CN109307661A - A Fluorescence Imaging Method of Cr3+, Al3+ or Zn2+ in Living Cells - Google Patents
A Fluorescence Imaging Method of Cr3+, Al3+ or Zn2+ in Living Cells Download PDFInfo
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- CN109307661A CN109307661A CN201710615551.2A CN201710615551A CN109307661A CN 109307661 A CN109307661 A CN 109307661A CN 201710615551 A CN201710615551 A CN 201710615551A CN 109307661 A CN109307661 A CN 109307661A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention discloses Cr in a kind of living cells3+、Al3+、Zn2+Fluorescence imaging method, with compound N, N ', N "-three [4- (benzothiazole -2- base) -2,5- 4-dihydroxy benzaldehyde] contracting-(3- aminoethyl) amine is as trace metal ion Cr in living cells3+、Al3+、Zn2+Fluorescence imaging probe, probe is compatible with living cells, penetrate into it is intracellular, by probe respectively to intracellular Cr3+、Al3+Or Zn2+After dyeing, fluorecyte image is detected with fluorescence inverted microscope.Probe of the present invention is able to achieve to Cr in living cells3+、Al3+、Zn2+Single channel, the binary channels image checking of three kinds of ions, detection efficiency is high, and testing cost is low.
Description
Technical field
The present invention relates to a kind of fluorescence imaging method of ions in living cells, Cr in especially a kind of living cells3+、Al3+Or
Zn2+Fluorescence imaging method.
Background technique
Fluorescence imaging analysis is a kind of highly sensitive visualization analysis technique for being now widely used for live cell assays.With
The development of life science, the information research of intracellular active specy, cellular signal transduction and Apoptosis etc. is got over
Come more deep.Fluorescent marker living cells imaging technique is more and more used for life science as a kind of strong research means
Field promotes Neuscience and stem-cell research to explain a variety of life and the physiological phenomenon in living cells, for understand cancer and
The molecule reason of other diseases and the breakthrough development of biomethanics bring hope.Fluorescence currently used for living cells imaging analysis is visited
Needle is mostly small organic molecule fluorescent dye, these organic fluorescent dye molecules overcome mostly the service life it is short, be easy quenching, biofacies
The disadvantage of capacitive and stability difference can be realized visualization, high selection, real-time, dynamic METHOD FOR CONTINUOUS DETERMINATION.
Some heavy metals and transition metal element are due to participating in the processes such as cell development, genetic transcription, neurotransmission
Have the function of very important physiological action and, but when content is more than its corresponding threshold value, they send out the growth of organism
It educates, physiological metabolism even morphosis etc. will cause obvious damage.Therefore influence of the specific ion to organism is studied, it is non-
One of normal necessary method is exactly to be detected using fluorescence imaging of the fluorescence probe to ion in living cells.
Can as the small organic molecule probe of cell imaging, because its molecular structure is simple, synthesize it is relatively easy, detection at
This is cheap, and spectrum property is superior, low to cell hypotoxicity, good cell membrane permeability and be concerned.Utilize fluorescence probe
The relevant report that technology detects trace metal ion in living cells is existing many, but can be realized simultaneously to work using a kind of probe
Intracellular Cr3+、Al3+、Zn2+Single channel, the binary channels image checking of three kinds of ions have not been reported.
Therefore, in the prior art, existing cannot realize simultaneously to Cr in living cells3+、Al3+、Zn2+The single-pass of three kinds of ions
The problem of road, binary channels image checking, detection efficiency is low, and testing cost is high.
Summary of the invention
The object of the present invention is to provide Cr3 in a kind of living cells+、Al3+And/or Zn2+Fluorescence imaging method, this hair
The bright probe is able to achieve to Cr in living cells3+、Al3+、Zn2+Single channel, the binary channels image checking of three kinds of ions, detection effect
Rate is high, and testing cost is low.
A kind of technical solution of the present invention: Cr in living cells3+、Al3+Or Zn2+Fluorescence imaging method, with compound N,
N ', N "-three [4- (benzothiazole -2- base) -2,5- 4-dihydroxy benzaldehyde] contracting-(3- aminoethyl) amine is as gold micro in living cells
Belong to ion Cr3+、Al3+Or Zn2+Fluorescence imaging probe, probe is compatible with living cells, penetrates into intracellular, is distinguished by probe
To intracellular Cr3+、Al3+Or Zn2+After dyeing, fluorecyte image is detected with fluorescence inverted microscope；The change of the probe
Learn structural formula are as follows:
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, it is described by probe respectively to cell
Interior Cr3+、Al3+Or Zn2+After dyeing, fluorecyte image is detected with fluorescence inverted microscope；It is to carry out as follows:
(1) cell culture: living cells is inoculated in through recovery containing 10% fetal calf serum and containing in 1% dual anti-culture medium,
It cultivates in the incubator, after passage, is inoculated in 12 orifice plates and cultivates, density is 2 × 104A/ml, secondary daily culture medium cleaning are thin
Born of the same parents when passage, were passed on 1 time every 2-3 days；
(2) probe is to cell incubation: the culture solution that concentration and probe concentration is 10~40 μM, incubator will be added in the cell of (1)
30~80min of interior incubation is sucked out the culture solution containing probe, cleans cell with culture medium, is placed under fluorescence inverted microscope respectively
It carries out light field and dark field is taken pictures, cell clearly image is observed under light field, cell image is not observed under dark field；The training
It is 98% culture medium and 2%N, dinethylformamide that the group of nutrient solution, which becomes volume ratio,；
(3)Cr3+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (2)2The Cr of O composition3+
Concentration is 60~120 μM of solution, is incubated for 30~60min in incubator, clean cell with culture medium, and it is micro- to be placed in fluorescence inversion
Probe is observed under the blue channel of mirror to intracellular Cr3+Fluorescence picture after dyeing, cell are presented bright blue-fluorescence, shoot
Trace Cr is detected in living cells to clearly blue-fluorescence cell outline image, probe3+Ion；
(4)Al3+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (2)2The Al of O composition3+
Concentration is 60~120 μM of solution, is incubated for 30~60min in incubator, clean cell with culture medium, and it is micro- to be placed in fluorescence inversion
Probe is observed under the blue channel of mirror to intracellular Al3+Fluorescence picture after dyeing, cell are presented bright blue-fluorescence, shoot
Trace of Al is detected in living cells to clearly blue-fluorescence cell outline image, probe3+Ion；
(5) probe is to cell incubation: the culture solution that concentration and probe concentration is 10~40 μM, culture solution will be added in the cell of (1)
Group to become volume ratio be 98% culture medium, 2%1,4- dioxane are incubated for 20~60min in incubator, are sucked out containing probe
Culture solution cleans cell with culture medium, is placed under fluorescence inverted microscope and carries out field light field respectively and dark field is taken pictures, sees under light field
Cell clearly image is observed, cell image is not observed under dark field；
(6)Zn2+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (5)2The Zn of O composition2+
Concentration is 60~120 μM of solution, is incubated for 30~80min in incubator, clean cell with culture medium, and it is micro- to be placed in fluorescence inversion
Probe is observed under the green channel of mirror to intracellular Zn2+Green fluorescence is presented in fluorescence picture after dyeing, cell, and shooting obtains clear
Green cells contour image, probe detects trace Zn in living cells2+Ion；Above-mentioned cell fluorescence is placed in again to fall
It sets and observes probe under microscopical red channel to intracellular Zn2+Fluorescence picture after dyeing, cell are presented red fluorescence, shoot
To clearly red fluorescent cell contour image, probe detects trace Zn in living cells2+Ion.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, the culture medium；It is RPMI-1640
