CN108329301A - A kind of two-photon pH ratios metering fluorescence probe and its preparation method and application monitoring cell autophagy - Google Patents
A kind of two-photon pH ratios metering fluorescence probe and its preparation method and application monitoring cell autophagy Download PDFInfo
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Abstract
The invention discloses a kind of two-photon pH ratios of monitoring cell autophagy to measure fluorescence probe and its preparation method and application, wherein the structure of the two-photon pH ratios metering fluorescence probe of monitoring cell autophagy is as follows：Fluorescence probe of the present invention can have pH the fluorescence signal of specificity to respond.The present invention by cell common location experiment firmly believe its can specificity target Cytolysosome, cytotoxicity test shows the present invention for cell almost without what toxic side effect, the experiment of two-photon confocal fluorescent micro-imaging shows that the present invention is good for the permeability of 7 cells of MCF, simultaneously, the pKa for being computed fluorescence probe of the present invention is 3.88, it is very suitable for the monitoring of Cytolysosome pH variation ranges, the case where cell autophagy process carries out can be monitored in real time by detecting the variation of Cytolysosome pH.
Description
Technical field
The present invention relates to a kind of two-photon Ratiometric fluorescent probe, specifically a kind of two-photon of monitoring cell autophagy
PH ratios measure fluorescence probe and its preparation method and application.
Background technology
Autophagy be cell swallowed when by destructive stimulus own cells matter albumen or organelle and invaginate at vesica into
And autophagy lysosome is formed with lysosome fusion, the process for the content that it is wrapped up of degrading.Cell autophagy is cell component drop
Solution and the basis recycled, play a part of " street cleaner " in cell, natural as intracellular organelle and other structures
When histocyte is injured by various chemical factors, autophagy lysosome increases significantly the usual channel of metabolism, to the damage of cell
A kind of protective effect is hindered.Traditional autophagy monitoring includes transmission electron microscope (TEM), western blot (Atg8/LC3)
With GFP-Atg8/LC3 fluorescence microscopes.However these monitoring means have its limitation, as scanning electron microscope and protein immunization print
Mark, they can not monitor the autophagy state in living cells, meanwhile, the result visualization effect of these methods monitoring is simultaneously paid no attention to
Think, therefore, it is a kind of can it is vivid and it is easy monitoring autophagy method it is very necessary to the research of cell autophagy process.
Lysosome is a kind of organelle of monofilm cystic structures in eukaryocyte, a variety of hydrolases is included, in cell
Main function is to decompose local cells matter or organelle that intracellular substance and vitellophag itself are entered from the external world, when thin
When born of the same parents' aging, lysosome rupture releases hydrolase, digests entire cell and make its death.Due to cell micro-environment (such as pole
Property, pH, viscosity) it is very different in lysosome and autophagosome, so when cell occur autophagy when both fusion formed autophagy
Microenvironment in lysosome certainly will change, therefore, we can by detect the variation of cell micro-environment during this come
Monitor cell autophagy there is a situation where.
PH is the important factor in order of cell mesophytization reaction balance, as an importance in biosystem microenvironment
Matter affects the operation of cell normal activities mechanism and the smooth conversion of biomolecule.Abnormal cellular pH variation
Have with a variety of diseases and physiology course in biosystem and closely contacts.
Two-photon fluorescence probe is as a kind of testing tool, the fluorescence when test substances property or local environment change
Signal will occur accordingly to change, and high sensitivity is easy to operate, and stability is high, and photobleaching is small, and Cell permeable is by force thin
Superior performance is shown when born of the same parents' level qualitative analysis.It is thereby possible to select two-photon fluorescence probe is by detecting cytase
The variation of internal pH monitors cell autophagy process.
Invention content
The present invention is intended to provide a kind of two-photon pH ratios metering fluorescence probe and preparation method thereof of monitoring cell autophagy
And purposes.The present invention selects a kind of suitable fluorescence probe structure by MOLECULE DESIGN, to realize the qualitative inspection of two photon imaging
Survey Cytolysosome pH variations.Fluorescence probe specificity of the present invention is strong, and high sensitivity, cytotoxicity experiment shows fluorescence of the present invention
Probe is to cell almost without toxic side effect.
