CN102146284B - Ratiometric fluorescent probe and application thereof - Google Patents
Ratiometric fluorescent probe and application thereof Download PDFInfo
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- CN102146284B CN102146284B CN201010108366.2A CN201010108366A CN102146284B CN 102146284 B CN102146284 B CN 102146284B CN 201010108366 A CN201010108366 A CN 201010108366A CN 102146284 B CN102146284 B CN 102146284B
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 32
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 14
- 150000002910 rare earth metals Chemical class 0.000 claims abstract description 11
- 239000003446 ligand Substances 0.000 claims abstract description 10
- -1 rare-earth ions Chemical class 0.000 claims abstract description 6
- 239000013110 organic ligand Substances 0.000 claims abstract description 4
- 230000008859 change Effects 0.000 claims description 9
- 238000003384 imaging method Methods 0.000 claims description 6
- 238000013329 compounding Methods 0.000 claims description 4
- 229910021645 metal ion Inorganic materials 0.000 claims description 3
- 238000001685 time-resolved fluorescence spectroscopy Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 14
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 abstract 3
- 238000000034 method Methods 0.000 description 21
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- 238000003756 stirring Methods 0.000 description 18
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Abstract
The invention relates to a novel rare-earth complex ratiometric fluorescent probe which can be used for measuring peroxynitrite in a biosystem. The probe is complexes formed by two trivalent rare-earth ions Eu<3+> and Tb<3+> and an organic ligand [4'-(2,4-dimethoxyphenyl)-2,2':6'2''-terpyridine-6,6''-dimethylamino]tetraacethyl (DTTA for short), wherein the structural formula of the ligand is disclosed in the specification. The probe can specifically act with the peroxynitrite, so that the two rare-earth complexes are changed at the ratio (F612/F541) of the fluorescence intensity of 612nm and 541nm, thereby implementing the selective fluorescent measurement of the peroxynitrite in a biosystem.
Description
Technical field
The present invention relates to the determination techniques of peroxynitrite negatively charged ion, specifically a kind of peroxynitrite negatively charged ion ratio type fluorescent probe and application based on rare earth compounding is a kind of fluorimetric Ratio-type rare-earth fluorescent of peroxynitrite anion-selective probe in living things system that can be used for.
Background technology
Peroxynitrite negatively charged ion (ONOO in living things system
-) be by nitrogen protoxide (NO) and ultra-oxygen anion free radical (O
2 -) rapidly in conjunction with a kind of important nitrogen oxygen activity compound generated.It has been generally acknowledged that ONOO
-than NO and O
2 -oxygenizement is stronger, effect a kind of potent cytotoxic substance (document 1:J.S.Beckman, T.W.Beckman, Proc.Natl.Acad.Sci.USA.1990,87,1620 more widely; Document 2:G.Ferrer-Sueta, R.Radi, ACS Chem.Biol.2009,4,161).As a kind of strong oxidizer, due to ONOO
-can produce the oxidative damage of cell and tissue, therefore with various disease conditions: as (document 3:C.Szabo that is associated such as shock, acute or chronic inflammation, septicemia, traumatic local asphyxia and arteriosclerosis, H.Ischiropoulos, R.Radi, Nat.Rev.Drug Discov.2007,6,662).Simultaneously, it can also the oxidation cytolemma, mercapto functional group and the biomacromolecules such as enzyme, albumen, lipid and DNA, is the important factor that causes cell injury, energy exhaustion and necrocytosis.Due to ONOO
-have very high mobility, can penetrate at an easy rate the lipid bilayer, this makes it not only can destroy cell, and near the cell being penetrated into, and these character have all been aggravated ONOO
-destruction to organism.