Culture medium；Wherein RPMI is the abbreviation of English Roswell Park Memorial Institute, and generation refers to that Loews Wei Pake records
Research institute is read, RPMI is a kind of cell culture medium of research institute research and development, and 1640 be culture medium code name.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, the living cells；It is human prostate
Cancer cell, abbreviation PC3 cell.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, the fluorescence inverted microscope；It is blue
The excitation wavelength of chrominance channel is 330~385nm, and the excitation wavelength of green channel is 450nm~490nm, the excitation of red channel
Wavelength is the fluorescence inverted microscope of 510nm~550nm.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, the probe, be by following routes close
At:
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, the probe；It is with three (2- ammonia second
Base) amine, o-amino thiophenol and terephthaldehyde's ether be primary raw material, be prepared.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, it is described with three (2- aminoethyl) amine, neighbour
Amino thiophenol, terephthaldehyde's ether are primary raw material, are prepared；It is to be prepared according to the following steps:
(1)N2Under protection in dry three-necked flask, terephthaldehyde's ether, tetrabutyl ethylenediamine and dried is added
Ether obtains A product；
(2) A product are cooled to 0 DEG C hereinafter, obtaining B product；
(3) n-BuLi is added in B product, after adding after 0 DEG C of the reaction was continued 5-15min, reaction solution is flowed back 18-22h, to
After reaction solution is cooled to room temperature, C product are obtained；
(4) n,N-Dimethylformamide is added in C product, is stirred overnight at room temperature, obtain D product；
(5) water is added in D product, and chloroform extraction, organic phase is dry with anhydrous magnesium sulfate, and solvent, silica gel column chromatography is removed under reduced pressure
Separation, eluent is n-hexane/toluene that volume ratio is 6/4, obtains yellow solid intermediate c, i.e. E product；
(6) in dry three-necked flask, E product, o-amino thiophenol, potassium metabisulfite and N, N- dimethyl formyl is added
Amine, N2Under protection, back flow reaction 2-4h is cooled to room temperature after reaction, obtains F product；
(7) F product are placed in ice water, and after yellow solid is precipitated completely, filtering is dried in vacuum overnight, silica gel column chromatography separation,
Eluent is toluene, obtains yellow intermediate d, i.e. G product；
(8) G product and methylene chloride are added in three-necked flask, are cooled to -30 DEG C, obtain H product；
(9) Boron tribromide is added in H product, -30 DEG C of reaction 0.5-1.5h, obtain J product under nitrogen protection；
(10) after reacting 20-28h under J product room temperature, distilled water is added and reacts 1-3h, is extracted with ethyl acetate, silica gel column layer
Analysis separation, eluent is n-hexane/chloroform that volume ratio is 6/4, obtains yellow intermediate e to get I product；
(11) in there-necked flask, intermediate compound I product are added, dehydrated alcohol, reflux keeps intermediate e completely molten under nitrogen protection
Solution is injected three (2- aminoethyl) amine with micro-sampling pin, is refluxed overnight, cooled and filtered, and ethyl alcohol recrystallization obtains yellow solid,
To obtain the final product.
Cr in living cells above-mentioned3+、Al3+Or Zn2+Fluorescence imaging method in, it is described with three (2- aminoethyl) amine, neighbour
Amino thiophenol and terephthaldehyde's ether are primary raw material, are prepared；It is to be prepared according to the following steps:
(1)N2Under protection in dry 500ml three-necked flask, terephthaldehyde's ether 40mmol, tetrabutyl ethylenediamine is added
200mmol and ether 140ml, obtains A product；
(2) A product are cooled to 0 DEG C hereinafter, obtaining B product；
(3) the n-BuLi 100ml that concentration is 1.6M is added in B product, the reaction was continued 10min under the conditions of 0 DEG C after adding will
Reaction solution reflux 20h obtains C product after reaction solution is cooled to room temperature；
(4) n,N-Dimethylformamide 220mmol is added in C product, is stirred overnight at room temperature；Obtain D product；
(5) 100ml water is added in D product, chloroform extracts (3 × 100ml), and organic phase is dry with anhydrous magnesium sulfate, and decompression removes
Solvent is removed, silica gel column chromatography separation, eluent is n-hexane/toluene that volume ratio is 6/4, obtains yellow solid intermediate c, i.e. E
Product；
(6) in dry 250ml three-necked flask, E product, o-amino thiophenol 23.8mmol, potassium metabisulfite are added
23.84mmol and 150mlN, dinethylformamide, N2Under protection, back flow reaction 3h is cooled to room temperature after reaction, is obtained
F product；
(7) F product are placed in the ice water of 250mml 0-4 DEG C, and after yellow solid is precipitated completely, filtering, filter cake was dried in vacuo
At night, silica gel column chromatography separation, eluent is toluene, obtains yellow midbody compound d, i.e. G product；
(8) G product and methylene chloride 50ml are added in 250ml three-necked flask, are cooled to -30 DEG C, obtain H product；
(9) Boron tribromide 14.04mmol is added in H product, -30 DEG C of reaction 1h, obtain J product under nitrogen protection；
(10) after being reacted for 24 hours under J product room temperature, the distilled water reaction 2h of 70ml is added, is extracted with ethyl acetate (3 × 100ml)
It takes, silica gel column chromatography separation, eluent is n-hexane/chloroform that volume ratio is 6/4, obtains yellow intermediate e to get I product；
(11) in the there-necked flask of 50ml, I product and dehydrated alcohol 30ml is added, reflux keeps I product completely molten under nitrogen protection
Solution is injected three (2- aminoethyl) amine 23.3mg, is refluxed overnight, cooled and filtered, ethyl alcohol recrystallization obtains probe.
Inventor has carried out a large amount of experimental study, and part test is as follows:
1, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, under the excitation of 320nm wavelength, probe emits faint glimmering at 470nm and 610nm
Light is separately added into 200 μM of metal ion Cr3+, Al3+, Zn2+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Hg2+, Sr2+, Cd2+, Ni2+,
Co2+, Pb2+, Cu2+, Mn2+, Fe3+, Ag+；It is placed at room temperature for 45min, sees Fig. 1.Cr3+、Al3+It can make fluorescence of the probe at 480nm
It significantly increases, Zn2+Make probe fluorescence enhancement at 480nm and 540nm, probe is not observed in the addition of remaining metal ion
Fluorescence spectrum has significant change；Show that probe is to Cr with this condition3+, Al3+, Zn2+There is recognition detection effect.
2, it is molten to be dissolved in n,N-Dimethylformamide/water probe that volume ratio is 96/4 for probe prepared by embodiment 1
In liquid, it is configured to the solution that concentration and probe concentration is 10 μM, the Cr of various concentration is added3+, it is placed at room temperature for 45min, with Cr3+Plus
Enter, point measurement fluorescence spectroscopic titration curve.The excitation wavelength of test is 320nm.It is specifically shown in Fig. 2.
3, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, is separately added into various concentration Cr3+, measure fluorescence intensity level at 480nm.Ordinate is
Fluorescence intensity, abscissa Cr3+Concentration.The excitation wavelength of test is 320nm, is specifically shown in Fig. 3.
4, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, is separately added into 200 μM of metal ion Al3+, Cr3+, Zn2+, Li+, Na+, K+, Mg2+,
Ca2+, Ba2+, Hg2+, Sr2+, Cd2+, Ni2+, Co2+, Pb2+, Cu2+, Mn2+, Ag+, Fe3+It is placed at room temperature for 45min, detects Cr3+?
Fluorescence intensity level at 480nm, then divide to probe-Cr3+After other coexistent metallic ions are added in mixed solution, then measure
The variation of fluorescence intensity level at 480nm.It is specifically shown in Fig. 4, after black bar expression is separately added into metal ion in probe solution
Fluorescence intensity level at 480nm；White bars are indicated in probe-Cr3+Mixed solution is separately added into above-mentioned other again and metal coexists
The variation of fluorescence intensity level after ion at 480nm.Show probe in detecting Cr3+Fluorescence intensity do not coexisted by above-mentioned ion
It influences.The excitation wavelength of test is 320nm, fluorescence emission wavelengths 480nm.
5, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, is separately added into various concentration Al3+, measure fluorescence intensity level at 480nm.Ordinate is
Fluorescence intensity, abscissa Al3+Concentration.The excitation wavelength of test is 320nm.It is specifically shown in Fig. 5.
6, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, is separately added into various concentration Al3+, measure fluorescence intensity level at 480nm.Ordinate is
Fluorescence intensity, abscissa Al3+Concentration.The excitation wavelength of test is 320nm.It is specifically shown in Fig. 6.
7, probe prepared by embodiment 1 is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4,
It is configured to the solution that concentration and probe concentration is 10 μM, is separately added into 200 μM of metal ion Al3+, Cr3+, Zn2+, Li+, Na+, K+, Mg2+,
Ca2+, Ba2+, Hg2+, Sr2+, Cd2+, Ni2+, Co2+, Pb2+, Cu2+, Mn2+, Ag+, Fe3+；Al is detected after being placed at room temperature for 45min3+?
Fluorescence intensity level at 480nm, then divide to probe-Al3+After other coexistent metallic ions are added in mixed solution, then measure
The variation of fluorescence intensity level at 480nm.It is specifically shown in Fig. 7, after black bar expression is separately added into metal ion in probe solution
Fluorescence intensity level at 480nm；White bars are indicated in probe-Al3+Mixed solution is separately added into above-mentioned other again and metal coexists
The variation of fluorescence intensity level after ion at 480nm.Show probe in detecting Al3+Fluorescence intensity do not coexisted by above-mentioned ion
It influences.The excitation wavelength of test is 320nm, fluorescence emission wavelengths 480nm.
8, probe prepared by embodiment 1 is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, is configured to
The solution that concentration and probe concentration is 10 μM, under the excitation of 340nm wavelength, the fluorescence of probe is very weak, is separately added into 200 μM of metal ion
Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Hg2+, Sr2+, Cd2+, Ni2+, Co2+, Pb2+, Cu2+, Mn2+, Ag+, Fe3+, Zn2+, Cr3+, Al3 +Into probe solution, it is placed at room temperature for 25min, only Zn2+Significantly increase fluorescence of the probe at 540nm, remaining metal ion
Addition fluorescence spectrum that probe is not observed have significant change, show that probe is to Zn with this condition2+There is recognition detection work
With.It is specifically shown in Fig. 8.
9, probe prepared by embodiment 1 is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, is configured to
The solution that concentration and probe concentration is 10 μM, is separately added into various concentration Zn2+, measure fluorescence intensity level at 540nm.Ordinate is that fluorescence is strong
Degree, abscissa Zn2+Concentration.The excitation wavelength of test is 340nm.It is specifically shown in Fig. 9.
10, probe prepared by embodiment 1 is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, prepares
The solution for being 10 μM at concentration and probe concentration, is separately added into various concentration Zn2+, measure fluorescence intensity level at 540nm.Ordinate is fluorescence
Intensity, abscissa Zn2+Concentration.The excitation wavelength of test is 340nm.It is specifically shown in Figure 10.
11, probe prepared by embodiment 1 is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, prepares
The solution for being 10 μM at concentration and probe concentration, is separately added into 200 μM of configuration metal ions Zns2+, Al3+, Cr3+, Li+, Na+, K+, Mg2+, Ca2+,
Ba2+, Hg2+, Sr2+, Cd2+, Ni2+, Co2+, Pb2+, Cu2+, Mn2+, Ag+, Fe3+；Zn is detected after being placed at room temperature for 25min2+In 540nm
The fluorescence intensity level at place, then divide to probe-Zn2+After other coexistent metallic ions are added in mixed solution, then measure at 540nm
Fluorescence intensity level variation.It is specifically shown in Figure 11, black bar expression is separately added into after metal ion in 540nm in probe solution
The fluorescence intensity level at place；White bars are indicated in probe-Zn2+Mixed solution is separately added into again after other above-mentioned coexistent metallic ions
The variation of fluorescence intensity level at 540nm.Except Cu2+, Hg2+, Al3+Outside slightly influencing, other metal ions are to probe in detecting Zn2+
Fluorescence intensity influence it is smaller.The excitation wavelength of test is 340nm, fluorescence emission wavelengths 540nm.
12, a.PC3 cell is incubated for 65min through the probe (being prepared by embodiment 1) that concentration is 20 μM, cleans, and use is glimmering
The photograph via bright field of light inverted microscope shooting, is specifically shown in Figure 12-a.Cell is adherent normal, is in full state, it was demonstrated that probe is at this
There is no toxicity to PC3 cell under test condition；
B. the above-mentioned PC3 cell being incubated for through probe, the dark field photo shot with fluorescence inverted microscope, cell-free fluorescence
Imaging, is specifically shown in Figure 12-b；
C. the above-mentioned probe through 20 μM is incubated for the PC3 cell of 65min, then with 100 μM of Cr3+After being incubated for 45min, in fluorescence
The cell picture shot under inverted microscope blue channel, is specifically shown in Figure 12-c, observes clearly blue-fluorescence cell distribution,
Prove probe and Cr3+Ion realizes dyeing in the cell, and cell dyeing fluorescence imaging is presented；Fluorescence for shooting is inverted micro-
The excitation wavelength of mirror blue channel is 330~385nm.
13, a.PC3 cell is incubated for 65min through the probe (being prepared by embodiment 1) that concentration is 20 μM, cleans, and use is glimmering
The photograph via bright field of light inverted microscope shooting.It is specifically shown in Figure 13-a, cell is adherent normal, is in full state, it was demonstrated that probe is at this
There is no toxicity to PC3 cell under test condition；
B. the above-mentioned PC3 cell being incubated for through probe, the dark field photo shot with fluorescence inverted microscope, cell-free fluorescence
Imaging is specifically shown in Figure 13-b；
C. the above-mentioned probe (being prepared by embodiment 1) through 20 μM is incubated for the PC3 cell of 65min, then with 100 μM
Al3+After being incubated for 50min, the cell picture shot under fluorescence inverted microscope blue channel is specifically shown in Figure 13-c, observes clear
Clear blue-fluorescence cell distribution, it was demonstrated that probe and Al3+Ion realizes dyeing in the cell, present cell dyeing fluorescence at
Picture；The excitation wavelength of fluorescence inverted microscope blue channel for shooting is 330~385nm.
14, a.PC3 cell is incubated for 30min, cleaning through the probe (being prepared by embodiment 1) that concentration is 20 μM, and use is glimmering
The photograph via bright field of light inverted microscope shooting.It is specifically shown in Figure 14-a, cell is adherent normal, is in full state, it was demonstrated that probe is at this
There is no toxicity to PC3 cell under test condition；
B. the above-mentioned PC3 cell being incubated for through probe, with the dark field photo shot under fluorescence inverted microscope green channel,
Cell-free fluorescence imaging is specifically shown in Figure 14-b；
C. the above-mentioned probe through 20 μM is incubated for the PC3 cell of 30min, then with 100 μM of Zn2+After being incubated for 60min, in fluorescence
The cell picture shot under inverted microscope green channel, is specifically shown in Figure 14-c, observes clearly green cells distribution,
Prove probe and Zn2+Ion realizes dyeing in the cell, and cell dyeing fluorescence imaging is presented；Fluorescence for shooting is inverted micro-
The excitation wavelength of mirror green channel is 450nm~490nm.