The present invention monitors the two-photon pH ratios metering fluorescence probe (Lyso-MPBC) of cell autophagy, and referred to as fluorescence is visited
Needle is using carbazole as parent, and 4- Methoxy-phenylacetylenes are electron-donating group, and lysosome seeking group is morpholine, benzimidazole pH
Response group, structural formula is as follows：
The present invention monitors the preparation method of the two-photon pH ratios metering fluorescence probe of cell autophagy, includes the following steps：
Compound A1g, o-phenylenediamine 0.46g, p-methyl benzenesulfonic acid 0.0082g and N,N-dimethylformamide 30mL are added
Enter into three-necked flask, 120 DEG C and logical argon gas protection reaction 12h are warming up in oil bath pan；Liquid is spin-dried for after reaction,
It is extracted with dichloromethane and water, collect upper organic phase and is spin-dried for obtaining crude product；By gained crude product sample preparation column chromatography color
Post separation is composed, target product 0.7g (1.28mmoL), yield 60% are obtained.
The structural formula of the compound A is：
Eluent used is dichloromethane and methanol by volume 100 when column chromatography chromatogram post separation:1 is mixed to get.
The building-up process that the present invention monitors the two-photon pH ratios metering fluorescence probe Lyso-MPCB of cell autophagy is as follows：
The present invention monitors the purposes of the two-photon pH ratios metering fluorescence probe Lyso-MPCB of cell autophagy, is to detect
It is used as detection reagent when lysosomal pH in cell.Fluorescence probe (Lyso-MPCB) of the present invention can be by detecting lysosome
PH changes to monitor cell autophagy process.
Fluorescence probe of the present invention is dissolved in the mother liquor that 1mM is made in DMSO, takes the mother liquor of 100 μ L in 10mL volumetric flasks
In, then with the solution to be measured of different pH value (phosphoric acid-acetic acid-boric acid solution system, it is molten by the way that 1mM hydrochloric acid or sodium hydroxide is added
Liquid is adjusted to different pH value) constant volume, it is configured to 10 μM.The excitation wavelength of fluorescence probe single photon and two-photon be respectively 370nm and
760nm detects the fluorescence spectrum variation in 390-700nm wave-length coverages, it can be seen that as the pH of buffer solution is from alkalinity
9.60 drop to acidity 3.20, and fluorescence maximum emission peak is from 410nm red shifts to 475nm, total 65nm, and fluorescence intensity red shift enhances.
The mechanism of action of fluorescence probe of the present invention is that the three-level nitrogen of the benzimidazole group in fluorescent probe molecule contains one
To lone electron pair, when test system pH value is smaller and larger, fluorescent probe molecule can exist in different forms, when probe point
Electronics can be different to the required excitation of excitation state by ground state transition when sub- light excitation-emission fluorescence, obtained fluorescence spectrum
Great changes have taken place.Lone pair electrons are combined to form a kind of organic salt structure with the proton in system in low ph value system, at this point, glimmering
The electron-withdrawing ability of light probe molecule enhances, and pushes and pulls the performance enhancement of electronics, it is required sharp that ground state electron transits to excitation state
Hair can reduce, and fluorescence emission spectrum Fa Sheng Red move fluorescence intensity and increase；Lone electron pair can not be with proton knot in high ph-values system
It closes, the conjugacy of entire fluorescent probe molecule is poor, pushes and pulls the reduced capability of electronics, and ground state electron transits to required for excitation state
Excitation can enhance, fluorescence intensity reduce.
Fluorescence probe of the present invention is simple in structure, and synthesis is convenient, easy to operate, is quick on the draw.With fluorescence power and color
Change the pH variations that can be used in detecting to detect the variation of microenvironment pH, and to cytotoxicity very little in cytase body,
And then monitor cell autophagy process.
