CN108181272A - Micro Fe in a kind of binary channels fluorescence imaging detection living cells3+、Hg2+And Cu2+Method - Google Patents

Micro Fe in a kind of binary channels fluorescence imaging detection living cells3+、Hg2+And Cu2+Method Download PDF

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CN108181272A
CN108181272A CN201710836801.5A CN201710836801A CN108181272A CN 108181272 A CN108181272 A CN 108181272A CN 201710836801 A CN201710836801 A CN 201710836801A CN 108181272 A CN108181272 A CN 108181272A
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曾晞
张红
方浚安
廖贤
牟兰
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Guizhou University
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Abstract

The present invention discloses micro Fe in a kind of binary channels fluorescence imaging detection living cells3+、Hg2+And Cu2+Method, be with triamine ethylamine derivative, abbreviation probe, as binary channels fluorescence imaging detect competent cell in micro Fe3+、Hg2+、Cu2+Fluorescence imaging probe, pass through binary channels fluorescence imaging detect competent cell in micro Fe3+、Hg2+And Cu2+;The method of the present invention can realize the detection to multiple target ion, probe and Fe in the present invention3+、Hg2+And Cu2+It is respectively formed probe Fe in the cell3+, probe Hg2+, probe Cu2+Complex emits different wave length intense fluorescence, easy to detect, visual good, and high sensitivity, selectivity are good, can realize that real-time online detects intracellular Fe3+、Hg2+、Cu2+Ion.

Description

Micro Fe in a kind of binary channels fluorescence imaging detection living cells3+、Hg2+And Cu2+'s Method
Technical field
The present invention relates to a kind of method that binary channels fluorescence imaging detects micro ion in living cells, particularly a kind of bilaterals Road fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method.
Background technology
In cell biology and medical research, fluorescence probe is because of its high-space resolution ability and minimum limit disturbance live body Feature, researcher can allow for real-time monitor the event betided in living cells and biological living, as the most powerful One of tool is increasingly paid attention to and is used.Probe is as research metal ion transport vehicle and in drug delivery system The leap behavior of cell membrane is particularly important.Fluorescence imaging detection intracellular ion is capable of providing the when space division of life system intermediate ion The most direct information of cloth is tracking metal ion in biosystem due to features such as the non-invasive, highly sensitive of it, high selections One of most reliable technology.
Iron is basic element important in human body, be that most of organisms are essential and human body in it is most abundant Transition metal.The transmission of oxygen is played in physiology course, the important function such as electronics transfer and enzymatic are capable of providing ferrous blood red The carrying capacity of oxygen in element is used as a kind of co-factor in many enzyme reactions.The level and the state of oxidation of in situ detection iron are to explaining Its bright effect is significantly.Due to Fe3+Paramagnetism lead to that fluorescent quenching, therefore Fe can be caused3+Fluorescence imaging lead to Often it is difficult.
Mercury pollution extends over the entire globe, and residual influences health and Environmental security very big.Either Elemental Mercury or nothing The bacilli shift that machine mercury can be transferred through in environment generates biological accumulation into methyl mercury, and then by food chain.Moreover, mercury and its spreading out The extreme toxicity of biology is attributed to its high mercapto compatibility in protein and enzyme, makes cell functional disorders that health be caused to be asked Topic.Due to the harmfulness of mercury, simply, the analysis method of the sensitive and quick mercury for measuring minor levels is necessary.
Copper is located at the most abundant basic element after iron, zinc as content in human body, is played in various physiology courses Important function.Copper ion, as cofactors, has the ability that other metal ions are replaced in substitution, when it in enzymic catalytic reaction Contents level is more than that will generate toxicity to biosystem needed for cell.Copper ion more than normal level content can cause to be poisoned And it generates oxidative stress and phrenoblabia etc. and includes Alzheimer's disease, op parkinson's disease.
Probe as cell imaging detection trace metal ion has many reports, but be Single probe mostly to single mesh Ion detection is marked, there is detection inconvenience, poor selectivity, it is impossible to realize efficient inspection of the single probe to multiple target ion in cell The problems such as survey.
Invention content
The object of the present invention is to provide a kind of binary channels fluorescence imagings to detect micro Fe in living cells respectively3+、Hg2+With Cu2+Method, the method for the present invention can realize the detection to multiple target ion, probe and Fe in the present invention3+、Hg2+、Cu2+Thin Intracellular is respectively formed probe-Fe3+, probe-Hg2+, probe-Cu2+Complex emits different wave length intense fluorescence, easy to detect, Visual good, high sensitivity, selectivity are good, can realize that real-time online detects intracellular Fe3+、Hg2+、Cu2+Ion.
