incubator
20min；
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 3, wherein the fluorescence co-focusing imaging that a is the cervical carcinoma HeLa crossed through 0 hour Nature enemy
Schematic diagram, b are the fluorescence co-focusing imaging schematic diagram of the cervical carcinoma HeLa crossed through 0.5 hour Nature enemy, and c is through 1 hour famine
The fluorescence co-focusing imaging schematic diagram of processed cervical carcinoma HeLa is starved, with the increase of starvation time, fluorescence probe (3) is thin
The fluorescence signal of intracellular gradually increases, and shows to increase with starvation time, and cell hunger intensity enhancing, intracellular autophagy lysosome increases
It is more.The experimental results showed that fluorescence probe 3 can detect the variation of hungry induction living cells autophagy lysosome.
Embodiment 4
Fluorescence probe (4) the monitoring Bafilomycin A1 for being utilized respectively the embodiment of the present invention inhibits intracellular lysosome autophagy
The fluorescence imaging of process, it is specific as follows：
(1) fluorescence probe (4) is dissolved respectively with a small amount of acetonitrile；
(2) the fluorescence probe solution of step (1) is added in culture medium, is configured to the fluorescent dye containing 20 μM of concentration
4 culture solution；
(3) culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun, is separately added into 0,200nM and 400nM bars
In the culture dish of the human fibrosarcoma cell HT1080 of Buddhist Lip river mycin A1 processing, and put it into 37 DEG C, 5%CO2Incubator
Middle culture 2h；
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 4, wherein a is the human fibrosarcoma cell handled without Ba Foluo mycins (i.e. 0nM) A1
The fluorescence co-focusing imaging schematic diagram of HT1080, b are the human fibrosarcoma cell HT1080 handled through 200nM Bafilomycin A1s
Fluorescence co-focusing imaging schematic diagram, c is the glimmering of the human fibrosarcoma cell HT1080 handled through 400nM Bafilomycin A1s
Light co-focusing imaging schematic diagram.With the increase of Ba Foluo mycin concentration, the fluorescence signal of fluorescence probe (4) in the cell is gradual
Weaken, shows that intracellular autophagy lysosome is reduced.The experimental results showed that fluorescence probe 4, which can monitor Bafilomycin A1, inhibits thin
The variation of intracellular autophagy lysosome.
Embodiment 5
It is utilized respectively lysosome autophagy process in fluorescence probe (5) the monitoring rapamycin inducing cell of the embodiment of the present invention
Fluorescence imaging, it is specific as follows：
(1) fluorescence probe (5) is dissolved respectively with a small amount of dimethyl sulfoxide (DMSO)；
(2) the fluorescence probe solution of step (1) is added in culture medium, is configured to the fluorescent dye containing 15 μM of concentration
5 culture solution；
(3) culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun, is added separately to mould with 0,2 and 5 μM of thunder pa
In the culture dish of the human breast cancer cell line Bcap-37 of element processing, and put it into 37 DEG C, 5%CO210min is cultivated in incubator；
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 5, wherein a is that the fluorescence of the human breast cancer cell line Bcap-37 through 0 μM of rapamycin treatment is copolymerized
Burnt imaging schematic diagram, b are the fluorescence co-focusing imaging schematic diagram of the human breast cancer cell line Bcap-37 through 2 μM of rapamycin treatments, c
For the fluorescence co-focusing imaging schematic diagram of the human breast cancer cell line Bcap-37 through 5 μM of rapamycin treatments, with rapamycin concentrations
Increase, the fluorescence signal of fluorescence probe (5) in the cell gradually increases, and shows intracellular autophagy lysosome increasing.Experiment knot
Fruit shows that fluorescence probe 5 has good membrane permeability, can monitor the process of autophagy lysosome in rapamycin inducing cell.
Embodiment 6
Fluorescence probe (6) the monitoring chloroquine for being utilized respectively the embodiment of the present invention inhibits the glimmering of intracellular lysosome autophagy process
Light is imaged, specific as follows：
(1) fluorescence probe (6) is dissolved respectively with a small amount of ethyl alcohol；
(2) the fluorescence probe solution of step (1) is added in culture medium, is configured to the fluorescent dye 6 containing 8 μM of concentration
Culture solution；
(3) culture solution for pipetting 1mL steps (2) preparation respectively with liquid-transfering gun is added separately to containing 0,2 and 5 μM of chloroquine
In the culture dish of human laryngeal cancer cell Hep-2, and put it into 37 DEG C, 5%CO24h is cultivated in incubator
(4) cell after culture is washed three times with PBS respectively, it is total adds 1mL blank mixed culture mediums progress fluorescence
Focal imaging, the results are shown in Figure 6, wherein the fluorescence co-focusing imaging signal that a is the human laryngeal cancer cell Hep-2 through 0 μM of chloroquine
Figure, b are the fluorescence co-focusing imaging schematic diagram of the human laryngeal cancer cell Hep-2 through 2 μM of chloroquines, and c is that the human laryngeal cancer through 5 μM of chloroquines is thin
The fluorescence co-focusing imaging schematic diagram of born of the same parents Hep-2, with the increase of chloroquine concentration, the fluorescence letter of fluorescence probe (6) in the cell
Number gradually weaken, shows that intracellular autophagy lysosome is reduced.The experimental results showed that fluorescence probe 6 has good membrane permeability,
The process that chloroquine inhibits intracellular autophagy lysosome can be monitored.
