CN102146284A - Ratiometric fluorescent probe and application thereof - Google Patents
Ratiometric fluorescent probe and application thereof Download PDFInfo
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- CN102146284A CN102146284A CN2010101083662A CN201010108366A CN102146284A CN 102146284 A CN102146284 A CN 102146284A CN 2010101083662 A CN2010101083662 A CN 2010101083662A CN 201010108366 A CN201010108366 A CN 201010108366A CN 102146284 A CN102146284 A CN 102146284A
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 27
- 239000000523 sample Substances 0.000 claims abstract description 31
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 14
- 239000003446 ligand Substances 0.000 claims abstract description 11
- 150000002910 rare earth metals Chemical class 0.000 claims abstract description 11
- -1 rare-earth ions Chemical class 0.000 claims abstract description 6
- 239000013110 organic ligand Substances 0.000 claims abstract description 3
- 230000008859 change Effects 0.000 claims description 10
- 238000003384 imaging method Methods 0.000 claims description 6
- 238000013329 compounding Methods 0.000 claims description 4
- 238000005558 fluorometry Methods 0.000 claims description 3
- 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
- 244000005700 microbiome Species 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
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- 238000000034 method Methods 0.000 description 21
- 238000000799 fluorescence microscopy Methods 0.000 description 17
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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 anionic determination techniques of peroxynitrite, specifically a kind of peroxynitrite negatively charged ion ratio type fluorescent probe and application based on rare earth compounding are a kind of fluorimetric ratio type of peroxynitrite anion-selective rare-earth fluorescent probes in the living things system that can be used for.
Background technology
Peroxynitrite negatively charged ion (ONOO in the 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 that generates.It has been generally acknowledged that ONOO
-Be than NO and O
2 -Oxygenizement is stronger, a kind of more widely potent cytotoxic substance of effect (document 1:J.S.Beckman, T.W.Beckman, Proc.Natl.Acad.Sci.USA.1990,87,1620; Document 2:G.Ferrer-Sueta, R.Radi, ACS Chem.Biol.2009,4,161).As a kind of strong oxidizer, because ONOO
-Can produce the oxidative damage of cell and tissue, therefore with various disease conditions: as be associated (document 3:C.Szabo, H.Ischiropoulos such as shock, acute or chronic inflammation, septicemia, traumatic local asphyxia and arteriosclerosis, R.Radi, Nat.Rev.Drug Discov.2007,6,662).Simultaneously, it can also the oxidation cytolemma, mercapto functional group and biomacromolecules such as enzyme, albumen, lipid and DNA, is the important factor that causes cell injury, energy exhaustion and necrocytosis.Because ONOO
-Have very high mobility, can penetrate the lipid bilayer at an easy rate, this makes it not only can destroy cell, and porous is near cell, and these character have all been aggravated ONOO
-Destruction to organism.
In order to be familiar with ONOO from molecular level
-The oxygenizement mechanism of pair cell, tissue and whole organism, the nineties in last century, the biochemist is for ONOO in the living things system
-Generation, metabolism with and pathology damage mechanism carried out big quantity research.Yet, because ONOO in vivo
-Have extremely strong reactive behavior and extremely short lifetime (transformation period has only 0.8s), and can with many different compound generation chemical reactions, generate various reaction product, this just gives ONOO in the living things system
-Mensuration bring many difficulties.At present, ONOO in the living things system
-Mensuration mainly contain following two kinds of methods: (1) is based on ONOO
-Change with the signal that reaction produced of probe molecule and to come method for measuring, comprise 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 measuring the method (document 7:D.A.Richards, M.A.Silva, Anal.Biochem.2006,351,77) of tyrosine nitration product in the protein molecule.The method that adopts fluorescent molecular probe in the aforesaid method is a kind of simple, sensitive and special analytical procedure, and up to the present, existing several fluorescent molecular probes successfully are used 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 this method has higher sensitivity, this type of probe specificity is relatively poor, and other biological oxidant such as hypochlorous acid, sulfydryl and peroxidase also can oxidation DCFH and luminous dichlorofluorescein and the rhodamines of DHR generation hyperfluorescence.In recent years, two kinds of New O NOO based on aromatic ring nitration and ketone oxidizing reaction
-Fluorescent probe: NiSPYs and HKGreens successively have been synthesized (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 takes place and cause the remarkable enhancing of fluorescence intensity, but probe is water-soluble relatively poor, easily little by photobleaching, Stokes displacement, and retention time is short in the cell, is unfavorable for anionic long-term observation of peroxynitrite and determination of trace in the cell.In addition, present existing ONOO
-Fluorescent probe all with fluorescence intensity as characterization parameter, though it is simple to operate, its result's accuracy but is subjected to outside atmosphere and instrument condition to change influence as factors such as optical path, photobleaching, scattering and bias lights easily.And the ratio fluorescent method can address these problems preferably, this method is by measuring the fluorescence intensity at two different wave length places, with a kind of measuring method of its ratio as characterization parameter, compare with traditional fluorescent probe, ratio type 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 has synthesized a kind of ONOO in the living things system (viable cell) that can be used on the basis to the ligand structure design
-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 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 the living things system
-Novel rare-earth title complex ratio type 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, the 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 described peroxynitrite negatively charged ion ratio type fluorescent probe based on above-mentioned rare earth compounding is: in all kinds of biologies and abiotic environment, utilize described rare earth coordination compound fluorescent probe DTTA-Eu
3+/ Tb
3+With ONOO in the system
-Interaction, cause the ratio (F of probe in 612nm and 541nm wavelength place fluorescence intensity
612/ F
541) significantly strengthen, determine ONOO by the fluorescence ratio method then
-Generation and growing amount.Described fluorometry also comprises time-resolved fluorometry and time resolution fluorescent microscope imaging assay method except the fluorometry of routine.