Technical scheme of the present invention:A kind of binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+ Method, be using a kind of triamine ethylamine derivative as probe, detected in competent cell respectively by binary channels fluorescence imaging Micro Fe3+、Hg2+And Cu2+;The chemical structural formula of the probe is:
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Using a kind of triamine ethylamine derivative as probe, micro Fe in competent cell is detected by binary channels fluorescence imaging respectively3+、 Hg2+And Cu2+;That first to enter probe with probe and cell hatching intracellular, then by hatch have probe cell respectively with Fe3+、 Hg2+、Cu2+Hatching, make probe respectively with Fe3+、Hg2+、Cu2+The spy of characteristic wavelength fluorescence can be emitted with reference to generation in the cell Needle-Fe3+, probe-Hg2+, probe-Cu2+Complex is realized that probe dyes intracellular ion and is imaged, uses fluorescence inverted microscope Competent cell fluorescence picture after observation hatching;Specifically include following steps:
(1) cell culture:Competent cell is inoculated in through recovery in culture solution, is 37 DEG C in temperature, 5%CO2And saturation is wet It spends in the incubator for 100% and cultivates, after passage, be inoculated in 12 orifice plates and cultivate, density is 2 × 104A/ml is secondary daily new Fresh culture solution cleans cell;The culture solution is containing 10% fetal calf serum, 1% dual anti-and 89% modified form RPMI1640 cultures Base;
(2) probe is to cell incubation:It will be added in the incubation culture solution containing 5~40 μM of probes in the cell of step (1), it should The composition of culture solution is incubated as 90% culture solution, 8%H2O, 2% acetonitrile, incubator is interior to be incubated 30~80min, is sucked out containing probe Incubation culture solution, clean cell with fresh culture solution, be placed under fluorescence inverted microscope and carry out light field and details in a play not acted out on stage, but told through dialogues respectively and clap According to observing cell clearly image under light field, cell image be not observed under details in a play not acted out on stage, but told through dialogues;
(3)Fe3+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Fe3+Dyeing culture solution, the dye The composition of color culture solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Fe3+Dyeing culture Liquid cleans cell with fresh culture solution, is placed under the red channel of fluorescence inverted microscope and observes probe to intracellular Fe3+Dye Bright red fluorescence is presented in fluorescence picture after color, cell, and shooting obtains clearly red fluorescent cell contour image;It again will be upper It states through probe and Fe3+Cell after dyeing respectively, which is placed under the green channel of fluorescence inverted microscope, observes probe to intracellular Fe3+Fluorescence picture after dyeing, cell are in green fluorescence, and shooting obtains green cells contour image, and probe is in competent cell In detect micro Fe3+Ion;
(4)Hg2+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Hg2+Dyeing culture solution, the dye The composition of color culture solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Hg2+Dyeing culture Liquid cleans cell with fresh culture solution, is placed under the red channel of fluorescence inverted microscope and observes probe to intracellular Hg2+Dye Bright red fluorescence is presented in fluorescence picture after color, cell, and shooting obtains clearly red fluorescent cell contour image;It again will be upper It states through probe and Hg2+Cell after dyeing respectively, which is placed under the green channel of fluorescence inverted microscope, observes probe to intracellular Hg2+Fluorescence picture after dyeing, cell are in green fluorescence, and shooting obtains green cells contour image, and probe is in competent cell In detect micro Hg2+Ion;
(5)Cu2+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Cu2+Dyeing culture solution, the dye The composition of color culture solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Cu2+Dyeing culture Liquid cleans cell with fresh culture solution, is placed under the green channel of fluorescence inverted microscope and observes probe to intracellular Cu2+Dye Fluorescence picture after color, cell obtain clearly green cells contour image in bright green fluorescence, shooting, and probe is in work Micro Cu is detected in property cell2+Ion.
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Fluorescence inverted microscope;Be green channel excitation wavelength for 450nm~490nm, the excitation wavelength of red channel for 510nm~ The fluorescence inverted microscope of 550nm.
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Modified form RPMI1640 culture mediums;Be RPMI be English RoswellParkMemorialInstitute abbreviation, acute pyogenic infection of finger tip Loews Parker's memorial institute is tieed up, RPMI is a kind of cell culture medium of research institute research and development, and 1640 be culture medium code name.
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Competent cell is;Human prostate's cancer cell, abbreviation PC3 cells.
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Probe;It is to be synthesized by following synthetic routes:
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Probe;It is prepared:In the three-necked flask of 100ml, three (2- aminoethyls) amine 25-30mmol, rhodamine B 2- are added in The absolute ethyl alcohol of 5mmol and 55-65ml, the lower reflux 33-39h of nitrogen protection, decompression boils off ethyl alcohol, respectively with 100ml dichloromethanes Alkane extracts 3 times, and organic phase is dried overnight with anhydrous magnesium sulfate, boils off solvent, obtains red thick shape object, and silica gel column chromatography detaches, Eluent is methanol/chloroform/triethylamine that volume ratio is 9/1/1, obtains colorless viscous shape intermediate 1;At three mouthfuls of 250ml In bottle, intermediate 1,115-125ml chloroforms and the chloro- 7- nitros benzofuraxans of 2-8mmol4- are added in, nitrogen is protected under ice bath 1.5-2.5h is reacted, boils off solvent, silica gel column chromatography separation, eluent is chloroform/methanol that volume ratio is 100/3, is obtained Light yellow solid intermediate 2;In the there-necked flask of 100ml, intermediate 2,0.66-0.85mmol7- hydroxyl -8- cumarins are added in Formaldehyde and 55-65ml methanol, the lower back flow reaction 7-9h of nitrogen protection, boil off solvent, silica gel column chromatography separation, eluent is volume Than chloroform/methanol for 100/2, orange solids probe is obtained.
Aforementioned binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method in, it is described Probe;It is prepared:In the three-necked flask of 100ml, three (2- aminoethyls) amine 27.36mmol, rhodamine are added in The absolute ethyl alcohol of B3.42mmol and 60ml, the lower reflux 36h of nitrogen protection, decompression boils off ethyl alcohol, respectively with 100ml dichloromethane Extraction 3 times, organic phase is dried overnight with anhydrous magnesium sulfate, boils off solvent, obtains red thick shape object, and silica gel column chromatography separation is washed De- liquid is methanol/chloroform/triethylamine that volume ratio is 9/1/1, obtains 1.71g colorless viscous shapes intermediate 1;250ml's In there-necked flask, 6.01mmol intermediates 1,120ml chloroforms and the chloro- 7- nitros benzofuraxans of 5.01mmol4-, nitrogen are added in 2h is reacted under protection ice bath, boils off solvent, silica gel column chromatography separation, eluent is chloroform/first that volume ratio is 100/3 Alcohol obtains light yellow solid intermediate 2;In the there-necked flask of 100ml, intermediate 2,0.748mmol7- hydroxyl -8- cumarins are added in Formaldehyde and 60ml methanol, the lower back flow reaction 8h of nitrogen protection, boil off solvent, silica gel column chromatography separation, eluent is that volume ratio is 100/2 chloroform/methanol, obtains orange solids probe.
Inventor has carried out the method for the present invention long-term a large amount of experimental study, is part test below:
1st, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is not added with metal ion or adds in 400 μM of metal ion Al respectively3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Sr2+, Hg2+, Pb2+, Cd2+, Zn2+, Co2+, Ni2+, Cu2+, Ag+, Fe3+Fluorescence spectrum afterwards.See Fig. 1, Fe3+、Hg2+Plus Enter to make probe to occur new fluorescence peak at 585nm, and fluorescence significantly increases;Cu2+Addition make fluorescence of the probe at 525nm It significantly increases, and the addition of other above-mentioned Experiment Metal ions does not change the fluorescence spectrum and intensity of probe, shows at this Under part, probe energy selective enumeration method Fe3+、Hg2+And Cu2+.The excitation wavelength of test be 470nm, launch wavelength 585nm, 525nm。
2nd, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Fe3+The fluorescence spectroscopic titration curve measured.With Fe3+Concentration increases, Fluorescence intensity at 585nm linearly enhances.The excitation wavelength of test is 470nm.See Fig. 2.