D.PC3 cell is incubated for 30min, cleaning, the light field shot with fluorescence inverted microscope through the probe that concentration is 20 μM
Photo is specifically shown in Figure 14-d.Cell is adherent normal, is in full state, it was demonstrated that probe does not in this test condition have PC3 cell
Toxicity；
E. the above-mentioned PC3 cell being incubated for through probe, with the dark field photo shot under fluorescence inverted microscope red channel,
It is specifically shown in Figure 14-e, cell-free fluorescence imaging；
F. the above-mentioned probe through 20 μM is incubated for the PC3 cell of 30min, then with 100 μM of Zn2+After being incubated for 60min, in fluorescence
The cell picture shot under inverted microscope red channel, is specifically shown in Figure 14-f, observes clearly red fluorescent cell distribution,
Prove probe and Zn2+Ion realizes dyeing in the cell, and cell dyeing fluorescence imaging is presented；Fluorescence for shooting is inverted micro-
The excitation wavelength of mirror red channel is 510nm~550nm.
Compared with prior art, the invention has the following advantages:
(1) using probe in n,N-Dimethylformamide/aqueous solution, under the excitation of 320nm wavelength, Cr is detected3+、Al3+
The 480nm wavelength fluorescent of transmitting, at this time Zn2+Not Interference Detection；Using probe in Isosorbide-5-Nitrae-dioxane/water solution, in 340nm
Under wavelength excitation, Zn is detected2+The 540nm wavelength fluorescent of transmitting, at this time Cr3+、Al3+Not Interference Detection utilizes solvent effect and wave
It is long to differentiate, the selectivity of detection is improved, interference is reduced；
(2) probe additionally depends on incubation time in imaging process, concentration to the imaging effect of intracellular specific ion, right
Different solvents, different ions have optimal imaging condition；
(3) probe as cell imaging reagent have it is good it is water-soluble, there is good stability in physiological conditions
With membrane penetrating, to cytotoxic, thus the fluorescence imaging of micro special metal in living cells can be used for；
(4) many reports existing to the cell imaging probe of single ionic, but by control test condition, it is imitated using solvent
It should be with fluorescence emission wavelengths difference, while trace Cr in highly sensitive, high selection detection cell3+、Al3+And Zn2+Method have no
Report；
(5) imaging agents as lot of trace metal ion in living cells, easy to detect, visual strong, high sensitivity,
Selectivity is good, it can be achieved that real-time online detects.
Therefore, probe of the present invention is able to achieve to Cr in living cells3+、Al3+、Zn2+The single channel of three kinds of ions, bilateral
Road image checking, detection efficiency is high, and testing cost is low.
Detailed description of the invention:
Fig. 1 is the fluorescence that the N,N-dimethylformamide/aqueous solution middle probe for being 96/4 in volume ratio detects metal ion
Spectrogram；
Fig. 2 is the Cr of the various concentration in N,N-dimethylformamide/aqueous solution that volume ratio is 96/43+To the glimmering of probe
Light spectrum drips curve graph；
Fig. 3 is that the N,N-dimethylformamide/aqueous solution middle probe for being 96/4 in volume ratio detects Cr3+Fluorescent spectrometry
Correction graph；
It is coexistent metallic ion in 96/4 N,N-dimethylformamide/aqueous solution to fluorescence probe that Fig. 4, which is in volume ratio,
Method detects Cr3+Fluorescence intensity influence diagram；
Fig. 5 is the Al of the various concentration in N,N-dimethylformamide/water solution that volume ratio is 96/43+To probe
Fluorescence spectrum drips curve graph；
Fig. 6 is that the N,N-dimethylformamide/aqueous solution middle probe for being 96/4 in volume ratio detects Al3+Fluorescent spectrometry
Calibration curve；
It is coexistent metallic ion in 96/4 N,N-dimethylformamide/aqueous solution to fluorescence probe that Fig. 7, which is in volume ratio,
Method detects Al3+Fluorescence intensity influence diagram；
Fig. 8 is the fluorescence spectrum that the 1,4- dioxane/water solution middle probe for being 1/1 in volume ratio detects metal ion
Figure；
Fig. 9 is the Zn of the various concentration in the 1,4- dioxane/water solution that volume ratio is 1/12+To the fluorescence light of probe
Compose titration curve；
Figure 10 is that the 1,4- dioxane/water solution middle probe for being 1/1 in volume ratio detects Zn2+Fluorescent spectrometry correction
Curve；
Figure 11 is that coexistent metallic ion examines fluorescence probe method in the 1,4- dioxane/water solution that volume ratio is 1/1
Survey Zn2+Fluorescence intensity influence diagram；
Figure 12 is probe to Cr in PC3 cell3+Fluorescent microscopic imaging photo；It is 20 μM that 12-a, which is PC3 cell through concentration,
Probe be incubated for 65min, cleaning, with fluorescence inverted microscope shoot photograph via bright field；12-b is above-mentioned to be incubated for through probe
PC3 cell, the dark field photo shot with fluorescence inverted microscope；12-c is that the PC3 that the above-mentioned probe through 20 μM is incubated for 65min is thin
Born of the same parents, then with 100 μM of Cr3+After being incubated for 45min, the cell picture that is shot under fluorescence inverted microscope blue channel；
Figure 13 is probe to Al in PC3 cell3+Fluorescent microscopic imaging photo；It is 20 μM that 13-a, which is PC3 cell through concentration,
Probe be incubated for 65min, cleaning, with fluorescence inverted microscope shoot photograph via bright field；13-b is above-mentioned to be incubated for through probe
PC3 cell, the dark field photo shot with fluorescence inverted microscope；13-c is that the PC3 that the above-mentioned probe through 20 μM is incubated for 65min is thin
Born of the same parents, then with 100 μM of Al3+After being incubated for 50min, the cell picture that is shot under fluorescence inverted microscope blue channel；
Figure 14 is probe to Zn in PC3 cell2+Fluorescent microscopic imaging photo；It is 20 μM that 14-a, which is PC3 cell through concentration,
Probe be incubated for 30min, cleaning, with fluorescence inverted microscope shoot photograph via bright field；14-b is above-mentioned to be incubated for through probe
PC3 cell, with the dark field photo shot under fluorescence inverted microscope green channel；14-c is that the above-mentioned probe through 20 μM is incubated for
The PC3 cell of 30min, then with 100 μM of Zn2+After being incubated for 60min, the cell that is shot under fluorescence inverted microscope green channel
Picture；14-d is the probe incubation 30min that PC3 cell is 20 μM through concentration, cleaning, the light field shot with fluorescence inverted microscope
Photo；Figure 14-e is the above-mentioned PC3 cell being incubated for through probe, is shone with the dark field shot under fluorescence inverted microscope red channel
Piece；Figure 14-f is the PC3 cell that the above-mentioned probe through 20 μM is incubated for 30min, then with 100 μM of Zn2+After being incubated for 60min, glimmering
The cell picture shot under light inverted microscope red channel.