In order from molecular level, to be familiar with ONOO
-to the oxygenizement mechanism of cell, tissue and whole organism, the nineties in last century, the biochemist is for ONOO in living things system
-generation, metabolism with and pathology damage mechanism carried out large quantity research.Yet, due to ONOO in vivo
-there is extremely strong reactive behavior and extremely short lifetime (transformation period only has 0.8s), and can from many different compound generation chemical reactions, generate various reaction product, this just gives ONOO in living things system
-mensuration bring many difficulties.At present, ONOO in living things system
-mensuration mainly contain following two kinds of methods: (1) is based on ONOO
-the signal intensity produced with reacting of probe molecule carrys out method for measuring, comprises ultraviolet-visible luminosity method, fluorescent method, electrochemical process and chemoluminescence method (document 4:S.B.Digerness, K.D.Harris, J.W.Kirklin, Free Radic.Biol.Med.1999,27,1386; Document 5:J.Xue, X.Ying, J.Chin, Anal.Chem.2000,72,5313; Document 6:S.Dikalov, M.Skatchkov, E.Basseneg, Biochem.Biophys.Res.Commun.1997,230,54); (2) by measuring ONOO
-infer ONOO with the reaction product of biomolecules
-method, as measured the method (document 7:D.A.Richards, M.A.Silva, Anal.Biochem.2006,351,77) of tyrosine nitration product in protein molecule.Adopting the method for fluorescent molecular probe in aforesaid method is a kind of simple, sensitive and special analytical procedure, and up to the present, existing several fluorescent molecular probes are by successfully for ONOO
-mensuration, as dichloro-dihydro fluorescein (DCFH) and dihydro rhodamine (DHR) (document 8:H.Possel, H.Noack, FEBS Lett.1997,416,175; Document 9:N.W.Kooy, J.A.Royall, Free Radic.Biol.Med.1994,16,149), though the method has higher sensitivity, this type of probe specificity is poor, and other biological oxidant also can oxidation DCFH and luminous dichlorofluorescein and the rhodamine of DHR generation hyperfluorescenceZeng Yongminggaoyingguang as hypochlorous acid, sulfydryl and peroxidase.In recent years, two kinds of New O NOO based on aromatic ring nitration and ketone oxidizing reaction
-fluorescent probe: NiSPYs and HKGreens, successively be synthesized out (document 10:T.Ueno, T.Nagano, J.Am.Chem.Soc.2006,128,10640; Document 11:D.Yang, H.Wang, J.Am.Chem.Soc.2006,128,600; Document 12:Z.Sun, H.Wang, D.Yang, Org.Lett.2009,11,1887), these two kinds of probes can with ONOO
-specific reaction occurs and cause the remarkable enhancing of fluorescence intensity, but probe is water-soluble poor, easily little by photobleaching, Stokes displacement, and in cell, retention time is short, is unfavorable for long-term observation and the determination of trace of peroxynitrite negatively charged ion in cell.In addition, current existing ONOO
-fluorescent probe is all usingd fluorescence intensity as characterization parameter, although it is simple to operate, the accuracy of its result but easily is subject to outside atmosphere and instrument condition to change the impact as factors such as optical path, photobleaching, scattering and bias lights.And the ratio fluorescent method can address these problems preferably, this method is by measuring the fluorescence intensity at two different wave length places, using a kind of measuring method of its ratio as characterization parameter, with traditional fluorescent probe, compare, Ratiometric fluorescent probe generally has higher selectivity and sensitivity, and (J.Phys.Chem.C 2009 for document 13:H.Li, H.Yan, 113,7526).
The present invention, on the basis to the ligand structure design, has synthesized a kind of ONOO in living things system (viable cell) that can be used for
-the Ratio-type rare earth coordination compound fluorescent probe that specificity fluorescent is measured, and utilize this probe binding time to differentiate the fluorometric assay technology and set up ONOO in a kind of viable cell
-the real time measure technology.
Summary of the invention
The purpose of this invention is to provide a kind of highly sensitive, selectivity and good water solubility, applied widely, can be used for ONOO in living things system
-novel rare-earth title complex Ratiometric fluorescent probe and the application thereof measured.
Technical scheme of the present invention is as follows:
With two kinds of trivalent rare earth ions Eu
3+and Tb
3+with organic ligand [4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] title complex that forms of tetraacethyl (be called for short DTTA) (is called for short DTTA-Eu
3+/ Tb
3+) be fluorescent probe, wherein said ligand structure formula is:
The mol ratio of ligand and metal ion is 1: 1, DTTA-Eu
3+with DTTA-Tb
3+molar ratio in 1: 5 to 5: 1 scope, change, be preferably 1: 2.
The application process of the described peroxynitrite negatively charged ion ratio type fluorescent probe based on above-mentioned rare earth compounding is: in different kind organism and abiotic environment, utilize described rare earth coordination compound fluorescent probe DTTA-Eu
3+/ Tb
3+with ONOO in system
-interaction, cause the ratio (F of probe in 612nm and 541nm wavelength place fluorescence intensity
612/ F
541) significantly strengthen, then by the ratio fluorescent method, determine ONOO
-generation and growing amount.Described fluorometry, except conventional fluorometry, also comprises time-resolved fluorometry and time resolution fluorescent microscope imaging assay method.
ONOO in described solution system
-concentration best>=10
-6mol/L.
Fluorescent probe of the present invention has following advantage:
1. have well water-solublely, be applicable to ONOO in biology and abiotic environment
-mensuration.