3rd, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Fe3+, measure fluorescence intensity at 585nm and obtain calibration curve.Ordinate is Fluorescence intensity level, abscissa Fe3+Concentration.Excitation wavelength is 470nm.See Fig. 3.
4th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into 400 μM of metal ion Al3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Sr2+, Hg2+, Pb2+, Cd2+, Zn2+, Co2+, Ni2+, Cu2+, Ag+, Fe3+Afterwards, the fluorescence intensity level at 585nm is measured.Again respectively to probe-Fe3+ After adding in 400 μM of other above-mentioned metal ions in mixed solution, the variation of the fluorescence intensity level at 585mn is measured.See Fig. 4, it is black Vitta is represented in probe-Fe3+The fluorescence intensity level after metal ion at 585mn is separately added into mixed solution again;White bars Represent the variation of the fluorescence intensity level after probe solution is separately added into other above-mentioned coexistent metallic ions at 585nm.Show Probe in detecting Fe3+Fluorescence intensity remove by Hg2+Outside influencing, not coexisted by other above-mentioned ions is influenced.The excitation wavelength of test For 470nm.
5th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Hg2+The fluorescence spectroscopic titration curve measured.With Hg2+Concentration increases, Fluorescence intensity at 585nm linearly enhances.The excitation wavelength of test is 470nm.See Fig. 5.
6th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Hg2+, measure fluorescence intensity at 585nm and obtain calibration curve.Ordinate is Fluorescence intensity level, abscissa Hg2+Concentration.Excitation wavelength is 470nm.See Fig. 6.
7th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into 400 μM of metal ion Al3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Sr2+, Hg2+, Pb2+, Cd2+, Zn2+, Co2+, Ni2+, Cu2+, Ag+, Fe3+Afterwards, the fluorescence intensity level at 585nm is measured.Again respectively to probe-Hg2+ After adding in 400 μM of other above-mentioned metal ions in mixed solution, the variation of the fluorescence intensity level at 585mn is measured.See Fig. 7, it is black Vitta is represented in probe-Hg2+The fluorescence intensity level after metal ion at 585mn is separately added into mixed solution again;White bars Represent the variation of the fluorescence intensity level after probe solution is separately added into other above-mentioned coexistent metallic ions at 585nm.Show Probe in detecting Hg2+Fluorescence intensity do not coexisted and influenced by above-mentioned ion.The excitation wavelength of test is 470nm.
8th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Cu2+The fluorescence spectroscopic titration curve measured.With Cu2+Concentration increases, Fluorescence intensity at 525nm linearly enhances.The excitation wavelength of test is 470nm.See Fig. 8.
9th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into various concentration Cu2+, measure fluorescence intensity at 525nm and obtain calibration curve.Ordinate is Fluorescence intensity level, abscissa Cu2+Concentration.Excitation wavelength is 470nm.See Fig. 9.
10th, the probe prepared by embodiment 1 is dissolved in the acetonitrile/water solution that volume ratio is 97/3, is configured to probe A concentration of 20 μM of solution is separately added into 400 μM of metal ion Al3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Sr2+, Hg2+, Pb2+, Cd2+, Zn2+, Co2+, Ni2+, Cu2+, Ag+, Fe3+Afterwards, the fluorescence intensity level at 525nm is measured.Again respectively to probe-Cu2+ After adding in 400 μM of other above-mentioned metal ions in mixed solution, the variation of the fluorescence intensity level at 525mn is measured.See Figure 10, it is black Vitta is represented in probe-Cu2+The fluorescence intensity level after metal ion at 525mn is separately added into mixed solution again;White bars Represent the variation of the fluorescence intensity level after probe solution is separately added into other above-mentioned coexistent metallic ions at 525nm.Show Probe in detecting Cu2+Fluorescence intensity do not coexisted and influenced by above-mentioned ion.The excitation wavelength of test is 470nm.
11st, probe is to Fe in active PC3 cells3+Fluorescence imaging detection photo, see Figure 11;Figure 11-a, Figure 11-d be through The photograph via bright field of the fluorescence inverted microscope of PC3 cells after a concentration of 20 μM of probe incubation 50min, cell is adherent normal, In full state, it was demonstrated that probe does not have toxicity to PC3 cells in this test condition;Figure 11-b, Figure 11-e are above-mentioned through probe The details in a play not acted out on stage, but told through dialogues fluorescence micrograph for the PC3 cells being incubated, shows acellular fluorescence imaging;Figure 11-c are above-mentioned first through 20 μM of spy Needle is incubated the PC3 cells of 50min, then with 50 μM of Fe3+After being incubated 50min, shot under fluorescence inverted microscope red channel Cell picture, observe clearly red fluorescent cell distribution, it was demonstrated that probe and Fe3+Ion realizes dyeing in the cell, Cell dyeing fluorescence imaging is presented, red channel excitation wavelength is 510nm~550nm.Figure 11-f are above-mentioned first through 20 μM of spy Needle is incubated the PC3 cells of 50min, then with 50 μM of Fe3+After being incubated 50min, shot under fluorescence inverted microscope green channel Cell picture, observe clearly green cells distribution, it was demonstrated that probe and Fe3+Ion realizes dyeing in the cell, Cell dyeing fluorescence imaging is presented, the excitation wavelength of green channel is 450nm~490nm.
