CN1986550A - Singlet oxygen fluorescence probe based on europium complex and its application - Google Patents
Singlet oxygen fluorescence probe based on europium complex and its application Download PDFInfo
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Abstract
The present invention relates to new europium complex fluorescent probe for the imaging determination of singlet oxygen in live cell and its application. The fluorescent probe is complex formed with trivalent europium ion Eu3+ and organic ligand [4'-(10-methyl-9-anthryl)-2, 2':6'2''-tripyridyl-6, 6''-dimethyl amino] tetracetic acid as shown. The complex can enter to live cell simply in the presence of photosensitizer and capture singlet oxygen in cell specifically, leading to obviously enhanced fluorescence strength of the complex for the fluorescent determination of singlet oxygen in live cell.
Description
Technical field
The present invention relates to the determination techniques of singlet oxygen in the viable cell, specifically a kind of europium coordination compound fluorescent probe and application thereof that can be used for singlet oxygen imaging mensuration in the viable cell.
Background technology
Singlet oxygen (
1O
2) be a kind of unsettled existence form that is in high-energy excited state of oxygen molecule.It is widespread in nature, and in many photochemistry and optical-biological reaction process, in the processes such as phototransformation, chemoluminescence, organism deterioration by oxidation or even photocarcinogenesis as photodegradation, pollutent, is all playing the part of crucial role.The application of singlet oxygen aspect organic synthesis had many reports, but its physiology oxygenizement in living things system more and more receives investigator's concern, it is containing the great effect to biology and life system, various biomolecules comprise that membrane lipid, protein, amino acid, Nucleotide, carbohydrate and mercaptan all can be by the oxidations of the singlet oxygen of strong oxidizing property institute.On the other hand, people also can utilize the strong oxidizing property matter of singlet oxygen to remove to kill malignant cell or tissue, reach the purpose of curing cancer, be referred to as the luminous energy therapy (photodynamictherapy, PDT).
Because
1O
2Have so important effect, detect
1O
2, particularly in the living things system
1O
2More and more cause people's attention.Begin till now the seventies from eighties of last century,
1O
2Detection mainly contain following several method: (1) utilizes
1O
2Self cancellation
1Δ
g→
3∑
g -The phosphorescence that produces detects
1O
2(document 1:A.A., Jr.Krasnovsky, Biofzika, 1976,21,748).This method specificity is very high, but at the bottom of the sensitivity, a little less than the detecting signal, can't be used for lower concentration
1O
2Detection.At present, along with the raising of detecting instrument performance, this method has been used in some living things system
1O
2Research (document 2:C.Kiryu, M.Makiuchi, J.Miyazaki, T.Fujinaga, K.Kakinuma, FEBS Lett.1999,443,154 of generation, physiological action and spatial distribution; Document 3:S.Oelckers, T.Ziegler, I.Michler, B.R der, J.Photochem.Photobiol.B:Biol.1999,53,121; Document 4:L.K.Andersen, P.R.Ogilby, Photochem.Photobiol.2001,73,489; Document 5:L K.Andersen, Z.Gao, P.R.Ogilby, L.Poulsen, I.Zebger, J.Phys.Chem.A 2002,106, and 8488).(2) utilize
1O
2Fluoresceins probe molecule specificity reaction with having anthracene nucleus makes probe become the hyperfluorescence molecule by original non-fluorescent molecule, thereby is used for
1O
2Detection (document 6:N.Umezawa, K.Tanaka, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Angew.Chem.Int.Ed.Engl.1999,38,2899; Document 7:K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, J.Am.Chem.Soc.2001,123,2530).This method is short, highly sensitive detection time, but is not suitable for low ph environment and detection in real time.(3) utilize 9,10-diphenylanthrancene (DPA) with
1O
2Characteristic reaction generate stable inner oxide, measure by the change of measuring the DPA absorption spectrum
1O
2(document 8:M.J.Stenbeck, A.U.Khan, M.J.Kamovsy, J.Biol.Chem.1992,267,13425; Document 9:M.J.Stenbeck, A.U.Khan, M.J.Kamovsy, J.Biol.Chem.1993,268,15649).This method has been used to measure and has produced in the phagocytic cell
1O
2, but owing to detect based on absorption spectrum, so sensitivity is lower.(4) utilize
1O
2And the transmission ofenergy between the probe molecule excites probe molecule to send strong delayed fluorescence, and then is used for
1O
2Detection (document 10:A.A.Jr.Krasnovsky, C.Schweitzer, H.Lesmann, C.Tanielian, E.A.Luk ' yanets, Quantum Electron.2000,30,445; Document 11:A.A.Jr.Krasnovsky, M.E.Bashtanov, N.N.Drozdova, O.A.Yuzhakova, E.A.Luk ' yanets, QuantumElectron.2002,32,83).This method is applicable to multiple system, and the luminescent quantum productive rate is
1O
2The 3-5 of phosphorescence doubly.But detect the sensitization simultaneously of used probe
1O
2Generation, make measurement result produce deviation.