2. stability is high, can prolonged preservation uses, and is applicable to slightly acidic, neutrality and the multiple environment such as alkaline.
3. with the ratio F of two different emission place fluorescence intensities
612/ F
541for signal parameter, the specificity and the sensitivity that detect have been improved.
4. to ONOO
-good selectivity is arranged, with other active oxygen species, make used time fluorescence intensity ratio value F
612/ F
541almost unchanged.
5. the time-resolved fluorometry that can be used for hundreds of Microsecond grades, to eliminate the interference of background fluorescence to measuring.
6. can be used for the highly sensitive fluorescence imaging of peroxynitrite negatively charged ion in viable cell measures.
The accompanying drawing explanation
Fig. 1 is the synthetic route of ligand DTTA.
In Fig. 2, (A) is DTTA-Eu
3+(solid line, 2.0 μ mol/L) and DTTA-Tb
3+(dotted line, 2.0 μ mol/L) time resolution fluorescence spectral in the 0.05mol/L of pH value 7.4 Tris-HCl buffered soln; (B) be different Eu
3+/ Tb
3+dTTA-Eu during mol ratio
3+/ Tb
3+the time resolved fluorescence emmission spectrum of mixture (total concn is 2.0 μ mol/L); (C) be a series of DTTA-Eu under the 365nm ultra violet lamp
3+/ Tb
3+the fluorescence radiation photo of mixture.
In Fig. 3 (A) and (B) be respectively DTTA-Eu
3+(1.0 μ mol/L) and DTTA-Tb
3+(1.0 μ mol/L) fluorescence intensity (■) and fluorescence lifetime (zero) in the 0.05mol/L of different pH values Tris-HCl buffered soln.
In Fig. 4 (A) and (B) be respectively DTTA-Eu
3+(2.0 μ mol/L) and DTTA-Tb
3+(2.0 μ mol/L) and different concns ONOO
-various photoluminescent properties (: fluorescence intensity after reaction; △: fluorescence lifetime; Zero: changing conditions time resolved fluorescence intensity).
In Fig. 5 (A) and (B) be respectively DTTA-Tb
3+(2.0 μ mol/L) and DTTA-Eu
3+(2.0 μ mol/L) and different concns ONOO
-reacted time resolution fluorescence spectral.
Fig. 6 is DTTA-Eu
3+/ Tb
3+(2.0 μ mol/L, Eu
3+/ Tb
3+=1/2) with different concns ONOO
-reacted time-resolved emission spectrum, the ratio F that illustration is two transmitted wave strong point fluorescence intensities
612/ F
541with ONOO
-the changing conditions of concentration.
In Fig. 7 (A) and (B) be respectively DTTA-Tb
3+(2.0 μ mol/L) and DTTA-Eu
3+/ Tb
3+(2.0 μ mol/L, Eu
3+/ Tb
3+=1/2) in the 0.05mol/L of pH value 7.4 Tris-HCl buffered soln with the reacted fluorescence response situation of various active oxygen species.
Fig. 8 is DTTA-Tb
3+the HeLa cell of mark is at different concns ONOO
-(A: negative control under donor SIN-1 exists; B:500 μ mol/L; C:1.0mmol/L) a fluorescence imaging measurement result (left side: light field imaging; In: common fluorescence imaging; Right: the time resolved fluorescence imaging).Scale: 10 microns.
Fig. 9 is DTTA-Eu
3+/ Tb
3+the HeLa cell of mark is at different concns ONOO
-(A: negative control under donor SIN-1 exists; B:100 μ mol/L; C:200 μ mol/L; D:500 μ mol/L) a fluorescence imaging measurement result (left side: light field; Right: common fluorescence imaging).Scale: 10 microns.
Figure 10 is DTTA-Eu
3+the fluorescence imaging measurement result of the HeLa cell of mark after placing different time.Scale: 10 microns.
Embodiment
Below by embodiment, the invention will be further described.The present embodiment is only for the present invention will be described, and the method based on same principle and similar raw material also belongs to scope of the present invention.
Embodiment 1: ligand [4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] tetraacethyl (be called for short DTTA) and methyl acetic acid ester (being called for short AM-DTTA) thereof synthetic.
1.DTTA synthetic
As shown in Figure 1, the elementary operation process is as follows for synthetic route:
(1) 4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 1) synthetic
By 3.32 grams 2,4-dimethoxy benzaldehyde (20mmol), 4.84 gram 2-acetopyridine (40mmol), and 2.64 gram KOH (85%, 40mmol) mix and be dissolved in the 100mL dehydrated alcohol, then add the 50mL strong aqua, stir 10 hours under 50 ℃, filter collecting precipitation, thick product ethyl alcohol recrystallization, obtain target compound 3.61 grams, productive rate: 48.9%.