12nd, probe is to Hg in active PC3 cells2+Fluorescence imaging detection photo, see Figure 12;Figure 12-a, Figure 12-d be through The photograph via bright field of the fluorescence inverted microscope of PC3 cells after a concentration of 20 μM of probe incubation 50min, cell is adherent normal, In full state, it was demonstrated that probe does not have toxicity to PC3 cells in this test condition;Figure 12-b, 12-e are incubated to be above-mentioned through probe The details in a play not acted out on stage, but told through dialogues fluorescence micrograph for the PC3 cells educated, shows acellular fluorescence imaging;Figure 12-c are above-mentioned first through 20 μM of probe It is incubated the PC3 cells of 50min, then with 50 μM of Hg2+After being incubated 40min, shot under fluorescence inverted microscope red channel Cell picture observes clearly red fluorescent cell distribution, it was demonstrated that probe and Hg2+Ion realizes dyeing in the cell, is in Existing cell dyeing fluorescence imaging, red channel excitation wavelength are 510nm~550nm.Figure 12-f are above-mentioned first through 20 μM of probe It is incubated the PC3 cells of 50min, then with 50 μM of Hg2+After being incubated 40min, shot under fluorescence inverted microscope green channel Cell picture observes clearly green cells distribution, it was demonstrated that probe and Hg2+Ion realizes dyeing in the cell, is in Existing cell dyeing fluorescence imaging, the excitation wavelength of green channel is 450nm~490nm.
13rd, probe is to Cu in active PC3 cells2+Fluorescence imaging detection photo, see Figure 13;Figure 13-a are through a concentration of 20 μM probe be incubated the PC3 cells after 50min fluorescence inverted microscope photograph via bright field, cell is adherent normal, in full shape State, it was demonstrated that probe does not have toxicity to PC3 cells in this test condition;13-b is the above-mentioned PC3 cells being incubated through probe Details in a play not acted out on stage, but told through dialogues fluorescence micrograph shows acellular fluorescence imaging;13-c is that the above-mentioned PC3 that 50min is first incubated through 20 μM of probe is thin Born of the same parents, then with 50 μM of Cu2+After being incubated 50min, the cell picture that is shot under fluorescence inverted microscope green channel observes clear Clear green cells distribution, it was demonstrated that probe and Cu2+Ion realizes dyeing in the cell, present cell dyeing fluorescence into Picture, the excitation wavelength of green channel is 450nm~490nm.
Compared with prior art, the invention has the advantages that:
(1) selection is in acetonitrile/water blending agent, using 470nm as excitation wavelength, probe Selective recognition Fe3+、Hg2+、 Cu2+.Probe-Fe3+, probe-Hg2+Emit 585nm fluorescent oranges, probe-Cu2+Emit 525nm green fluorescences, utilize spy accordingly Needle realizes intracellular Fe3+、Hg2+、Cu2+Fluorescence imaging;
(2) excitation wavelength is 470nm and launch wavelength is 585nm, 525nm, Stokes shift (Stokesshift), Greatly, influence of the excitation wavelength to launch wavelength is not only eliminated, while makes the wavelength of fluorescence of imaging cells close near infrared band And it can realize Dual channel detection;
(3) it is used as a kind of intracellular micro Fe3+、Hg2+、Cu2+Fluorescence imaging method, can simultaneously in fluorescence exciting wavelength Fluorescence imaging is realized under the green channel that red channel and fluorescence exciting wavelength for 510nm~550nm are 450nm~490nm. As micro Fe in a kind of living cells3+、Hg2+、Cu2+Imaging agents, there is easy to detect, visual strong, high sensitivity, choosing Selecting property feel better other ions interference it is small, it can be achieved that real-time online detect.
(4) it is synthesized by inventor a kind of to Fe3+、Hg2+、Cu2+The fluorescence probe of highly sensitive, highly selective detection is visited Needle elder generation and cell incubation make probe penetrate into the cell, then have the cell of probe respectively to Fe with incubation3+、Hg2+、Cu2+Into Row is incubated, and makes probe and Fe3+、Hg2+、Cu2+Complex is formed in the cell, and fluorescence imaging is carried out under fluorescence inverted microscope Detection, lot of trace metal cations Fe in being realized under two excitation channels to living cells respectively3+、Hg2+、Cu2+Difference The fluorescent staining of color and image checking.
Therefore, the method for the present invention middle probe and Fe3+、Hg2+、Cu2+Mating capability it is good, easy to detect, visual good, spirit Sensitivity is high, selectivity is good, and the interference by other ions is small, and can realize that real-time online detects.
Description of the drawings:
Fig. 1 is probe in detecting Fe3+、Hg2+、Cu2+Fluorescence spectra;
Fig. 2 is the Fe of various concentration3+To the fluorescence titration spectrogram of probe;
Fig. 3 is probe in detecting Fe3+Fluorescent spectrometry calibration curve;
Fig. 4 is coexistent metallic ion to probe in detecting Fe3+Fluorescence intensity influence figure;
Fig. 5 is the Hg of various concentration2+To the fluorescence titration spectrogram of probe;
Fig. 6 is probe in detecting Hg2+Fluorescent spectrometry calibration curve;
Fig. 7 is coexistent metallic ion to probe in detecting Hg2+Fluorescence intensity influence figure;
Fig. 8 is the Cu of various concentration2+To the fluorescence titration spectrogram of probe;
Fig. 9 is probe in detecting Cu2+Fluorescent spectrometry calibration curve;
Figure 10 is coexistent metallic ion to probe in detecting Cu2+Fluorescence intensity influence figure;
Figure 11 is probe to Fe in active PC3 cells3+Fluorescence imaging detection photo;A, d is that cell is incubated through probe solution Photograph via bright field after educating;B corresponds to fluorescence photo of the cell of a under fluorescence inverted microscope red channel;E is corresponded to Fluorescence photo of the cell of d under fluorescence inverted microscope green channel;C is the cell being first incubated through probe, then through Fe3+It is incubated Afterwards, the cell photo shot under fluorescence inverted microscope red channel;F is the cell being first incubated through probe, then through Fe3+It is incubated Afterwards, the cell photo shot under fluorescence inverted microscope green channel.
Figure 12 is probe to Hg in active PC3 cells2+Fluorescence imaging detection photo;A, d is that cell is incubated through probe solution Photograph via bright field after educating;B corresponds to fluorescence photo of the cell of a under fluorescence inverted microscope red channel;E is corresponded to Fluorescence photo of the cell of d under fluorescence inverted microscope green channel;C is the cell being first incubated through probe, then through Hg2+It is incubated Afterwards, the cell photo shot under fluorescence inverted microscope red channel;F is the cell being first incubated through probe, then through Hg2+It is incubated Afterwards, the cell photo shot under fluorescence inverted microscope green channel.