Specific embodiment
Embodiment 1:
1, the preparation of probe:
A kind of chemical name is N, N ', N "-three [4- (benzothiazole -2- base) -2,5- 4-dihydroxy benzaldehyde] contracting-(3- ammonia
Ethyl) amine compound, as trace metal ion Cr in living cells3+、Al3+、Zn2+Fluorescence imaging probe, chemical structure
Formula are as follows:
Its synthetic route is as follows:
It is specific the preparation method comprises the following steps:
(1)N2Under protection in dry 500ml three-necked flask, terephthaldehyde's ether 40mmol, tetrabutyl ethylenediamine is added
200mmol and ether 140ml, obtains A product；
(2) A product are cooled to 0 DEG C hereinafter, obtaining B product；
(3) the n-BuLi 100ml that concentration is 1.6M is added in B product, the reaction was continued 10min under the conditions of 0 DEG C after adding will
Reaction solution reflux 20h obtains C product after reaction solution is cooled to room temperature；
(4) n,N-Dimethylformamide 220mmol is added in C product, is stirred overnight at room temperature；Obtain D product；
(5) 100ml water is added in D product, chloroform extracts (3 × 100ml), and organic phase is dry with anhydrous magnesium sulfate, and decompression removes
Solvent is removed, silica gel column chromatography separation, eluent is n-hexane/toluene that volume ratio is 6/4, obtains yellow solid intermediate c, i.e. E
Product；Yield 35.8%.1H NMR(500MHz,CDCl3, ppm) and δ: 10.484 (s, 2H ,-CHO), 7.440 (S, 2H, ArH),
3.932(s,6H,-OCH3)；
(6) in dry 250ml three-necked flask, E product, o-amino thiophenol 23.8mmol, potassium metabisulfite are added
23.84mmol and 150mlN, dinethylformamide, N2Under protection, back flow reaction 3h is cooled to room temperature after reaction, is obtained
F product；
(7) F product are placed in the ice water of 250mml 0-4 DEG C, and after yellow solid is precipitated completely, filtering, filter cake was dried in vacuo
At night, silica gel column chromatography separation, eluent is toluene, obtains yellow midbody compound d, i.e. G product；Yield 65.8%.1H NMR
(500MHz,CDCl3, ppm) δ: 10.517 (s, 1H ,-CHO), 8.266 (s, 1H, ArH), 8.136 (d, J=8.0Hz, 1H,
ArH), 7.970 (d, J=7.0Hz, 1H, ArH), 7.529 (t, J=9.0Hz, 1H, ArH), 7.529 (s, 1H, ArH), 7.427
(t, J=7.5Hz, 1H, ArH), 4.080 (s, 6H ,-OCH3)；
(8) the dry CH of compound d and 50ml is added in 250ml three-necked flask in G product2Cl2, -30 DEG C are cooled to, H product are obtained；
(9) BBr is added in H product3(14ml, 14.04mmol), -30 DEG C of reaction 1h, obtain J product under nitrogen protection；
(10) it is reacted for 24 hours under J product room temperature, the distilled water that 70ml is then added reacts 2h, with ethyl acetate (3 × 100ml)
Extraction, silica gel column chromatography separate (n-hexane/chloroform, 6:4, v/v), obtain 420mg yellow midbody compound e, i.e. I product, produce
Rate is 44.1%.1H NMR(500MHz,CDCl3, ppm) and δ: 12.089 (s, 1H ,-OH), 10.366 (s, 1H ,-CHO), 9.926
(s, 1H ,-OH), 8.076 (d, J=8.5Hz, 1H, ArH), 7.977 (d, J=8.0Hz, 1H, ArH), 7.573 (t, J=
7.5Hz, 1H, ArH), 7.497 (t, J=8.0Hz, 1H, ArH), 7.365 (s, 1H, ArH), 7.324 (s, 1H, ArH)；
(11) in the there-necked flask of 50ml, intermediate e is added, the dehydrated alcohol of 30ml, reflux makes centre under nitrogen protection
Body e is completely dissolved, with micro-sampling pin inject three (2- aminoethyl) amine 23.3mg (146.24,24 μ l, 4.5mmol, ρ=
0.975), be refluxed overnight, cooled and filtered, ethyl alcohol recrystallization, obtain yellow solid product 110mg to get；Yield 76%.
M.p.264.4-265.3 DEG C, IR (KBr, ν cm-1): 3346 (OH), 1633 (C=N), 1563 (C=C), 1485 (C=C), 1294
(C-N), 864 (Ar-H), 758 (Ar-H).1H NMR(500MHz,DMSO-d6-CDCl3,ppm)δ:13.050(s,3H,-OH×
3), 11.941 (s, 3H ,-OH × 3), 8.551 (s, 3H ,-CH=N × 3), 8.143 (t, J=5.0Hz, 3H, ArH), 7.893
(t, J=4.0Hz, 3H, ArH), 7.658 (t, J=4.0Hz, 6H, ArH), 7.437 (s, 3H, ArH), 7.078 (s, 3H, ArH),
4.018 (t, J=5.0Hz, 6H ,-N=CH2CH2× 3), 3.256 (t, J=5.0Hz, 6H ,-N=CH2CH2×3)。MS
(MALDI-TOF) calculated value [C48H39N7O6S3]: m/z 906.22. measured value: m/z 906.322 [M+H]+。
2, the preparation of each solution, reagent
(1) n,N-Dimethylformamide probe stock solution is prepared: 9.06mg probe (being prepared according to the above method) is weighed,
It is dissolved with n,N-Dimethylformamide, is configured to the solution that 10mL concentration and probe concentration is 1mM.With containing 10% fetal calf serum and containing
The culture medium of 1% dual anti-RPMI-1640 is diluted to 20 μM for cell dyeing.
(2) Isosorbide-5-Nitrae-dioxane probe stock solution is prepared: 9.06mg probe (being prepared according to the above method) is weighed, with 1,
The dissolution of 4- dioxane is configured to the solution that 10mL concentration and probe concentration is 1mM.With containing 10% fetal calf serum and containing 1% it is dual anti-
The culture medium of RPMI-1640 is diluted to 20 μM for cell dyeing.
(3)Al3+Sample solution is prepared: being weighed nine perchloric acid hydrate aluminium 34.33mg, is settled to 100ml physiological saline, is made into
Al3+Concentration is 1mM metal ion stock solution.It is dilute with the culture medium containing 10% fetal calf serum and containing 1% dual anti-RPMI-1640
Releasing to 100 μM can be used for cell dyeing.Al can be adjusted according to dye levels3+Sample diluted concentration.
(4)Cr3+Sample solution is prepared: being weighed chromic nitrate 23.8mg and is settled to 100ml physiological saline, is made into Cr3+Concentration is
1mM metal ion stock solution.100 μM are diluted to the culture medium containing 10% fetal calf serum and containing 1% dual anti-RPMI-1640
It can be used for cell dyeing.Al can be adjusted according to dye levels3+Sample diluted concentration.
(5)Zn2+Sample solution is prepared: being weighed six perchloric acid hydrate zinc 37.24mg and is settled to 100ml physiological saline, is made into
Cr3+Concentration is 1mM metal ion stock solution.It is dilute with the culture medium containing 10% fetal calf serum and containing 1% dual anti-RPMI-1640
Releasing to 100 μM can be used for cell dyeing.Zn can be adjusted according to dye levels2+Sample diluted concentration.
(6) other coexistent metallic ion solution: taking the corresponding perchlorate of each metal, and preparation method is same as above.
(7) 75% ethanol solution: dehydrated alcohol 75mL adds water to 100mL, mixes, and room temperature preservation is spare.
(8) phosphate buffer solution (D-hanks balanced salt solution): 0.4g KCl, 0.06g KH2PO4、8.0g NaCl、
1.0g C6H12O6、0.35gNaHCO3、0.152g Na2HPO4·12H2O, 100,000 IU are dual anti-, and adjustment pH is 7.2~7.4, ultrapure
Water water is settled to 1000mL, and pin type filter (0.22 μm of import miillpore filter) filtration sterilization dispenses spare.
(9) 1 ten thousand unit (IU)/dual anti-solution of mL: 800,000 units of Penicillin sodium are dissolved in 40mL D-hanks solution, are matched
At 20,000 units of final concentration/mL；1,600,000 unit streptomycin sulphates are dissolved in 80mL D-hanks solution, final concentration 20,000 is made into
Unit/mL.Isometric Benzylpenicillin sodium salt solution and streptomycin sulfate solution mixing are taken respectively, obtain Benzylpenicillin sodium salt and sulfuric acid strepto-
The final concentration of element is 10,000 units/mL solution；Pin type filter (0.22m import miillpore filter) filtration sterilization dispenses 1mL/
Branch, -20 DEG C save backup.
(10) 0.25% trypsase: weighing 0.25g trypsase, is dissolved in the D-hanks liquid of 100mL, pin type filter
(0.22 (m import miillpore filter) filtration sterilization dispenses 1mL/ branch to device, and -20 DEG C save backup.