1h NMR (CDCl
3) measurement result: δ=8.71 (d, J, 4.8Hz, 2H); (8.66 d, J, 7.6Hz, 2H); (8.62 s, 2H); (7.86 t, J, 7.6Hz, 2H); (7.49 d, J, 8.4Hz, 1H); (7.33 t, J, 6.0Hz, 2H); (6.61-6.58 m, 2H); (3.88 s, 3H); (3.85 s, 3H); Results of elemental analyses: press C
23h
19n
3o
2calculated value (%): C, 74.78; H, 5.18; N, 11.37; Measured value (%): C, 74.31; H, 5.23; N, 11.46.
(2) 6,6 "-dinitrile-4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 2) synthetic
By 3.69 digest compound 1 (10.0mmol) and 8.63 gram m-chloro-benzoic acid peroxides (80%, 40.0mmol) be dissolved in the 200mL methylene dichloride, under room temperature, stir 24 hours.The Na of 150mL10% for organic phase
2cO
3solution washing twice, after anhydrous sodium sulfate drying, remove solvent under reduced pressure, vacuum-drying.Product obtained above is dissolved in to 250mL CH
2cl
2in, add 7.93 gram (CH
3)
3siCN (80mmol), stir after 2 hours under room temperature, slowly is added dropwise to 7.03 gram Benzoyl chlorides (50mmol), under room temperature, stirs 24 hours.Remove solvent under reduced pressure, add 250mL10%K
2cO
3solution, stir 1 hour under room temperature.Filter collecting precipitation, water and acetonitrile washing final vacuum drying.Obtain target compound 1.89 grams, productive rate: 45.2%.
1h NMR (CDCl
3) measurement result: δ=8.84 (d, J, 8.0Hz, 2H); (8.73 s, 2H); (8.00 t, J, 8.0Hz, 2H); (7.74 d, J, 7.6Hz, 2H); (7.50 d, J, 8.8Hz, 2H); (6.69-6.63 m, 2H); (3.91 s, 6H).
(3) 4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dioctyl phthalate dimethyl ester (compound 3) synthetic
Digest compound 2 (8.0mmol) by 3.35 and be dissolved in the mixing solutions that the 16mL vitriol oil, 48mL Glacial acetic acid and 12mL water is made into, stir 12 hours under 93 ℃.Reaction solution, to entering in the 500mL frozen water, filters collecting precipitation, washing final vacuum drying.
By 8.24 gram SOCl
2(69.2mmol) slowly join under ice bath in the methyl alcohol of 250mL drying, after stirring 30 minutes, add the said hydrolyzed product, stirring and refluxing 24 hours.Remove solvent under reduced pressure, add 200mL10%Na
2cO
3the solution neutralization, filter collecting precipitation, vacuum-drying.Thick product separates with silica gel column chromatography, with the chloroform-methanol wash-out of 95: 5, obtains target compound 2.90 grams, productive rate: 74.7%.
1h NMR (CDCl
3) measurement result: δ=8.81 (d, J, 7.6Hz, 2H); (8.72 s, 2H); (8.15 d, J, 7.6Hz, 2H); (8.00 t, J, 8.0Hz, 2H); (7.51 d, J, 8.4Hz, 2H); (6.64-6.60 m, 2H); (4.03 s, 6H); (3.89 s, 3H); (3.86 s, 3H).
(4) 6,6 "-dihydroxymethyl-4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 4) synthetic
Digest compound 3 (8.9mmol) by 4.31 and be dissolved in the 150mL dehydrated alcohol, stir and form suspension liquid.Add 1.35 gram NaBH
4(46.4mmol), under room temperature, stir 2 hours, then return stirring 6 hours.Remove solvent under reduced pressure, add the saturated NaHCO of 25mL
3solution is heated to boil.Add 100mL water after cooling, in the reaction solution refrigerator, (～4 ℃) are placed and are spent the night, and filter collecting precipitation, thick product water and acetonitrile washing, vacuum-drying.Obtain target compound 3.58 grams, productive rate: 93.8%.
1h NMR (CDCl
3) measurement result: δ=8.61 (s, 2H); (8.53 d, J, 8.0Hz, 2H); (7.84 t, J, 7.6Hz, 2H); (7.46 d, J, 8.4Hz, 2H); (7.24 d, J, 5.6Hz, 2H); (6.66-6.63 m, 2H); (4.84 s, 4H); (3.89 s, 3H); (3.87 s, 3H).