Figure 13 is probe to Cu in active PC3 cells2+Fluorescence imaging detection photo;A is that cell is incubated through probe solution Photograph via bright field afterwards;B corresponds to fluorescence photo of the cell of a under fluorescence inverted microscope green channel;C is first through probe The cell of incubation, then through Cu2+After incubation, the cell photo that is shot under fluorescence inverted microscope green channel.
Specific embodiment
Embodiment 1:
The synthetic route of probe:
The specific synthetic method of probe:
1) synthesis of intermediate 1:In the three-necked flask of 100ml, add in 27.36mmol three (2- aminoethyls) amine, 3.42mmol rhodamine B and 60ml absolute ethyl alcohol, the lower reflux 36h of nitrogen protection, decompression boils off ethyl alcohol, uses 100ml respectively Dichloromethane extracts 3 times, and organic phase is dried overnight with anhydrous magnesium sulfate, boils off solvent, obtains red thick shape object, silica gel column chromatography Separation, eluent is methanol/chloroform/triethylamine that volume ratio is 9/1/1, obtains 1.71g colorless viscous shapes intermediate 1;
2) synthesis of intermediate 2:In the there-necked flask of 250ml, intermediate 1,120ml chloroforms and 5.01mmol are added in The chloro- 7- nitros benzofuraxans of 4-, reacts 2h under nitrogen protection ice bath, boils off solvent, silica gel column chromatography separation, eluent is body Product obtains light yellow solid intermediate 2 than chloroform/methanol for 100/3;
3) synthesis of probe:In the there-necked flask of 100ml, the 7- hydroxyl -8- cumarin first of intermediate 2,0.748mmol is added in Aldehyde, 60ml methanol, the lower back flow reaction 8h of nitrogen protection boil off solvent, silica gel column chromatography separation, eluent is that volume ratio is 100/ 2 chloroform/methanol, obtains orange solids probe.Structural characterization data are as follows:IR(KBr,νcm-1):3422(-OH),2025 (C=O), 1599 (C=C), 1388 (N-CH3),1360(C-O-C),764(Ar-H),624(Ar-H),1HNMR(500M, CDCl3,ppm)δ:10.60 (s, 1H ,-OH), 8.68 (s, 1H ,-CH=N), 8.32 (s, 1H, Ar-H), 8.02 (s, 1H, Ar- ), H 7.66 (d, J=10Hz, 1H, Ar-H), 7.52 (m, J=6Hz, 2H, Ar-H), 7.45 (d, J=9.5Hz, 2H, Ar-H × 2), 7.1 (d, J=6.5,1H, Ar-H), 7.08 (d, J=9Hz, 1H, Ar-H), 6.4 (m, J=8.5Hz, 2H, Ar-H), 6.3 (m, J=10.5Hz, 4H, Ar-H), 6.1 (d, J=9.5Hz, 1H, Ar-H), 3.34~3.29 (m, 8H, NCH2CH3×4),3.25 (s,2H,NCH2CH2-),2.85(s,2H,CH2CH2N),2.70(s,2H,-CH2CH2N),1.75(bs,CH2NH2).13CNMR (500M,CDCl3,ppm)δ:206.83,190.61,166.68,159.85,159.49,156.30,148.22,145.47, 144.86,144.20,143.62,137.60,135.75,133.65,130.96,128.67,123.89,122.42,120.59, 119.30,108.29,107.68,104.96,104.78,103.08,98.94,97.13,78.98,64.42,52.20, (51.57,50.76,49.24,45.72,43.73.MS ESI/TOF-Q) calculated value [C50H51N9O8]:M/z905.39215 is measured Value:m/z906.39436[M+H]+
2nd, the preparation of reagent:
(1) preparation of probe solution:The probe 9.1mg prepared as stated above is weighed, is dissolved, is configured to acetonitrile 100mL(CH3CN/H2O, 1/4) concentration and probe concentration is 1.00 × 10-4The solution of M.
(2)Hg2+Ion stock liquid is prepared:Three perchloric acid hydrate mercury 45.35mg are weighed, with ultrapure water dissolution, are configured to dense Spend the solution 100mL for 1mM.
(3)Cu2+Ion stock liquid is prepared:Cupric diperchlorate hexahydrate 37.05mg is weighed, with ultrapure water dissolution, is configured to dense Spend the solution 100mL for 1mM.
(4)Fe3+Ion stock liquid is prepared:Ferric perchlorate 35.41mg is weighed, with ultrapure water dissolution, is configured to a concentration of 1mM Solution 100mL.
(5) 75% ethanol solution:Absolute ethyl alcohol 75mL adds ultra-pure water to 100mL, mixing, and room temperature preservation is spare.
(6) phosphate buffer solution (D-hanks balanced salt solutions):0.4gKCl、0.06gKH2PO4、8.0gNaCl、1.0g Glucose, 0.35gNaHCO3、0.152gNa2HPO4·12H2O, 100,000 IU are dual anti-, and adjustment pH is 7.2~7.4, and ultra-pure water is gone to determine Hold to 1000mL, pin type filter (0.22um imports miillpore filter) filtration sterilization, dispense spare.
(7) 1 ten thousand unit (IU)/dual anti-solution of mL:800,000 units of Penicillin sodium are dissolved in 40mLD-hanks solution, are matched Into 20,000 units of final concentration/mL;1,600,000 unit streptomycin sulphates are dissolved in 80mLD-hanks solution, it is single to be made into final concentration 20,000 Position/mL.Isometric Benzylpenicillin sodium salt solution and streptomycin sulfate solution is taken to mix respectively, obtains Benzylpenicillin sodium salt and streptomycin sulphate Final concentration be 10,000 units/mL solution;Pin type filter (0.22um imports miillpore filter) filtration sterilization dispenses 1mL/ Branch, -20 DEG C save backup.
(8) 0.25% trypsase:0.25g trypsase is weighed, is dissolved in the D-hanks liquid of 100mL, pin type filter (0.22um imports miillpore filter) filtration sterilization dispenses 1mL/ branch, and -20 DEG C save backup.
(9) 0.02% ethylenediamine tetra-acetic acids (EDTA):By 0.02gEDTA, it is dissolved in the D-hanks liquid of 100mL, pin type Filter (0.22 μm of import miillpore filter) filtration sterilization dispenses 1mL/ branch, and -20 DEG C save backup.