(11) culture solution: with Sterile pipette measure 10mL inactivated fetal calf serum, 90mLRPMI-1640 culture medium with
And the dual anti-liquid of 1mL is mixed in the sterile culture flask of 100mL, 2~8 DEG C save backup.The RPMI is English Roswell
In the abbreviation of Park Memorial Institute, generation, refer to that Loews Wei Pake memorial institute, RPMI are research institute research and development
A kind of cell culture medium, 1640 be culture medium code name.
Sepectrophotofluorometer model Cary Eclipse sepectrophotofluorometer used in the present invention, U.S. VARIAN are public
Department's production；8000 water storage type CO of ThermoFisher2Cell incubator；IX-71 type fluorescence inverted phase contrast microscope, Japan
Olympus company；AR1530/C electronic balance；25cm2Tissue Culture Flask, Corning company of U.S. vertical pressure steam sterilizing
Device (LS-B75)；DHG-9230A electric heating constant-temperature blowing drying box, the upper macro experimental facilities Co., Ltd of Nereid；Spectrum experiment water
For ultrapure water.
3 probes are to Cr3+、Al3+、Zn2+The fluorescence detection of ion
After n,N-Dimethylformamide probe stock solution (1mM, 0.1mL) is added in 10mL volumetric flask, with N, N- diformazan
Base formamide/water solution is diluted to scale, makes n,N-Dimethylformamide/water volume ratio 96/4 in probe test solution, shakes
It is even to be placed at room temperature for 45min, it takes solution 3mL in the cuvette of 1cm, carries out fluorescence spectrometry under the excitation of 320nm wavelength.
After being separately added into n,N-Dimethylformamide probe stock solution (0.1mM, 1mL) in a series of 10mL volumetric flasks,
Being separately added into 0.1mL concentration again is 20mM metal ion Cr3+、Al3+、Zn2+、Li+、Na+、K+、Mg2+、Ca2+、Ba2+、Hg2+、Sr2 +、Cd2+、Ni2+、Co2+、Pb2+、Cu2+、Mn2+、Ag+、Fe3+Sample solution makes to test n,N-Dimethylformamide/water-soluble in solution
Liquid volume ratio is 96/4, shakes up and is placed at room temperature for 45min, solution 3mL is taken to carry out fluorescence spectrometry in the cuvette of 1cm.
The probe prepared according to the above method is taken, is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4, matches
The solution that concentration and probe concentration is 10 μM is made, under the excitation of 320nm wavelength, emits hypofluorescence in 470nm and 610nm, is separately added into
200 μM of metal ion Cr3+、Al3+、Zn2+、Li+、Na+、K+、Mg2+、Ca2+、Ba2+、Hg2+、Sr2+、Cd2+、Ni2+、Co2+、Pb2+、
Cu2+、Mn2+、Ag+、Fe3+After survey its fluorescence spectrum, Cr3+, Al3+Significantly increase fluorescence of the probe at 480nm blue glimmering
Light, Zn2+Enhance probe fluorescence peak at 480nm, and have new fluorescence peak at 540nm, is in yellow-green fluorescence, remaining metal
The fluorescence spectrum that probe is not observed in the addition of ion has significant change (see Fig. 1).Show that probe is to Cr with this condition3+,
Al3+, Zn2+There is recognition detection effect.
The probe prepared according to the above method is taken, is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4, matches
The solution that concentration and probe concentration is 10 μM is made, setting fluorescence exciting wavelength is 320nm, is separately added into various concentration Cr3+It is molten to probe
In liquid, with Cr3+Addition, the fluorescence spectroscopic titration curve measured respectively.With Cr3+Concentration increases, the fluorescence at 480nm
Intensity linearly enhances (see Fig. 2).Measure Cr3+Fluorescence intensity of the probe at 480nm when concentration changes, obtains fluorescence correction curve
(see Fig. 3).
Under test condition same as described above, probe in detecting Cr3+Fluorescence intensity level at 480nm wavelength is in other above-mentioned gold
Belong to ion and is present in probe-Cr respectively as coexisting ion3+In mixed solution, as coexistent metallic ion concentration and Cr3+Ion phase
At that time, probe in detecting Cr3+Fluorescence intensity do not coexisted and influenced (see Fig. 4) by other metal ions.
The probe prepared according to the above method is taken, is dissolved in n,N-Dimethylformamide/aqueous solution that volume ratio is 96/4, matches
The solution that concentration and probe concentration is 10 μM is made, setting fluorescence exciting wavelength is 320nm, is separately added into various concentration Al3+It is molten to probe
In liquid, with Al3+Addition, the fluorescence spectroscopic titration curve measured respectively.With Al3+Concentration increases, the fluorescence at 480nm
Intensity linearly enhances (see Fig. 5).Measure Cr3+Fluorescence intensity of the probe at 480nm when concentration changes, obtains fluorescence correction curve
(see Fig. 6).
Under test condition same as described above, probe in detecting Al3+Fluorescence intensity level at 480nm wavelength is in other above-mentioned gold
Belong to ion and is present in probe-Al respectively as coexisting ion3+In mixed solution, as coexistent metallic ion concentration and Al3+Ion phase
At that time, probe in detecting Al3+Fluorescence intensity do not coexisted and influenced (see Fig. 7) by other metal ions.
4 probes are to Zn2+The fluorescence detection of ion
In 10mL volumetric flask be added Isosorbide-5-Nitrae-dioxane probe stock solution (1mM, 0.1mL) after, with Isosorbide-5-Nitrae-dioxane/
Aqueous solution is diluted to scale, makes Isosorbide-5-Nitrae-dioxane/water volume ratio 1/1 in probe test solution, shakes up and be placed at room temperature for
25min takes solution 3mL in the cuvette of 1cm, carries out fluorescence spectrometry under the excitation of 340nm wavelength.
After being separately added into Isosorbide-5-Nitrae-dioxane probe stock solution (0.1mM, 1mL) in a series of 10mL volumetric flasks, then divide
Not Jia Ru 0.1mL concentration be 20mM metal ion Cr3+、Al3+、Zn2+、Li+、Na+、K+、Mg2+、Ca2+、Ba2+、Hg2+、Sr2+、Cd2 +、Ni2+、Co2+、Pb2+、Cu2+、Mn2+、Ag+、Fe3+Solution makes to test Isosorbide-5-Nitrae-dioxane/water solution volume ratio 1/ in solution
1, it shakes up and is placed at room temperature for 25min, solution 3mL is taken to carry out fluorescence spectrometry in the cuvette of 1cm.
The probe prepared according to the above method is taken, is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, is configured to
The solution that concentration and probe concentration is 10 μM, under the excitation of 340nm wavelength, the fluorescence of probe is very weak, is separately added into 200 μM of metal ion
Cr3+、Al3+、Zn2+、Li+、Na+、K+、Mg2+、Ca2+、Ba2+、Hg2+、Sr2+、Cd2+、Ni2+、Co2+、Pb2+、Cu2+、Mn2+、Ag+、Fe3 +Into probe solution, its fluorescence spectrum, only Zn are surveyed2+Significantly increase fluorescence of the probe at 540nm in yellow fluorescence.Its
The fluorescence spectrum that probe is not observed in the addition of remaining metal ion has significant change (being specifically shown in Fig. 8).Show with this condition
Probe is to Zn2+There is recognition detection effect.
The probe prepared according to the above method is taken, is dissolved in Isosorbide-5-Nitrae-dioxane/water solution that volume ratio is 1/1, is configured to
The solution that concentration and probe concentration is 10 μM, setting fluorescence exciting wavelength are 340nm, and the probe that concentration is 10 μM is 1/1 in volume ratio
In Isosorbide-5-Nitrae-dioxane/water solution, it is separately added into various concentration Zn2+Into probe solution, with Zn2+Addition, measure respectively
Fluorescence spectroscopic titration curve.With Zn2+Concentration increases, and the fluorescence intensity at 540nm linearly enhances (see Fig. 9).Measure Zn2+
Fluorescence intensity of the probe at 540nm when concentration changes, obtains fluorescence correction curve (see Figure 10).