(5) 6,6 "-bis-brooethyls-4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 5) synthetic
Add 4.97 gram PBr in the anhydrous DMF of 130mL
3(18.5mmol), under room temperature, stir after 15 minutes and add 3.17 to digest compound 4 (7.4mmol), continue to stir 24 hours.Reaction adds saturated NaHCO after finishing
3the solution neutralization, filter collecting precipitation, thick product water and acetonitrile washing, vacuum-drying.Obtain target compound 3.86 grams, productive rate: 94.1%.
1h NMR (CDCl
3) measurement result: δ=8.66 (s, 2H); (8.55 d, J, 8.0Hz, 2H); (7.86 t, J, 7.6Hz, 2H); (7.51-7.47 m, 3H); (6.67-6.62 m, 2H); (4.65 s, 4H); (3.90 s, 3H); (3.88 s, 3H).
(6) 4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacethyl ethyl ester (compound 6) synthetic
Digest compound 5 (5.6mmol), 2.34 gram ethyl diacetate base amine (12.4mmol) and 7.70 gram anhydrous K by 3.10
2cO
3(56mmol) in the mixing solutions that adds the tetrahydrofuran (THF) of the acetonitrile of 350mL drying and 100mL drying to be made into, stirring and refluxing 24 hours.Remove by filter insolubles, remove solvent under reduced pressure.Resultant is dissolved in the 200mL ethyl acetate, with the 5%NaHCO of equal volume
3and water washing.The organic phase anhydrous sodium sulfate drying, remove under reduced pressure after solvent and separate with silica gel column chromatography, and eluent is ethyl acetate, removes product normal hexane recrystallization after solvent under reduced pressure, vacuum-drying.Obtain target compound 2.32 grams, productive rate: 53.3%.
1h NMR (CDCl
3) measurement result: δ=8.61 (s, 2H); (8.54 d, J, 7.6Hz, 2H); (7.88 t, J, 6.8Hz, 2H); (7.64 d, J, 6.8Hz, 2H); (7.52 d, J, 8.8Hz, 1H); (6.65-6.61 m, 2H); (4.20-4.12 m, 12H); (3.89 s, 3H); (3.86 s, 3H); (3.71 s, 8H); (1.22 t, J, 7.2Hz, 12H). results of elemental analyses: press C
41h
49n
5o
10calculated value (%): C, 63.80; H, 6.40; N, 9.07. measured value (%): C, 63.31; H, 6.37; N, 9.34.
(7) [4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] tetraacethyl (DTTA) synthetic
Digest compound 6 (1.0mmol) by 0.77 and be dissolved in the mixing solutions that 35mL ethanol and 6mL water is made into, then add 1.50 gram KOH, return stirring 2 hours.After removing solvent under reduced pressure, product is dissolved in 30mL water, and the hydrochloric acid adjust pH to 2 of use 1mol/L～3, stir 3 hours under room temperature, filters collecting precipitation, washes with water.After vacuum-drying, product is added in the 30mL acetonitrile, stirring and refluxing 30 minutes, filter collecting precipitation, obtains target compound 0.43 gram, productive rate: 65.3% after vacuum-drying.
1hNMR (DMSO-d
6) measurement result: δ=8.54-8.52 (m, 4H); (8.02 d, J, 7.6Hz, 2H); (7.62 d, J, 7.6Hz, 2H); (7.50 d, J, 8.4Hz, 2H); (6.77-6.72 m, 2H); (4.08 s, 4H); (3.86 d, J, 2.4Hz, 6H). results of elemental analyses, press C
33h
33n
5o
104H
2o calculated value (%): C, 54.18; H, 5.65; N, 9.57; Measured value (%): C, 53.69; H, 5.03; N, 9.56.Mass spectrometry results: molecular ion peak m/z=658.3 (relative intensity 100%).
2.DTTA methyl acetic acid ester (AM-DTTA) is synthetic
By 18.5 milligrams of DTTA (0.025mmol), 14.6 mixing, milligram triethylamine (1.3mmol) and 37.5mg brooethyl acetic ester (0.25mmol) be dissolved in the dimethyl sulfoxide (DMSO) of 0.5mL drying, stirred overnight at room temperature, obtain brown AM-DTTA dimethyl sulphoxide solution, test for cell imaging.Mass spectrometry results: molecular ion peak m/z=948.3 (relative intensity 25%).