(10) culture solution:Fetal calf serum, the 90mL modified forms RPMI-1640 trainings that 10mL has been inactivated are measured with Sterile pipette It supports base and the dual anti-liquid of 1mL is mixed in the sterile culture flask of 100mL, 2~8 DEG C save backup.
Sepectrophotofluorometer model CaryEclipse sepectrophotofluorometers used in the present invention, U.S. VARIAN are public Department's production;ThermoFisher8000 water storage types CO2Cell incubator;IX-71 type fluorescence inverted phase contrast microscopes, Japan Olympus companies;AR1530/C electronic balances;25cm2Tissue Culture Flask, Corning companies of U.S. vertical pressure steam sterilizing Device (LS-B75);DHG-9230A electric heating constant-temperature blowing drying boxes, the upper macro experimental facilities Co., Ltd of Nereid.
3rd, fluorescent spectrometry detection Fe3+、Cu2+、Hg2+
Probe solution (1mM, 200 μ L) is added in 10mL volumetric flasks, is diluted with acetonitrile/water, makes the composition of probe solution Volume ratio for acetonitrile/water is 97/3, is shaken up.The solution after 3ml dilutions is added in the cuvette of 1cm, using 470nm as fluorescence Excitation wavelength carries out fluorescence spectrometry.
In the acetonitrile/water solution for being 97/3 in volume ratio, a concentration of 20 μM of probe solution has weak glimmering at 525nm wavelength Light emitting.It is separately added into 400 μM of metal ion Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Sr2+, Zn2+, Cd2+, Ni2+, Co2+, Pb2 +, Al3+, Cr3+, Ag2+When, fluorescence spectrum is not observed and significantly changes, only adds in 400 μM of Fe3+、Hg2+Probe is made to exist The new fluorescence peak of appearance and fluorescence at 585nm significantly increase;Add in 400 μM of Cu2+Make Fluorescence Increasing of the probe at 525nm (see Fig. 1).
In the acetonitrile/water solution for being 97/3 in volume ratio, the Fe of various concentration is used a concentration of 20 μM of probe solutions respectively3+ Ion carries out fluorescence spectroscopic titration (see Fig. 2).Measure Fe3+Fluorescence intensity of the probe at 585nm when concentration changes, obtains glimmering Light calibration curve (see Fig. 3).By the standard deviation of 11 blank values of slope and measure of calibration curve, measure and spy is calculated Needle Fluorometric assay Fe3+The concentration range of linearity and detection limit be listed in table 1.
Probe in detecting Fe3+Fluorescence intensity at 585nm exists in other above-mentioned metal ions respectively as coexisting ion In probe-Fe3+In mixed solution, when concentration and Fe3+When identical, except Hg2+Outside having an impact, other coexistent metallic ions are to probe Detect Fe3+Fluorescence intensity do not interfere (see Fig. 4).
In the acetonitrile/water solution for being 97/3 in volume ratio, the Hg of various concentration is used a concentration of 20 μM of probe solutions respectively2+ Ion carries out fluorescence spectroscopic titration (see Fig. 5).Measure Hg2+Fluorescence intensity of the probe at 585nm when concentration changes, obtains glimmering Light calibration curve (see Fig. 6).By the standard deviation of 11 blank values of slope and measure of calibration curve, measure and spy is calculated Needle Fluorometric assay Hg2+The concentration range of linearity and detection limit be listed in table 1.
Probe in detecting Hg2+Fluorescence intensity at 585nm exists in other above-mentioned metal ions respectively as coexisting ion In probe-Hg2+In mixed solution, when concentration and Hg2+When identical, coexistent metallic ion is to probe in detecting Hg2+Fluorescence intensity It does not interfere (see Fig. 7).
In the acetonitrile/water solution for being 97/3 in volume ratio, the Cu of various concentration is used a concentration of 20 μM of probe solutions respectively2+ Ion carries out fluorescence spectroscopic titration (see Fig. 8).Measure Cu2+Fluorescence intensity of the probe at 525nm when concentration changes, obtains glimmering Light calibration curve (see Fig. 9).By the standard deviation of 11 blank values of slope and measure of calibration curve, measure and spy is calculated Needle Fluorometric assay Cu2+The concentration range of linearity and detection limit be listed in table 1.
Probe in detecting Cu2+Fluorescence intensity at 525nm exists in other above-mentioned metal ions respectively as coexisting ion In probe-Cu2+In mixed solution, when concentration and Cu2+When identical, coexistent metallic ion is to probe in detecting Cu2+Fluorescence intensity It does not interfere (see Figure 10).
1 fluorescence probe method of table detects Fe3+、Hg2+、Cu2+Analytical parameters
4th, the fluorescence imaging of activity PC3 cells
(1) recovery cell
PC3 cells are taken out out of -80 DEG C refrigerators, is placed in 37 DEG C of water and quickly shakes cell cryopreservation tube, in 1-2 minutes Completely thaw, in aseptic operating platform, sucked in centrifuge tube, add 11ml culture solutions, be mixed, and by this cell suspension in It centrifuges 5min on 1000r/min centrifuges, removes supernatant liquor, the cell of bottom precipitation is added into culture solution featheriness, breaks up, mix , it is transferred in culture bottle, nutrient solution volume in culture bottle is made in 5~7mL, 37 DEG C to be placed into, containing 5%CO2, saturated humidity To be cultivated in 100% incubator.
(2) observation → passage → fishplate bar
Replace a culture solution daily, and observe cell growth status under the microscope, until PC3 cells it is adherent cover in In entire culture bottle wall, you can old culture solution is outwelled in passage in aseptic operating platform, adds in the EDTA liquid intrusion cell 30s of 1mL After outwell, the trypsin solution for adding 1mL is digested, and after observation is until cell size is rounded under the microscope, pats training Foster bottle makes cell detachment and adds in appropriate culture solution prevention digestion immediately, it is divided into two and is incubated in 2 culture bottles, to be passed Cell after generation is adherent to be paved with when in entirely culture bottle wall, identical with passage operation, is made carefully after adding in EDTA and trypsase Born of the same parents' digestion comes off and adds in the culture solution prevention digestion of 3mL immediately, and preparation is inoculated in 12 orifice plates.It is added in each orifice plate The cell liquid for having prevented digestion of 200 μ L, then add appropriate new culture solution that orifice plate is placed in incubator after being mixed and cultivate.