The present invention by cell common location experiment firmly believe its can specificity target Cytolysosome, cytotoxicity test table
For the bright present invention for cell almost without what toxic side effect, the experiment of two-photon confocal fluorescent micro-imaging shows that the present invention is right
It is good in the permeability of MCF-7 cells, meanwhile, the pKa for being computed fluorescence probe of the present invention is 3.88, is very suitable for cytase
The monitoring of body pH variation ranges can monitor the feelings of cell autophagy process progress in real time by detecting the variation of Cytolysosome pH
Condition.
Description of the drawings
Fig. 1 is UV absorption light of the fluorescence probe Lyso-MPCB of the present invention (10 μM) in the buffer solution of different pH value
Spectrum.
Fig. 2 is fluorescence emissions of the fluorescence probe Lyso-MPCB of the present invention (10 μM) in the buffer solution of different pH value
Spectrogram, every line are all the tests immediately carried out after probe molecule is added.
Fig. 3 is that fluorescence probe Lyso-MPCB of the present invention is molten in the test adjusted back and forth with 1mM hydrochloric acid and sodium hydroxide solution
In liquid, measure fluorescent emission spectrogram respectively as pH=4.2 and pH=7.2, then calculate fluorescence emission wavelengths be 475nm and
Fluorescence intensity ratio (I under 410nm475nm/I410nm) cycle figure.
Fig. 4 is that fluorescence probe Lyso-MPCB (0.1mM) of the present invention wavelength different in the buffer solution of different pH value swashs
Give two photon absorption cross section value.
Fig. 5 is cell survival rates of the fluorescence probe Lyso-MPCB of the present invention after MCF-7 cell culture for 24 hours.
Fig. 6 is lysosome positioning images of the fluorescence probe Lyso-MPCB of the present invention in MCF-7 cells.Probe
Lyso-MPCB (10 μM), which is added in MCF-7 cells, to be cultivated 30 minutes, and lysosome dyestuff is then added thereto again
LysoTracker Red FM (0.5 μM) continue culture 10 minutes.Wherein figure (a) green channel (460-490nm), λex=
760nm；(b) red channel (580-620nm), λex=559nm；(c) be (a) and the channel (b) stacking chart；(d) be (a) and
(b) channel corresponds to the Discrete point analysis of fluorescence intensity.
Fig. 7 is lured with starvation after (10 μM) of fluorescence probe Lyso-MPCB molecules culture MCF-7 cells 0.5 hour of the present invention
Lead change in fluorescence situation of the autophagy after 4 hours.Wherein, figure (a1-2) blue channel (390-420nm)；(b1-2) green channel
(460-490nm)；(c1-2) it is light field；(d1) it is the stacking chart in the channel (a1), (b1) and (c1)；(d2) be (a2), (b2) and
(c2) stacking chart in channel.
Specific implementation mode
Technical scheme of the present invention is described further below by specific embodiment.
Embodiment 1：The synthesis of fluorescent probe molecule Lyso-MPCB
Compound A1g, o-phenylenediamine 0.46g, p-methyl benzenesulfonic acid 0.0082g and N,N-dimethylformamide 30mL are added
Enter into three-necked flask, 120 DEG C and logical argon gas protection reaction 12h are warming up in oil bath pan；Liquid is spin-dried for after reaction,
It is extracted with dichloromethane and water, collect upper organic phase and is spin-dried for obtaining crude product；By gained crude product sample preparation column chromatography color
Post separation is composed, target product is obtained and obtains target product 0.7g (1.28mmoL), yield 60%.The structural formula of the compound A
For：
1H NMR(400MHz,DMSO-d6) δ 12.84 (s, 1H), 9.04 (s, 1H), 8.41 (s, 1H), 8.33 (d, J=
8.6Hz, 1H), 7.84 (d, J=8.7Hz, 1H), 7.73 (d, J=8.5Hz, 1H), 7.65 (d, J=8.5Hz, 2H), 7.55 (t,
J=11.5Hz, 3H), 7.20 (dd, J=5.5,2.6Hz, 2H), 7.02 (d, J=8.3Hz, 2H), 4.49 (t, J=7.0Hz,
2H),3.82(s,3H),3.53(s,4H),2.30(s,6H),1.89-1.79(m,2H),1.53-1.47(m,2H).13C NMR
(101MHz,DMSO-d6)δ160.23,153.36,142.25,141.14,133.69,130.29,125.90,124.60,
123.28,122.87,122.62,120.14,115.89,115.39,114.33,111.23,111.09,90.22,88.74,
67.07,58.31,56.25,54.12,43.37,30.00,27.13.