2: two kinds of rare earth compounding DTTA-Eu of embodiment
3+and DTTA-Tb
3+property testing
1. spectral quality
With the 0.05mol/L borate buffer solution of pH value 9.1, be that solvent has been measured DTTA-Eu
3+and DTTA-Tb
3+ultraviolet-visible absorption spectroscopy, fluorescence spectrum, molar extinction coefficient (ε), fluorescence quantum yield (φ) and the fluorescence lifetime (τ) of (in two kinds of title complexs, the mol ratio of ligand and metal ion is 1: 1).It is Perkin Elmer Lambda 35 type spectrophotometers with instrument that ultraviolet-visible absorption spectroscopy is measured.The fluorometric assay instrument is Perkin Elmer LS 50B spectrophotofluorometer.Fluorescence quantum yield is measured and is used 4 '-phenyl-2,2 ': 6 ', 2 "-Lian tri-pyrroles-6,6 "-dimethylamine tetraacethyl and Eu
3+and Tb
3+title complex record (document 14:M.Latva, H.Takalo, J.Kankare, J.Lumin.1997,75,149) as standard substance by literature method, calculating formula is φ
1=I
1ε
2c
2φ
2/ I
2ε
1c
1, I in formula
2, ε
2, C
2, φ
2for fluorescence intensity, molar extinction coefficient, concentration and the fluorescent quantum yield of standard substance, I
1, ε
1, C
1, φ
1fluorescence intensity, molar extinction coefficient, concentration and fluorescence quantum yield for determinand.Measurement result is in Table 1.
Table 1. title complex DTTA-Eu
3+and DTTA-Tb
3+absorption in borate buffer solution and photoluminescent property
| Title complex | The maximum excitation wavelength, nm | Maximum emission wavelength, nm | Molar extinction coefficient, cm -1mol -1L | Fluorescence quantum yield, % | Fluorescence lifetime, ms |
| DTTA-Eu 3+ DTTA-Tb 3+ | 335 335 | 612 541 | 24200 25700 | 10.0 9.9 | 1.38 0.26 |
As can be seen from Table 1, DTTA-Eu
3+with DTTA-Tb
3+not only all there is higher fluorescence quantum yield, larger molar extinction coefficient, and also both have identical maximum excitation wavelength, so both are mixed and can obtain a series of different DTTA-Eu that form by different mol ratio
3+/ Tb
3+mixed ligand complex, the emmission spectrum of these mixed ligand complexs is with Eu
3+/ Tb
3+the variation of ratio presents regular variation, can launch the fluorescence (as shown in Figure 2) of different colours under ultraviolet excitation.
2. pH is to DTTA-Eu
3+with DTTA-Tb
3+the impact of photoluminescent property
In the 0.05mol/L of different pH values Tris-HCl buffered soln, measured DTTA-Eu
3+with DTTA-Tb
3+fluorescence intensity and fluorescence lifetime, the results are shown in Figure 3.From Fig. 3 result, can find out, in the scope of pH value 3～10, DTTA-Eu
3+with DTTA-Tb
3+fluorescence intensity and fluorescence lifetime substantially be not subject to the impact of pH value, show that these two kinds of title complexs all can be used in slightly acidic, neutrality and weakly alkaline environment.
Embodiment 3: with DTTA-Eu
3+/ Tb
3+for fluorescent probe is measured ONOO in the aqueous solution
-concentration
1.ONOO
-to DTTA-Eu
3+with DTTA-Tb
3+the impact of photoluminescent property
Add respectively DTTA-Ln at the 0.05mol/L of a series of pH values 7.4 Tris-HCl in buffered soln
3+(Ln
3+=Eu
3+or Tb
3+, 2.0 μ mol/L) and the ONOO of different concns
-, stirring reaction, after 15 minutes, is measured fluorescence intensity and the fluorescence lifetime of each solution.Measuring is PerkinElmer LS 50B spectrophotofluorometer with instrument.
As shown in Figure 4, along with ONOO
-the increase of concentration, DTTA-Eu
3+fluorescence intensity and fluorescence lifetime substantially do not change; But DTTA-Tb
3+fluorescence intensity reduce gradually, fluorescence lifetime obviously shortens, time resolved fluorescence intensity significantly weakens.Fig. 5 has provided DTTA-Eu
3+and DTTA-Tb
3+at different concns ONOO
-time resolution fluorescence spectral while existing, show title complex DTTA-Tb
3+can be used as the intensity type fluorescent probe for quantitative assay solution ONOO
-concentration.