Under test condition same as described above, probe in detecting Zn2+Fluorescence intensity level at 540nm wavelength is in other above-mentioned gold
Belong to ion and is present in probe-Zn respectively as coexisting ion2+In mixed solution, as coexistent metallic ion concentration and Zn2+Ion phase
At that time, Cu is removed2+、Hg2+、Al3+Slightly influence outer, probe in detecting Zn2+Fluorescence intensity do not coexisted and influence by other metal ions
(see Figure 11).
The fluorescent microscopic imaging of 5 activity PC3 cells
(1) recovery cell
The PC3 cell frozen is taken out out of -80 DEG C refrigerators, is placed in 37 DEG C of water and is quickly shaken cell cryopreservation tube, in 1~
It thaws in 2 minutes, in aseptic operating platform, is sucked in centrifuge tube completely, add 11ml culture solution, which contains 10% tire ox
Serum and 1% dual anti-liquid, are mixed, and this cell suspension is removed supernatant liquor in being centrifuged 2min on 1000r/min centrifuge,
Add culture solution featheriness to break up to be mixed and be transferred in culture bottle in the cell of bottom precipitation, make in culture bottle nutrient solution volume 5~
In 7mL, 37 DEG C are placed into, contains 5%CO2Incubator in cultivated.
(2) observation → passage → fishplate bar
Culture solution of replacement daily, and observes cell growth status under the microscope, until PC3 cell it is adherent cover in
In entire culture bottle wall, it can pass on, old culture solution is outwelled in aseptic operating platform, the EDTA liquid intrusion cell 30s of 1mL is added
After outwell, the trypsin solution for adding 1mL is digested, and after observation is until cell size is rounded under the microscope, pats training
Feeding bottle makes cell detachment and appropriate culture solution prevention digestion is added 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 as passage operation, is made carefully after EDTA and trypsase is added
Born of the same parents' digestion falls off and the culture solution prevention digestion of 3mL is added immediately, and preparation is inoculated in 12 orifice plates.It is added in each orifice plate
200 μ L prevented digestion cell liquid, then plus culture solution be mixed after by orifice plate be placed in 37 DEG C, contain 5%CO2Incubator in into
Row culture.
(3) cell dyeing
Cell growth condition in next day observation orifice plate discards old culture solution to the adherent generation of cell, is washed with new culture solution
It washs 2 times, it is spare.
A1 group: the probe of 20 μM of addition is molten in cell culture plate, and solution composition is the culture medium that volume ratio is 98%
N,N-Dimethylformamide with 2%, contains 5%CO at 37 DEG C2Incubator in be incubated for 65min, with fresh modified form RPMI-
1640 culture mediums are washed twice, are placed under fluorescence inverted microscope and are carried out light field and dark-field imaging and take pictures, cell unstressed configuration show (see
Figure 12-a and 12-b)；
A2 group: 20 μM of probe solution is added in cell culture plate, solution composition is the culture that volume ratio is 98%
Base and 2% n,N-Dimethylformamide, 37 DEG C contain 5%CO2Incubator in be incubated for 65min, with contain 10% fetal calf serum
And washed twice containing 1% dual anti-RPMI-1640 culture medium, it is placed under fluorescence inverted microscope and carries out light field and dark-field imaging bat
According to cell unstressed configuration is shown (see attached drawing 13-a and 13-b)；
20 μM of probe solution is added in A3 group in cell culture plate, and solution composition is the culture medium that volume ratio is 98%
Isosorbide-5-Nitrae-dioxane with 2%, contains 5%CO at 37 DEG C2Incubator in be incubated for 30min, with containing 10% fetal calf serum and containing
1% dual anti-RPMI-1640 culture medium is washed twice, is placed in progress light field and dark-field imaging under fluorescence inverted microscope and is taken pictures, carefully
Born of the same parents' unstressed configuration is shown.(see attached drawing 14-a, 14-b, 14-d and 14-e)
B1 group: passing through the processed PC3 cell of step A1 group, and 100 μM of Cr is added in cell culture plate3+Solution,
Solution composition is the water of the culture medium that volume ratio is 90% and 10%, at 37 DEG C, contains 5%CO2Incubator in be incubated for 45min
Afterwards, culture solution is sucked out, is washed twice with containing 10% fetal calf serum and containing 1% dual anti-type RPMI-1640 culture medium, it will be by visiting
Needle and Cr3+The cell that ionic dyeing is crossed observes clearly blue-fluorescence cell point under fluorescence inverted microscope green channel
Cloth takes cell fluorescence image (see Figure 12-c).
B2 group: passing through the processed PC3 cell of step A2 group, and 100 μM of Al is added in cell culture plate3+Solution,
Solution composition is the water of the culture medium that volume ratio is 90% and 10%, contains 5%CO at 37 DEG C2Incubator in be incubated for 50min after,
Culture solution is sucked out, with washing twice containing 10% fetal calf serum and containing 1% dual anti-RPMI-1640 culture medium, will by probe and
Al3+The cell that ionic dyeing is crossed observes clearly blue-fluorescence cell distribution under fluorescence inverted microscope green channel, claps
Cell fluorescence image is taken the photograph, (see Figure 13-c).
B3 group: passing through the processed PC3 cell of step A3, and 100 μM of Zn is added in cell culture plate2+Solution, it is molten
It is 90% culture medium and 10% water that liquid group, which becomes volume ratio, contains 5%CO at 37 DEG C2Incubator in be incubated for 60min after, inhale
Culture solution out is washed twice with containing 10% fetal calf serum and containing 1% dual anti-RPMI-1640 culture medium, will pass through probe and Zn2+
The cell that ionic dyeing is crossed observes that clearly green and red are glimmering under fluorescence inverted microscope green and red channel respectively
Photo-cell distribution, takes cell fluorescence image (see Figure 14-c and 14-f).