(3) cell dyeing
Cell growth condition in next day observation orifice plate, treats the adherent generation of cell, discards old culture solution, use fresh medium Washing 2 times, it is spare.
1)Fe3+Cell imaging
The dyeing culture solution containing 10 μM of probes will be added in above-mentioned PC3 cells orifice plate, the dyeing culture solution is by 90% culture Liquid, 8%H2O, 2% acetonitrile composition, is placed in incubator after being incubated 50min, with the cell in fresh culture solution cleaning orifice After three times, be respectively placed in fluorescence inverted microscope light field imaging take pictures (see Figure 11-a, 11-d) and excitation wavelength be 510nm~ The red channel details in a play not acted out on stage, but told through dialogues of 550nm is taken pictures (see Figure 11-b), excitation wavelength be 450nm~490nm green channel details in a play not acted out on stage, but told through dialogues take pictures (see Figure 11-e), cell unstressed configuration.
The Fe that 0.5mL contains 50 μM is added into above-mentioned orifice plate inner cell3+Dyeing culture solution, the group of the dyeing culture solution As 95% culture solution and 5%H2O is washed three times after being incubated 50min in incubator with fresh culture solution, is placed in fluorescence inversion Microscopical excitation wavelength is to be observed under the red channel of 510nm~550nm, the bright red fluorescence image of cell presentation (see Figure 11-c);The excitation wavelength for being placed in fluorescence inverted microscope is to observe to take pictures under the green channel of 450nm~490nm, and cell is in Bright green fluorescence image (see Figure 11-f).
2)Hg2+Cell imaging
The dyeing culture solution containing 10 μM of probes will be added in above-mentioned PC3 cells orifice plate, the dyeing culture solution is by 90% culture Liquid, 8%H2O, 2% acetonitrile composition, is placed in incubator after being incubated 50min, with the cell in fresh culture solution cleaning orifice After three times, be respectively placed in fluorescence inverted microscope light field imaging take pictures (see Figure 12-a, 12-d) and excitation wavelength be 510nm~ The red channel details in a play not acted out on stage, but told through dialogues of 550nm is taken pictures (see Figure 12-b), excitation wavelength be 450nm~490nm green channel details in a play not acted out on stage, but told through dialogues take pictures (see Figure 12-e), cell unstressed configuration.
The Hg that 0.5mL contains 50 μM is added into above-mentioned orifice plate inner cell2+Dyeing culture solution, the group of the dyeing culture solution As 95% culture solution and 5%H2O is washed three times after being incubated 40min in incubator with fresh culture solution, is placed in fluorescence inversion Microscopical excitation wavelength is to be observed under the red channel of 510nm~550nm, the bright red fluorescence image of cell presentation (see Figure 12-c);The excitation wavelength for being placed in fluorescence inverted microscope is to observe to take pictures under the green channel of 450nm~490nm, and cell is in Green fluorescence image (see Figure 12-f).
3)Cu2+Cell imaging
The dyeing culture solution containing 10 μM of probes will be added in above-mentioned PC3 cells orifice plate, the dyeing culture solution is by 90% culture Liquid, 8%H2O, 2% acetonitrile composition, is placed in incubator after being incubated 50min, with the cell in fresh culture solution cleaning orifice After three times, be respectively placed in fluorescence inverted microscope light field imaging take pictures (see Figure 13-a) and excitation wavelength be 450nm~490nm Green channel details in a play not acted out on stage, but told through dialogues take pictures (see Figure 13-b), cell unstressed configuration.
The Cu that 0.5mL contains 50 μM is added into above-mentioned orifice plate inner cell2+Dyeing culture solution, the group of the dyeing culture solution As 95% culture solution and 5%H2O is washed three times after being incubated 50min in incubator with fresh culture solution, is placed in fluorescence inversion Microscopical excitation wavelength is to observe to take pictures under the green channel of 450nm~490nm, and cell is in bright green fluorescence image (see Figure 13-c).

Claims (8)

1. a kind of binary channels fluorescence imaging detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method, it is characterised in that:It is Using a kind of triamine ethylamine derivative as probe, micro Fe in competent cell is detected by binary channels fluorescence imaging respectively3+、 Hg2+And Cu2+;The chemical structural formula of the probe is:
2. binary channels fluorescence imaging as described in claim 1 detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method, It is characterized in that:It is described using a kind of triamine ethylamine derivative as probe, work is detected by binary channels fluorescence imaging respectively Micro Fe in property cell3+、Hg2+And Cu2+;It is first probe to be made to enter into the cell with probe and cell hatching, then hatching is had probe Cell respectively with Fe3+、Hg2+、Cu2+Hatching, make probe respectively with Fe3+、Hg2+、Cu2+It can emit spy with reference to generation in the cell Levy the probe-Fe of wavelength fluorescent3+, probe-Hg2+, probe-Cu2+Complex is realized that probe dyes intracellular ion and is imaged, uses Competent cell fluorescence picture after fluorescence inverted microscope observation hatching;Specifically include following steps:
(1) cell culture:Competent cell is inoculated in through recovery in culture solution, is 37 DEG C in temperature, 5%CO2And saturated humidity is It is cultivated in 100% incubator, after passage, is inoculated in 12 orifice plates and cultivates, density is 2 × 104A/ml, secondary daily fresh training Nutrient solution cleans cell;The culture solution is containing 10% fetal calf serum, 1% dual anti-and 89% modified form RPMI, 1640 culture mediums;
(2) probe is to cell incubation:It will be added in the incubation culture solution containing 5~40 μM of probes in the cell of step (1), the incubation The composition of culture solution be 90% culture solution, 8%H2O, 2% acetonitrile, incubator is interior to be incubated 30~80min, and incubating containing probe is sucked out Culture solution is educated, cell is cleaned with fresh culture solution, is placed under fluorescence inverted microscope and carries out light field respectively and details in a play not acted out on stage, but told through dialogues is taken pictures, it is bright Cell clearly image is observed off field, and cell image is not observed under details in a play not acted out on stage, but told through dialogues;