ONOO in the 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 the abiotic environment
-Mensuration.
2. stability is high, can prolonged preservation use, and is applicable to multiple environment such as slightly acidic, neutrality and alkalescence.
3. with the ratio F of two different emission place fluorescence intensities
612/ F
541Be signal parameter, improved the specificity and the sensitivity that detect.
4. to ONOO
-Good selectivity is arranged, make time spent fluorescence intensity ratio value F with other active oxygen species
612/ F
541No change almost.
5. the time-resolved fluorometry that can be used for hundreds of microsecond levels is to eliminate the interference of background fluorescence to measuring.
6. can be used for the anionic highly sensitive fluorescence imaging mensuration of peroxynitrite in the viable cell.
Description of drawings
Fig. 1 is the synthetic route of ligand DTTA.
(A) is DTTA-Eu among Fig. 2
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.
Among 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.
Among 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
-Reaction back various photoluminescent properties (: fluorescence intensity; △: fluorescence lifetime; Zero: changing conditions time resolved fluorescence intensity).
Among 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, illustration are the ratio F of two transmitted wave strong point fluorescence intensities
612/ F
541With ONOO
-The changing conditions of concentration.
Among 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
-There is (A: negative control down in donor SIN-1; 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
-There is (A: negative control down in donor SIN-1; 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
The invention will be further described below by embodiment.Present embodiment only is used for that the present invention will be described, also belongs to scope of the present invention based on the method for same principle and similar raw material.
Embodiment 1: ligand [4 '-(2, the 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
Synthetic route as shown in Figure 1, the elementary operation process is as follows:
(1) 4 '-(2, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 1) synthetic
With 3.32 grams 2,4-dimethoxy benzaldehyde (20mmol), 4.84 gram 2-acetopyridine (40mmol), (85%, 40mmol) mixing is dissolved in the 100mL dehydrated alcohol 2.64 gram KOH, add the 50mL strong aqua then, 50 ℃ were stirred 10 hours down, filtered collecting precipitation, thick product ethyl alcohol recrystallization, get 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, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 2) synthetic
With 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, stirred under the room temperature 24 hours.The organic phase Na of 150mL10%
2CO
3Solution washing twice behind the anhydrous sodium sulfate drying, removes solvent under reduced pressure, vacuum-drying.The above-mentioned product that obtains is dissolved in 250mL CH
2Cl
2In, add 7.93 gram (CH
3)
3SiCN (80mmol) stirs after 2 hours under the room temperature, slowly is added dropwise to 7.03 gram Benzoyl chlorides (50mmol), stirs 24 hours under the room temperature.Remove solvent under reduced pressure, add 250mL10%K
2CO
3Solution stirred 1 hour under the room temperature.Filter collecting precipitation, water and acetonitrile washing final vacuum drying.Get 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, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dioctyl phthalate dimethyl ester (compound 3) synthetic
Digest compound 2 (8.0mmol) with 3.35 and be dissolved in the mixing solutions that the 16mL vitriol oil, 48mL Glacial acetic acid and 12mL water is made into, 93 ℃ were stirred 12 hours down.Reaction solution filters collecting precipitation to going in the 500mL frozen water, washing final vacuum drying.