2. with DTTA-Eu
3+/ Tb
3+for fluorescent probe is measured ONOO in the aqueous solution
-concentration
Fig. 5 experimental result shows, along with ONOO
-the increase of concentration, DTTA-Tb
3+fluorescence intensity reduce gradually, and DTTA-Eu
3+fluorescence intensity substantially constant.Therefore, by DTTA-Tb
3+and DTTA-Eu
3+mix by a certain percentage, can obtain a kind of ONOO of can be used for
-the Ratio-type rare earth coordination compound fluorescent probe DTTA-Eu of quantitative assay
3+/ Tb
3+(DTTA-Eu
3+with DTTA-Tb
3+molar ratio can in 1: 5 to 5: 1 scope, change, best than being 1: 2).Fig. 6 has provided Ratio-type probe DTTA-Eu
3+/ Tb
3+(2.0 μ mol/L, Eu
3+/ Tb
3+=1/2) at different concns ONOO
-time resolved fluorescence emmission spectrum while existing (excitation wavelength: 335nm), the ratio (F that illustration is two wavelength place fluorescence intensities
612/ F
541) with ONOO
-the change curve of concentration, show DTTA-Eu
3+/ Tb
3+can be used as the Ratio-type probe for quantitative assay solution ONOO
-concentration, detectability is about 7 * 10
-6mol/L.
Embodiment 4: probe DTTA-Tb
3+with DTTA-Eu
3+/ Tb
3+to ONOO
-the selectivity of measuring
Investigated respectively active specy H
2o
2, hydroxyl radical free radical (OH), ClO
-, singlet oxygen (
1o
2), NO
2 -, NO
3 -, NO, O
2 -and ONOO
-with DTTA-Tb
3+and DTTA-Eu
3+/ Tb
3+(Eu
3+/ Tb
3+=1/2) response situation.Same concentrations and reaction conditions (respond and all carry out under room temperature in the 0.05mol/L of pH value 7.4 Tris-HCl buffered soln, the reaction times is 30 minutes, reaction density is: DTTA-Tb
3+=2.0 μ mol/L; DTTA-Eu
3+/ Tb
3+=2.0 μ mol/L; Active specy=200 μ mol/L) lower 9 kinds of active species and probe DTTA-Tb
3+(or DTTA-Eu
3+/ Tb
3+) fluorescence intensity (or the ratio F of two wavelength place fluorescence intensities of reaction product
612/ F
541), measurement result is as shown in Figure 7.Can find out probe DTTA-Tb from Fig. 7 result
3+with H
2o
2, OCl
-,
1o
2, NO
3 -, O
2 -considerable change (<5%) does not occur in reacted fluorescence intensity; With ONOO
-after reaction, the fluorescence intensity of probe significantly reduces (98%); With NO, OH and NO
2 -after reaction, the fluorescence intensity of probe has reduced respectively approximately 20%, 30% and 50%.This illustrates ONOO
-to DTTA-Tb
3+the cancellation effect of fluorescence not only comes from the strong oxidizing property of himself, and with other degradation production as NO, OH, NO
2 -deng relation (document 15:C.Szabo, H.Ischiropoulos, R.Radi, Nat.Rev.Drug Discov.2007,6,662) is also arranged.Yet, with DTTA-Tb
3+compare Ratio-type probe DTTA-Eu
3+/ Tb
3+to ONOO
-selectivity greatly improve, work as DTTA-Eu
3+/ Tb
3+with NO,
1o
2, O
2 -, OH, ClO
-, H
2o
2and NO
3 -after reaction, F
612/ F
541substantially constant (variation is less than 0.1), with NO
2 -f after reaction
612/ F
541only increased by 0.7 times, this and ONOO
-the impact of (5.6 times) is compared or is much smaller.The above results shows Ratiometric fluorescent probe DTTA-Eu
3+/ Tb
3+to ONOO
-mensuration has good selectivity.
Embodiment 5: probe DTTA-Tb
3+and DTTA-Eu
3+/ Tb
3+for ONOO in viable cell
-fluorescence imaging measure
1. probe DTTA-Tb
3+for ONOO in viable cell
-the time resolved fluorescence imaging measure
The dimethyl sulphoxide solution and the TbCl that contain AM-DTTA (0.05mol/L) by new system
3(0.05mol/L) aqueous solution joins the HeLa cell (4 * 10 that 1.0mL cultivates with RPMI-1640
5cells/mL) in (DMSO concentration is 1.0%), at 5%CO
2cultivate under 37 ℃ after 2 hours in incubator, discard nutrient solution and ooze the abundant washed cell of salts solution to remove the probe molecule that does not enter cell by grade, to the ONOO that adds different concns in culturing bottle
-the grade of donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazoles (being called for short SIN-1) is oozed salts solution, at 5%CO
2cultivate after 0.5 hour and carry out fluorescence imaging mensuration under 37 ℃ in incubator.