(3)Fe3+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Fe3+Dyeing culture solution, the dyeing training The composition of nutrient solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Fe3+Dyeing culture solution, use Fresh culture solution cleaning cell, is placed under the red channel of fluorescence inverted microscope and observes probe to intracellular Fe3+After dyeing Fluorescence picture, bright red fluorescence is presented in cell, and shooting obtains clearly red fluorescent cell contour image;Again by above-mentioned warp Probe and Fe3+Cell after dyeing respectively, which is placed under the green channel of fluorescence inverted microscope, observes probe to intracellular Fe3+Dye Fluorescence picture after color, cell are in green fluorescence, and shooting obtains green cells contour image, and probe detects in competent cell To micro Fe3+Ion;
(4)Hg2+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Hg2+Dyeing culture solution, the dyeing training The composition of nutrient solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Hg2+Dyeing culture solution, use Fresh culture solution cleaning cell, is placed under the red channel of fluorescence inverted microscope and observes probe to intracellular Hg2+After dyeing Fluorescence picture, bright red fluorescence is presented in cell, and shooting obtains clearly red fluorescent cell contour image;Again by above-mentioned warp Probe and Hg2+Cell after dyeing respectively, which is placed under the green channel of fluorescence inverted microscope, observes probe to intracellular Hg2+Dye Fluorescence picture after color, cell are in green fluorescence, and shooting obtains green cells contour image, and probe detects in competent cell To micro Hg2+Ion;
(5)Cu2+To cell dyeing:It is added in the cell of step (2) containing 30~80 μM of Cu2+Dyeing culture solution, the dyeing training The composition of nutrient solution is 95% culture solution and 5%H2O, incubator is interior to be incubated 30~80min, is sucked out containing Cu2+Dyeing culture solution, use Fresh culture solution cleaning cell, is placed under the green channel of fluorescence inverted microscope and observes probe to intracellular Cu2+After dyeing Fluorescence picture, cell is in bright green fluorescence, and shooting obtains clearly green cells contour image, and probe is active thin Micro Cu is detected in born of the same parents2+Ion.
3. binary channels fluorescence imaging as claimed in claim 2 detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method, It is characterized in that:The fluorescence inverted microscope;The excitation wavelength for being green channel is 450nm~490nm, red channel Excitation wavelength is the fluorescence inverted microscope of 510nm~550nm.
4. binary channels fluorescence imaging as claimed in claim 2 detects micro Fe in living cells respectively3+、Hg2+And Cu2+Method, It is characterized in that:1640 culture mediums of modified form RPMI;Be RPMI be English Roswell Park Memorial The abbreviation of Institute, acute pyogenic infection of finger tip Loews Wei Pake memorial institutes, RPMI are a kind of cell culture of research institute research and development Base, 1640 be culture medium code name.
5. binary channels fluorescence imaging as claimed in claim 1 or 2 detects micro Fe in living cells respectively3+、Hg2+And Cu2+Side Method, it is characterised in that:The competent cell is;Human prostate's cancer cell, abbreviation PC3 cells.
6. binary channels fluorescence imaging as claimed in claim 1 or 2 detects micro Fe in living cells respectively3+、Hg2+And Cu2+Side Method, it is characterised in that:The probe;It is to be synthesized by following synthetic routes:
7. micro Fe in the binary channels fluorescence imaging detection living cells as described in any one of claim 1-63+、Hg2+、Cu2+'s Method, it is characterised in that:The probe;It is prepared:In the three-necked flask of 100ml, three (2- aminoethyls) are added in The absolute ethyl alcohol of amine 25-30mmol, rhodamine B 2-5mmol and 55-65ml, the lower reflux 33-39h of nitrogen protection, decompression boil off Ethyl alcohol is extracted 3 times respectively with 100ml dichloromethane, and organic phase is dried overnight with anhydrous magnesium sulfate, boils off solvent, is obtained red viscous Thick liquid, silica gel column chromatography separation, eluent is methanol/chloroform/triethylamine that volume ratio is 9/1/1, obtains colorless viscous Shape intermediate 1;In the there-necked flask of 250ml, intermediate 1,115-125ml chloroforms and the chloro- 7- nitre of 2-8mmol 4- are added in Base benzofuraxan, nitrogen is protected reacts 1.5-2.5h under ice bath, boils off solvent, silica gel column chromatography separation, eluent is volume ratio For 100/3 chloroform/methanol, light yellow solid intermediate 2 is obtained;In the there-necked flask of 100ml, addition intermediate 2, 0.66-0.85mmol 7- hydroxyls -8- cumarins formaldehyde and 55-65ml methanol, the lower back flow reaction 7-9h of nitrogen protection, boil off molten Agent, silica gel column chromatography separation, eluent is chloroform/methanol that volume ratio is 100/2, obtains orange solids probe.
8. micro Fe in binary channels fluorescence imaging detection living cells as claimed in claim 73+、Hg2+、Cu2+Method, it is special Sign is:The probe;It is prepared:In the three-necked flask of 100ml, three (2- aminoethyls) amine are added in The absolute ethyl alcohol of 27.36mmol, rhodamine B 3.42mmol and 60ml, the lower reflux 36h of nitrogen protection, decompression boil off ethyl alcohol, point Not Yong 100ml dichloromethane extract 3 times, organic phase is dried overnight with anhydrous magnesium sulfate, boils off solvent, obtains red thick shape object, Silica gel column chromatography detaches, and eluent is methanol/chloroform/triethylamine that volume ratio is 9/1/1, obtains 1.71g colorless viscous shapes Intermediate 1;In the there-necked flask of 250ml, 6.01mmol intermediates 1,120ml chloroforms and the chloro- 7- of 5.01mmol 4- are added in Nitro benzofuraxan, nitrogen is protected reacts 2h under ice bath, boils off solvent, silica gel column chromatography separation, eluent is that volume ratio is 100/3 chloroform/methanol, obtains light yellow solid intermediate 2;In the there-necked flask of 100ml, addition intermediate 2, 0.748mmol 7- hydroxyls -8- cumarins formaldehyde and 60ml methanol, the lower back flow reaction 8h of nitrogen protection, boil off solvent, silicagel column Chromatography, eluent are chloroform/methanol that volume ratio is 100/2, obtain orange solids probe.
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Application publication date: 20180619