Claims (9)
1. Cr in a kind of living cells3+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: with compound N, N ', N "-three
[4- (benzothiazole -2- base) -2,5- 4-dihydroxy benzaldehyde] contracting-(3- aminoethyl) amine is as trace metal ion in living cells
Cr3+、Al3+Or Zn2+Fluorescence imaging probe, probe is compatible with living cells, penetrate into it is intracellular, by probe to intracellular Cr3 +、Al3+Or Zn2+After dyeing, fluorecyte image is detected with fluorescence inverted microscope；The chemical structural formula of the probe are as follows:
2. Cr in living cells as described in claim 13+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
By probe respectively to intracellular Cr3+、Al3+Or Zn2+After dyeing, fluorecyte image is detected with fluorescence inverted microscope；Be by
Following methods carry out:
(1) cell culture: living cells is inoculated in through recovery containing 10% fetal calf serum and containing in 1% dual anti-culture medium, is being trained
It supports and is cultivated in case, after passage, be inoculated in 12 orifice plates and cultivate, density is 2 × 104A/ml, secondary daily culture medium clean cell；
(2) probe is to cell incubation: the culture solution that concentration and probe concentration is 10~40 μM will be added in the cell of (1), incubate in incubator
30~80min is educated, the culture solution containing probe is sucked out, cleans cell with culture medium, is placed under fluorescence inverted microscope and carries out respectively
Light field and dark field are taken pictures, and cell clearly image is observed under light field, cell image is not observed under dark field；The culture solution
Group become volume ratio be 98% culture medium and 2%N, dinethylformamide；
(3)Cr3+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (2)2The Cr of O composition3+Concentration
For 60~120 μM of solution, it is incubated for 30~60min in incubator, cleans cell with culture medium, is placed in fluorescence inverted microscope
Probe is observed under blue channel to intracellular Cr3+Bright blue-fluorescence is presented in fluorescence picture after dyeing, cell, and shooting obtains clear
Clear blue-fluorescence cell outline image, probe detect trace Cr in living cells3+Ion；
(4)Al3+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (2)2The Al of O composition3+Concentration
For 60~120 μM of solution, it is incubated for 30~60min in incubator, cleans cell with culture medium, is placed in fluorescence inverted microscope
Probe is observed under blue channel to intracellular Al3+Bright blue-fluorescence is presented in fluorescence picture after dyeing, cell, and shooting obtains clear
Clear blue-fluorescence cell outline image, probe detect trace of Al in living cells3+Ion；
(5) probe is to cell incubation: the culture solution that concentration and probe concentration is 10~40 μM, the group of culture solution will be added in the cell of (1)
It is 98% culture medium as volume ratio, 2%1,4- dioxane, incubator is interior to be incubated for 20~60min, and the culture containing probe is sucked out
Liquid cleans cell with culture medium, is placed under fluorescence inverted microscope and carries out field light field respectively and dark field is taken pictures, observes under light field
Cell image is not observed under dark field in cell clearly image；
(6)Zn2+To cell dyeing: the culture medium/H for being 9/1 by volume ratio will be added in the cell of (5)2The Zn of O composition2+Concentration
For 60~120 μM of solution, it is incubated for 30~80min in incubator, cleans cell with culture medium, is placed in fluorescence inverted microscope
Probe is observed under green channel to intracellular Zn2+Green fluorescence is presented in fluorescence picture after dyeing, cell, and shooting obtains clearly green
Color fluorecyte contour image, probe detect trace Zn in living cells2+Ion；It is aobvious that above-mentioned cell is placed in fluorescence inversion again
Probe is observed under the red channel of micro mirror to intracellular Zn2+Red fluorescence is presented in fluorescence picture after dyeing, cell, and shooting obtains clear
Clear red fluorescent cell contour image, probe detect trace Zn in living cells2+Ion.
3. Cr in living cells as claimed in claim 23+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
Culture medium；It is RPMI-1640 culture medium.
4. Cr in living cells as claimed in claim 1 or 23+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
Living cells；It is human prostate's cancer cell, abbreviation PC3 cell.
5. Cr in living cells as claimed in claim 1 or 23+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
Fluorescence inverted microscope；The excitation wavelength for being blue channel is 330~385nm, the excitation wavelength of green channel be 450nm~
490nm, the excitation wavelength of red channel are the fluorescence inverted microscope of 510nm~550nm.
6. Cr in the living cells as described in right 1 or 23+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: the spy
Needle is synthesized by following routes:
7. Cr in the living cells as described in right 1 or 23+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: the spy
Needle；It is to be prepared using three (2- aminoethyl) amine, o-amino thiophenol and terephthaldehyde's ether as primary raw material.
8. Cr in living cells as claimed in claim 73+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
Using three (2- aminoethyl) amine, o-amino thiophenol, terephthaldehyde's ether as primary raw material, it is prepared；It is to be made according to the following steps
It is standby:
(1)N2Under protection in dry three-necked flask, terephthaldehyde's ether, tetrabutyl ethylenediamine and dried ether is added,
Obtain A product；
(2) A product are cooled to 0 DEG C hereinafter, obtaining B product；
(3) n-BuLi is added in B product, after adding after 0 DEG C of the reaction was continued 5-15min, reaction solution is flowed back 18-22h, wait react
After liquid is cooled to room temperature, C product are obtained；
(4) n,N-Dimethylformamide is added in C product, is stirred overnight at room temperature, obtain D product；
(5) water is added in D product, and chloroform extraction, organic phase is dry with anhydrous magnesium sulfate, and solvent is removed under reduced pressure, and silica gel column chromatography separates,
Eluent is n-hexane/toluene that volume ratio is 6/4, obtains yellow solid intermediate c, i.e. E product；
(6) in dry three-necked flask, E product, o-amino thiophenol, potassium metabisulfite and n,N-Dimethylformamide, N is added2
Under protection, back flow reaction 2-4h is cooled to room temperature after reaction, obtains F product；
(7) F product are placed in ice water, and after yellow solid is precipitated completely, filtering is dried in vacuum overnight, silica gel column chromatography separation, elution
Liquid is toluene, obtains yellow intermediate d, i.e. G product；
(8) G product and methylene chloride are added in three-necked flask, are cooled to -30 DEG C, obtain H product；
(9) Boron tribromide is added in H product, -30 DEG C of reaction 0.5-1.5h, obtain J product under nitrogen protection；
(10) after reacting 20-28h under J product room temperature, distilled water is added and reacts 1-3h, is extracted with ethyl acetate, silica gel column chromatography point
From eluent is n-hexane/chloroform that volume ratio is 6/4, obtains yellow intermediate e to get I product；
(11) in there-necked flask, intermediate compound I product are added, dehydrated alcohol, reflux is completely dissolved intermediate e under nitrogen protection, uses
Micro-sampling pin inject three (2- aminoethyl) amine, be refluxed overnight, cooled and filtered, ethyl alcohol recrystallization, obtain yellow solid to get.
9. Cr in living cells as claimed in claim 83+、Al3+Or Zn2+Fluorescence imaging method, it is characterised in that: it is described
Using three (2- aminoethyl) amine, o-amino thiophenol and terephthaldehyde's ether as primary raw material, it is prepared；It is to be made according to the following steps
It is standby:
(1)N2Under protection in dry 500ml three-necked flask, terephthaldehyde's ether 40mmol, tetrabutyl ethylenediamine is added
200mmol and ether 140ml, obtains A product；
(2) A product are cooled to 0 DEG C hereinafter, obtaining B product；
(3) the n-BuLi 100ml that concentration is 1.6M is added in B product, the reaction was continued 10min under the conditions of 0 DEG C, will react after adding
Liquid reflux 20h obtains C product after reaction solution is cooled to room temperature；
(4) n,N-Dimethylformamide 220mmol is added in C product, is stirred overnight at room temperature；Obtain D product；
(5) 100ml water is added in D product, chloroform extracts (3 × 100ml), and organic phase is dry with anhydrous magnesium sulfate, is removed under reduced pressure molten
Agent, silica gel column chromatography separation, eluent is n-hexane/toluene that volume ratio is 6/4, obtains yellow solid intermediate c, i.e. E product；
(6) in dry 250ml three-necked flask, E product, o-amino thiophenol 23.8mmol, potassium metabisulfite are added
23.84mmol and 150mlN, dinethylformamide, N2Under protection, back flow reaction 3h is cooled to room temperature after reaction, is obtained
F product；
(7) F product are placed in the ice water of 250mml 0-4 DEG C, and after yellow solid is precipitated completely, filtering, filter cake is dried in vacuum overnight,
Silica gel column chromatography separation, eluent is toluene, obtains yellow midbody compound d, i.e. G product；
(8) G product and methylene chloride 50ml are added in 250ml three-necked flask, are cooled to -30 DEG C, obtain H product；
(9) Boron tribromide 14.04mmol is added in H product, -30 DEG C of reaction 1h, obtain J product under nitrogen protection；
(10) after being reacted for 24 hours under J product room temperature, the distilled water reaction 2h of 70ml is added, is extracted with ethyl acetate (3 × 100ml), silicon
Plastic column chromatography separation, eluent is n-hexane/chloroform that volume ratio is 6/4, obtains yellow intermediate e to get I product；
(11) in the there-necked flask of 50ml, I product and dehydrated alcohol 30ml is added, reflux is completely dissolved I product under nitrogen protection, infuses
Enter three (2- aminoethyl) amine 23.3mg, is refluxed overnight, cooled and filtered, ethyl alcohol recrystallization obtains probe.
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