With 8.24 gram SOCl
2(69.2mmol) under ice bath, slowly join in the 250mL exsiccant methyl alcohol, stir after 30 minutes, add the said hydrolyzed product, stirring and refluxing 24 hours.Remove solvent under reduced pressure, add 200mL10%Na
2CO
3The solution neutralization is filtered collecting precipitation, vacuum-drying.Thick product separates with silica gel column chromatography, with 95: 5 chloroform-methanol wash-out, gets 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, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 4) synthetic
Digest compound 3 (8.9mmol) with 4.31 and be dissolved in the 150mL dehydrated alcohol, stir and form suspension liquid.Add 1.35 gram NaBH
4(46.4mmol), stirred 2 hours under the room temperature, reflux again and stirred 6 hours.Remove solvent under reduced pressure, add the saturated NaHCO of 25mL
3Solution is heated to and boils.The cooling back adds 100mL water, and (～4 ℃) are placed and spent the night in the reaction solution refrigerator, filter collecting precipitation, thick product water and acetonitrile washing, vacuum-drying.Get 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 "-two brooethyls-4 '-(2, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 5) synthetic
At the anhydrous N of 130mL, add 4.97 gram PBr in the dinethylformamide
3(18.5mmol), stir under the room temperature and add 3.17 after 15 minutes and digest compound 4 (7.4mmol), continue to stir 24 hours.Reaction finishes the back and adds saturated NaHCO
3Collecting precipitation is filtered in the solution neutralization, thick product water and acetonitrile washing, vacuum-drying.Get 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, the 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 with 3.10
2CO
3(56mmol) in the mixing solutions that adding 350mL exsiccant acetonitrile and 100mL exsiccant tetrahydrofuran (THF) are 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 removes under reduced pressure behind the solvent and separates with silica gel column chromatography, and eluent is an ethyl acetate, removes product normal hexane recrystallization behind the solvent under reduced pressure, vacuum-drying.Get 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, the 4-Dimethoxyphenyl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] tetraacethyl (DTTA) synthetic
Digest compound 6 (1.0mmol) with 0.77 and be dissolved in the mixing solutions that 35mL ethanol and 6mL water is made into, add 1.50 gram KOH again, reflux and stirred 2 hours.After removing solvent under reduced pressure, product is dissolved in the 30mL water, with hydrochloric acid adjust pH to 2～3 of 1mol/L, stirs 3 hours under the room temperature, filters collecting precipitation, washes with water.After the vacuum-drying product is added in the 30mL acetonitrile, stirring and refluxing 30 minutes is filtered collecting precipitation, gets target compound 0.43 gram after the vacuum-drying, productive rate: 65.3%.
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). and 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
With 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 0.5mL exsiccant dimethyl sulfoxide (DMSO), stirred overnight at room temperature obtains brown AM-DTTA dimethyl sulphoxide solution, is used for the cell imaging experiment.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
0.05mol/L borate buffer solution with pH value 9.1 is 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 (mol ratio of ligand and metal ion is 1: 1 in two kinds of title complexs).It is Perkin Elmer Lambda 35 type spectrophotometers that ultraviolet-visible absorption spectroscopy is measured with instrument.The fluorometric assay instrument is a Perkin Elmer LS 50B spectrophotofluorometer.Fluorescence quantum yield is measured and is used 4 '-phenyl-2,2 ': 6 ', 2 "-Lian three 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 the formula
2, ε
2, C
2, φ
2Be 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 sees 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 have higher fluorescence quantum yield, bigger molar extinction coefficient, and both have identical maximum excitation wavelength can obtain a series of different DTTA-Eu that form so both are mixed 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. the pH value of solution value is to DTTA-Eu
3+With DTTA-Tb
3+The influence 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 as can be seen, in the scope of pH value 3～10, DTTA-Eu
3+With DTTA-Tb
3+Fluorescence intensity and the fluorescence lifetime influence that is not subjected to the pH value substantially, show that these two kinds of title complexs all can use 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 influence of photoluminescent property
Add DTTA-Ln at the 0.05mol/L of a series of pH values 7.4 Tris-HCl respectively in the buffered soln
3+(Ln
3+=Eu
3+Or Tb
3+, 2.0 μ mol/L) and the ONOO of different concns
-, stirring reaction was measured each fluorescence intensity of solution and fluorescence lifetime after 15 minutes.Measuring with instrument is PerkinElmer LS 50B spectrophotofluorometer.