Embodiment 2：The fluorometric investigation and two-photon of fluorescent probe molecule are tested
Fluorescence probe Lyso-MPCB of the present invention is dissolved in the mother liquor that 1mM is made in DMSO, take the mother liquor of 100 μ L in
In 10mL volumetric flasks, then with the buffer solutions of different pH value (phosphoric acid-acetic acid-boric acid solution system, by be added 1mM hydrochloric acid or
Sodium hydroxide solution is adjusted to different pH value) constant volume, it is configured to 10 μM.The excitation wavelength of fluorescence probe single photon and two-photon is distinguished
For 370nm and 760nm, the fluorescence spectrum variation in 390-700nm wave-length coverages is detected.And it is corresponding by different pH value
I475nm/I410nmCalculate the pKa value (3.88) of probe.
Fluorescence probe Lyso-MPCB works as pH=in the test solution adjusted back and forth with 1mM hydrochloric acid and sodium hydroxide solution
Fluorescent emission spectrogram is measured when 4.2 and pH=7.2 respectively, then it is fluorescence intensity under 475nm and 410nm to calculate fluorescence emission wavelengths
Ratio (I475nm/I410nm) cycle figure (Fig. 3), cycle-index be 6 times.
Using two-photon measuring technology, two-photon absorption of the test fluorescence probe (Lyso-MPCB) in pH=3.2 is cut
Face, from fig. 4, it can be seen that maximum two photon absorption cross section of the fluorescent probe molecule in pH=3.2 is 335GM, two-photon swashs
Wavelength is sent out in 760nm.
Embodiment 3：Cytotoxicity test
MTT (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides) experiments are the articles that basis has been reported,
Do some cytotoxicity tests.Be added 0,5,10,15,20 μM of fluorescence probe in same a collection of cell respectively, this condition be
37 DEG C, contain 5%CO2Cell incubator in be incubated 24 hours, according to the formula of cell survival degree：Cell survival rate %=
OD570 (samples)/OD570 (control groups)× 100, cell survival rate (Fig. 5) is obtained finally.It will be seen that a concentration of 20 μM from Fig. 5
When, cell survival rate also has 90% or so, illustrates that fluorescence probe of the present invention acts on cytotoxic, therefore can be used for examining
Survey the viscosity in cell.
Embodiment 4：Cellular localization is tested
MCF-7 cells are imaged the previous day, it is total that MCF-7 cells are put in laser by DEME (invitrogen) culture solution culture
Focus in ware, when imaging the DMSO solution of MCF-7 cells and 10 μM of fluorescence probe Lyso-MPCB in 37 DEG C, contain 5%CO2's
It is incubated 0.5 hour in cell incubator, after washing 3 times with neutral PBS buffer solutions, then 0.5 μM of quotient is added into culture dish
Product lysosome coloring agent LysoTracker Red FM solution continues to be incubated 0.5 hour, is washed with neutral PBS buffer solutions
3 times.With two-photon fluorescence co-focusing imaging, green channel tracker1, excitation wavelength 760nm are set, emission band is
460-490nm, this channel are used for receiving the fluorescence of probe molecule Lyso-MPCB transmittings.Red channel tracker2 is set, is swashed
Hair wavelength is 559nm, and launch wavelength 580-620nm, this channel is used for receiving commercialization lysosome coloring agent
The fluorescence (Fig. 6) of LysoTracker Red FM transmittings.