It summarizes
Integrated embodiment 1-6 can show that the fluorescence probe membrane permeability of the embodiment of the present invention is good, need not be carried out to cell
The processing such as fixed, penetrating carry out specific marker under conditions of keeping cell activity to intracellular autophagy lysosome；Simultaneously should
Probe has the advantages that good light stability and cytotoxicity are low, may be implemented effectively to observe the cell sample long period, and not
It is influenced by intracellular ph value.In addition, the probe ingredient is simple, detection is easy to operate, quick, is expected to become detection living cells certainly
Bite the Universal Dye of lysosome.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiments or example in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of being detached from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The range of invention is limited by claim and its equivalent.
Claims (10)
1. a kind of fluorescence probe, which is characterized in that there is structure or its stereoisomer shown in Formulas I,
Wherein,
R1For hydrogen, C1-6Alkyl, phenyl or alkyl-substituted phenyl；
R2-R9It independently is hydrogen atom, C1-6Alkyl, C1-6Halogenated alkyl, C1-6Alkoxy or halogen atom；
R10And R11It independently is sulfonic group or the C of sulfonic group substitution1-6Alkyl；
X1, X2It independently is carbon, oxygen, sulphur, selenium or tellurium.
2. fluorescence probe according to claim 1, which is characterized in that the alkyl is methyl, ethyl, n-propyl, isopropyl
Base, normal-butyl, isobutyl group, tertiary butyl, amyl, isopentyl, n-hexyl or isohesyl；
Optionally, the alkyl-substituted phenyl is aminomethyl phenyl or 3,5-dimethylphenyl.
3. fluorescence probe according to claim 1, which is characterized in that the halogenated alkyl be a fluoromethane, difluoromethane,
Fluoroform, a bromomethane, methylene bromide or bromoform.
4. fluorescence probe according to claim 1, which is characterized in that the alkoxy is methoxyl group, ethyoxyl or the third oxygen
Base.
5. fluorescence probe according to claim 1, which is characterized in that the halogen atom is fluorine, chlorine, bromine or iodine.
6. fluorescence probe according to claim 1, which is characterized in that R1For hydrogen or C1-3Alkyl；
R2-R9It independently is hydrogen atom, C1-3Alkyl, C1-3Halogenated alkyl, methoxyl group or halogen atom；
R10And R11It independently is sulfonic group, sulfonomethyl or sulfonic group ethyl.
7. fluorescence probe according to claim 1, which is characterized in that include the structure of one of：
8. purposes of the claim 1-7 any one of them fluorescence probe in targeting intracellular autophagy lysosome fluorescence imaging.
9. a kind of method of the fluorescence imaging of cell autophagy lysosome, which is characterized in that including：
Claim 1-7 any one of them fluorescence probes are mixed with solvent, to obtain fluorescence probe solution；
The fluorescence probe solution and cell are subjected to contact culture, to obtain the cell of fluorescent marker；And
Fluorescence imaging is carried out to the cell of the fluorescent marker.
10. according to the method described in claim 9, it is characterized in that, the solvent is selected from physiological saline, trihydroxy methyl amino
Methane-hydrochloric acid buffer solution, phosphate buffer solution, methanol solution, ethanol solution, acetonitrile solution, dimethyl sulfoxide solution and two
At least one of formamide solution.
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| CN109596585A (en) * | 2018-12-03 | 2019-04-09 | 贵州大学 | A kind of method of new high flux screening lysosome |
| CN110055054A (en) * | 2019-04-09 | 2019-07-26 | 中国科学院化学研究所 | It is a kind of target tetra- serobila of living cells mitochondria G- fluorescence probe and its application |
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| CN111039897A (en) * | 2019-12-26 | 2020-04-21 | 中国科学院化学研究所 | Fluorescent probes and their use in detection of autophagosomes |
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| CN111116573A (en) * | 2019-12-31 | 2020-05-08 | 中山大学 | Near-infrared fluorescent probe for simultaneously detecting DNA and RNA under dual channels and preparation method and application thereof |
| CN113636988A (en) * | 2020-04-26 | 2021-11-12 | 中国科学院理化技术研究所 | Seleno indocyanine green derivative and preparation method and application thereof |
| CN113636988B (en) * | 2020-04-26 | 2023-07-07 | 中国科学院理化技术研究所 | Seleno indocyanine green derivative and preparation method and application thereof |
| CN112898228A (en) * | 2021-01-18 | 2021-06-04 | 中国科学院化学研究所 | Mixed assembly based mixed aggregate FRET probes and their use in detecting mitochondrial autophagy |
| CN112898228B (en) * | 2021-01-18 | 2022-08-02 | 中国科学院化学研究所 | Mixed assembly based mixed aggregate FRET probes and their use in detecting mitochondrial autophagy |
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