As shown in Figure 4, along with ONOO
-The increase of concentration, DTTA-Eu
3+Fluorescence intensity and fluorescence lifetime do not change substantially; 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 when existing shows title complex DTTA-Tb
3+Can be used as the intensity type fluorescent probe and be used 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 and DTTA-Eu gradually
3+Fluorescence intensity constant substantially.Therefore, with 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
-(excitation wavelength: 335nm), illustration is the ratio (F of two wavelength place fluorescence intensities to time resolved fluorescence emmission spectrum when existing
612/ F
541) with ONOO
-The change curve of concentration shows DTTA-Eu
3+/ Tb
3+Can be used as ratio type probe and be used 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 active specy H respectively
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 in the 0.05mol/L of pH value 7.4 Tris-HCl buffered soln, carry out under the room temperature, 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) following 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.From Fig. 7 result as can be seen, probe DTTA-Tb
3+With H
2O
2, OCl
-,
1O
2, NO
3 -, O
2 -Considerable change (＜5%) does not take place in reacted fluorescence intensity; With ONOO
-After the reaction, the fluorescence intensity of probe significantly reduces (98%); With NO, OH and NO
2 -After the reaction, the fluorescence intensity of probe has reduced about 20%, 30% and 50% respectively.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 such 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 then improve greatly, work as DTTA-Eu
3+/ Tb
3+With NO,
1O
2, O
2 -, OH, ClO
-, H
2O
2And NO
3 -After the reaction, F
612/ F
541Substantially constant (changing less than 0.1) is with NO
2 -Reaction back F
612/ F
541Only increased by 0.7 times, this and ONOO
-The influence of (5.6 times) is compared or is much smaller.The above results shows ratio type 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+Be used for ONOO in the viable cell
-Fluorescence imaging measure
1. probe DTTA-Tb
3+Be used for ONOO in the viable cell
-The time resolved fluorescence imaging measure
The dimethyl sulphoxide solution and the TbCl that contain AM-DTTA (0.05mol/L) with 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 after 2 hours down for 37 ℃ in the incubator, discard nutrient solution and ooze salts solution thorough washing cell to remove the probe molecule that does not enter cell, in culturing bottle, add the ONOO of different concns with waiting
-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 under 37 ℃ in the incubator and carry out fluorescence imaging mensuration after 0.5 hour.
Fig. 8 has provided the DTTA-Tb behind the adding different concns SIN-1
3+The fluorescence imaging figure of labeled cell.From Fig. 8 result as can be seen, 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 employing time resolved fluorescence imaging pattern was measured, the blue background fluorescence of cell was eliminated fully, 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+Be the ONOO of probe in can pair cell
-Carrying out the time resolved fluorescence micro-imaging measures.
2. probe DTTA-Eu
3+/ Tb
3+Be used for ONOO in the viable cell
-Fluorescence imaging measure
The dimethyl sulphoxide solution and the EuCl that contain AM-DTTA (0.05mol/L) with 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 behind the adding different concns SIN-1
3+/ Tb
3+The fluorescence imaging figure of labeled cell.From Fig. 9 result as can be seen, 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, the fluorescence color that cell presents from the pearl opal to the redness thereupon changes, and 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 constant substantially and slowly highlight, and this result shows probe DTTA-Eu
3+/ Tb
3+Can be used as a kind of novel ratio type probe and be used for intracellular ONOO
-Fluorescent microscopic imaging measure.Owing to lack very color time resolved fluorescence microscope at present, use 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 an 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, from Figure 10 result as can be seen, cell is in 30 minutes put procedure, and its fluorescence intensity does not change, and shows esterification probe AM-DTTA-Eu
3+In case after entering cell, just can not pass cytolemma again and be diffused in the nutrient solution, also can in the cell washing process, do not washed out.Its reason is to enter esterification probe AM-DTTA-Eu behind the cell
3+Can be under the effect of cell lactonase hydrolysis and form the DTTA-Eu that can not pass cytolemma more rapidly
3+Thereby, stably being retained in the cell, this is that common organic fluorescence probe is incomparable.
Claims (4)
1. a ratio type 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, the 4-Dimethoxyphenyl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] title complex that tetraacethyl (be called for short DTTA) forms, the structural formula of wherein said ligand DTTA 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.
2. ratio type 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 ratio type of claim 1 fluorescent probe is characterized in that: in all kinds of biologies, microorganism or abiotic solution system, utilize the ONOO in described rare earth compounding ratio type fluorescent probe and the system
-Interact, make probe have significant change in the fluorescence intensity of 541nm, and constant substantially in the fluorescence intensity at 612nm place, therefore can pass through F
612/ F
541Fluorescence ratio is measured ONOO
-Generation and growing amount.
4. according to the application of the described ratio type of claim 3 fluorescent probe, it is characterized in that: described fluorescent probe has the fluorescence lifetime of Millisecond, and therefore available fluorometry is measured F
612/ F
541Place's fluorescence ratio can also be measured distribution and the intensity distribution of fluorescent probe in sample with time-resolved fluorometry or time resolved fluorescence microscope imaging assay method.
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