Embodiment 5：Cell autophagy monitors
MCF-7 cells are imaged the previous day by DEME (invitrogen) culture solution culture, and MCF-7 cells are put in flat table
In the ware of face, when imaging the DMSO solution of MCF-7 cells and 10 μM of fluorescence probe Lyso-MPCB in 37 DEG C, contain 5%CO2It is thin
It is incubated 0.5 hour in born of the same parents' incubator, after fully being washed with neutral PBS buffer solutions or culture solution, changes culture medium into HBSS
(starvation media of autophagy process occurs for inducing cell).Then, start when (0h) and processing a period of time (4h) after, use
Two-photon fluorescence co-focusing imaging, obtains Fig. 7.Wherein, figure (a1-2) blue channel (390-420nm)；(b1-2) green channel
(460-490nm)；(c1-2) it is light field；(d1) it is the stacking chart in the channel (a1), (b1) and (c1)；(d2) be (a2), (b2) and
(c2) stacking chart in channel.
Claims (7)
1. a kind of two-photon pH ratios of monitoring cell autophagy measure fluorescence probe, it is characterised in that：It is the 4- using carbazole as parent
Methoxy-phenylacetylene is electron-donating group, and lysosome seeking group is morpholine, and benzimidazole is the response group of pH.
2. fluorescence probe according to claim 1, it is characterised in that its structural formula is as follows：
3. a kind of preparation method of the two-photon pH ratios metering fluorescence probe of monitoring cell autophagy as claimed in claim 1 or 2,
It is characterized by comprising following steps：
Compound A 1g, o-phenylenediamine 0.46g, p-methyl benzenesulfonic acid 0.0082g and N,N-dimethylformamide 30mL are added
Into three-necked flask, it is warming up to 120 DEG C and logical argon gas protection reaction 12h；Liquid is spin-dried for after reaction, with dichloromethane and
Water extracts, and collects upper organic phase and is spin-dried for obtaining crude product；By gained crude product sample preparation column chromatography chromatogram post separation, obtain
Target product；
The structural formula of the compound A is：
4. preparation method according to claim 3, it is characterised in that：
Eluent used is dichloromethane and methanol by volume 100 when column chromatography chromatogram post separation:1 is mixed to get.
5. a kind of purposes of the two-photon pH ratios metering fluorescence probe of monitoring cell autophagy as claimed in claim 1 or 2, special
Sign is：When autophagy lysosomal pH in detecting cell changes as detection reagent application.
6. a kind of application of the two-photon pH ratios metering fluorescence probe of monitoring cell autophagy as claimed in claim 1 or 2, special
Sign is：The fluorescence probe monitors the journey of cell autophagy progress as detection reagent by detecting Cytolysosome pH variations
Degree.
7. application according to claim 6, it is characterised in that include the following steps：
The fluorescence probe is dissolved in the mother liquor that 1mM is made in DMSO, takes the mother liquor of 100 μ L in 10mL volumetric flasks, then use
Solution constant volume to be measured, passes through sense channel one：390~420nm and channel two：Fluorescence light in 460~490nm wave-length coverages
Spectrum peak ratiometer amount changes I475nm/I410nmRealize the pH variations of quantitatively detection cell autophagy lysosome, it is thin to reach monitoring
The purpose of born of the same parents' autophagy process.
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| CN111004246A (en) * | 2019-12-13 | 2020-04-14 | 山西大学 | Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy, preparation and application thereof |
| CN111100627A (en) * | 2019-12-20 | 2020-05-05 | 中国科学院化学研究所 | Fluorescent probe and application thereof |
| CN113429335A (en) * | 2021-06-25 | 2021-09-24 | 安徽大学 | Lysosome targeted dual-response two-photon fluorescence probe and preparation method and application thereof |
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Cited By (5)
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| CN111004246A (en) * | 2019-12-13 | 2020-04-14 | 山西大学 | Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy, preparation and application thereof |
| CN111004246B (en) * | 2019-12-13 | 2021-03-30 | 山西大学 | Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy, preparation and application thereof |
| CN111100627A (en) * | 2019-12-20 | 2020-05-05 | 中国科学院化学研究所 | Fluorescent probe and application thereof |
| CN113429335A (en) * | 2021-06-25 | 2021-09-24 | 安徽大学 | Lysosome targeted dual-response two-photon fluorescence probe and preparation method and application thereof |
| CN113429335B (en) * | 2021-06-25 | 2023-05-16 | 安徽大学 | Lysosome-targeted dual-response two-photon fluorescent probe and preparation method and application thereof |
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