Fig. 8 has provided the DTTA-Tb added after different concns SIN-1
3+the fluorescence imaging figure of labeled cell.From Fig. 8 result, can find out, when not adding SIN-1, DTTA-Tb
3+the HeLa cell of mark is launched bright cyan fluorescence (cell blue-fluorescence and DTTA-Tb
3+due to green fluorescence mixes), after adopting the time resolved fluorescence imaging pattern to measure, the blue background fluorescence of cell is completely eliminated, and only demonstrates DTTA-Tb
3+the feature green fluorescence.Along with the increase that adds SIN-1 concentration, the fluorescence that cell sends (comprising common fluorescence and time resolved fluorescence) dies down gradually, shows with DTTA-Tb
3+for probe can be to intracellular ONOO
-carry out time resolved fluorescence micro-imaging mensuration.
2. probe DTTA-Eu
3+/ Tb
3+for ONOO in viable cell
-fluorescence imaging measure
The dimethyl sulphoxide solution and the EuCl that contain AM-DTTA (0.05mol/L) by new system
3-TbCl
3(0.05mol/L, Eu
3+/ Tb
3+=1/2) aqueous solution joins the HeLa cell (4 * 10 that 1.0mL cultivates with RPMI-1640
5cells/mL) in (DMSO concentration is 1.0%), by above-mentioned DTTA-Tb
3+the fluorescence imaging experiment is carried out in the operation of mark HeLa cell.
Fig. 9 has provided the DTTA-Eu added after different concns SIN-1
3+/ Tb
3+the fluorescence imaging figure of labeled cell.From Fig. 9 result, can find out, when not adding SIN-1, DTTA-Eu
3+/ Tb
3+heLa cell emission pearl opal fluorescence (cell blue-fluorescence, the DTTA-Tb of mark
3+green fluorescence and DTTA-Eu
3+due to red fluorescence mixes); Along with the increase that adds SIN-1 concentration, cell presents from pearl opal thereupon and changes to red fluorescence color, traces it to its cause, and is because of the increase along with SIN-1 concentration, DTTA-Tb
3+green fluorescence dies down gradually, and DTTA-Eu
3+red fluorescence is substantially constant and slowly highlight, and this result shows probe DTTA-Eu
3+/ Tb
3+can be used as a kind of novel Ratio-type probe for intracellular ONOO
-fluorescent microscopic imaging measure.Owing to lacking at present very color time resolved fluorescence microscope, apply this Ratio-type probe to ONOO
-carrying out the experiment of time resolved fluorescence imaging mensuration fails to carry out.
3. the reservation experiment of probe in viable cell
For ease of observing, with DTTA-Eu
3+the HeLa cell of mark (concrete experimental implementation is the same) is example, has investigated probe at intracellular retention property.Figure 10 has provided DTTA-Eu
3+the fluorescence imaging measurement result of the HeLa cell of mark after placing different time can find out that from Figure 10 result cell is in the put procedure of 30 minutes, and its fluorescence intensity does not change, and shows esterification probe AM-DTTA-Eu
3+once after entering cell, just can not be diffused in nutrient solution through cytolemma again, also can not washed out in the cell washing process.Its reason is to enter esterification probe AM-DTTA-Eu after cell
3+can under the effect of cell lactonase, be hydrolyzed rapidly and form can not be again through the DTTA-Eu of cytolemma
3+thereby, stably being retained in cell, this is that common organic fluorescence probe is incomparable.
Claims (3)
1. a Ratiometric fluorescent probe, is characterized in that: be with two kinds of trivalent rare earth ions Eu
3+and Tb
3+with organic ligand [4 '-(2,4-Dimethoxyphenyl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] title complex that tetraacethyl forms, wherein said organic ligand is referred to as DTTA, its structural formula is:
The mol ratio of ligand and metal ion is 1:1, DTTA-Eu
3+with DTTA-Tb
3+molar ratio at 1:5, in the 5:1 scope, change.
2. Ratiometric fluorescent probe according to claim 1, is characterized in that: DTTA-Eu
3+with DTTA-Tb
3+molar ratio be 1:2.
3. the application of the described Ratiometric fluorescent probe of claim 1 is characterized in that: utilize the ONOO in described rare earth compounding Ratiometric fluorescent probe and system
-interact, make probe have significant change in the fluorescence intensity of 541nm, and substantially constant in the fluorescence intensity at 612nm place, therefore in abiotic solution system, pass through F
612/ F
541fluorescence ratio is measured ONOO
-generation and growing amount, measure distribution and the intensity distribution of fluorescent probe in sample by time-resolved fluorometry or time resolved fluorescence microscope imaging assay method in biological, microbial solution system.
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