(5) utilize the chemiluminescence probe method to detect
1O
2(document 12:X.H.Li, G.X.Zhang, H.M.Ma.D.Q.Zhang, J.Li, D.B.Zhu, J.Am.Chem.Soc.2004,126,11543; Document 13:L.A.MacManus-Spencer, D.E.Latch, K.M.Kroncke, K.McNeill, Anal.Chem.2005,77,1200).This method selectivity is good, highly sensitive, detection is rapid, but the probe poorly water-soluble is unfavorable in the living things system
1O
2Mensuration.
Though above-mentioned various measuring method exists
1O
2Mensuration in obtained good application, but since in the cell various compositions cause these methods can't be used for viable cell to the serious interference of method of optically measuring
1O
2The real time measure.The time resolved fluorescence detection technique that grows up based on the long lifetime fluorescent characteristics of rare-earth fluorescent title complex can be eliminated the interference of short-life sample background fluorescence to measuring very effectively, measure sensitivity and greatly improve, be subjected to extensive concern (document 14:J.Yuan at the high-sensitivity biological detection range in recent years, K.Matsumoto, H.Kimura, Anal.Chem., 1998,70,596-601; Document 15:I.Hemmil , V.-M.Mukkala, Crit.Rev.Clin.Lab.Sci., 2001,38,441-519).
The present invention successfully develops two kinds based on rare earth compounding recently at us
1O
2(document 16: Yuan Jingli, Song Bo, Wang Guilan, a kind of singlet oxygen europium coordination compound fluorescent probe and application thereof, Chinese invention patent, application number: 200510045768.1 on the basis of fluorescent probe; Document 17: Yuan Jingli, Tan Mingqian, Wang Guilan, a kind of terbium coordination compound singlet oxygen fluorescent probe and application thereof, Chinese invention patent, application number: 200510045767.7), improve by structure and to prepare a kind of can be used in the viable cell to ligand
1O
2The europium coordination compound fluorescent probe that fluorescence imaging is measured utilizes this fluorescent probe binding time to differentiate the fluorescence imaging determination techniques and has set up in a kind of viable cell
1O
2The 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 in the viable cell
1O
2Novel europium coordination compound fluorescent probe and application that imaging is measured are applied to time resolved fluorescence imaging determination techniques in the viable cell
1O
2Detection.
For achieving the above object, the technical scheme of invention employing is:
With trivalent europium ion Eu
3+With ligand [4 '-(10-methyl-9-anthryl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] title complex that forms of tetraacethyl (be called for short MTTA) (is called for short MTTA-Eu
3+) be fluorescent probe, wherein said ligand structure formula is:
The application process of described singlet oxygen fluorescence probe based on europium complex is: in all kinds of biologies and abiotic environment, utilize described europium coordination compound fluorescent probe to catch in the system
1O
2, make the fluorescence intensity of probe significantly strengthen, determine by fluorometry then
1O
2Generation and growing amount.Described fluorometry also comprises time-resolved fluorometry and time resolution fluorescent microscope imaging assay method except the fluorometry of routine.
Described singlet oxygen europium coordination compound fluorescent probe can be used for containing in the mensuration test kit and related reagent of its component.
Fluorescent probe of the present invention has following advantage:
1. have well water-solublely, be applicable in the living things system
1O
2Mensuration.
2. stability is high, can prolonged preservation use, and is applicable to multiple environment such as slightly acidic, neutrality and alkalescence.
3. have high
1O
2Measure sensitivity, be limited to 3.8 nmol/L under its lowest detection.
4. right
1O
2Good selectivity is arranged, with other active oxygen species effect fluorescent signal no change almost.
5. catching
1O
2After not only can cause fluorescence to strengthen 15 times, and the fluorescence lifetime of product reaches 1.29ms, can be used for the time-resolved fluorometry of hundreds of microsecond levels.
6. under the coexistence of photosensitization reagent, can enter viable cell simply, capture the singlet oxygen in the cell specifically, cause the fluorescence intensity of title complex significantly to strengthen, and then be used for the time-resolved fluorometry of viable cell singlet oxygen.
Description of drawings
Fig. 1 is the synthetic route of ligand MTTA.
Fig. 2 is MTTA-Eu
3+(solid line, 1.0 μ mol/L) and EP-MTTA-Eu
3+(dotted line, 1.0 μ mol/L) fluorescence spectrum in the 0.05mol/L of pH value 9.1 borate buffer solution.
Fig. 3 is EP-MTTA-Eu
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.
Fig. 4 is MTTA-Eu
3+Compare with the fluorescence intensity of various active oxygen species reaction product.
Fig. 5 is MTTA-Eu
3+With different concns
1O
2The changing conditions of its product fluorescence spectrum of reaction back.(A) excitation spectrum, (B) emmission spectrum.
Fig. 6 is to use MTTA-Eu
3+Detect Na
2MoO
4/ H
2O
2Quantitatively produce in the system
1O
2Working curve.
Fig. 7 is MTTA-Eu
3+Being used for neutral aqueous solution is produced under rayed by photosensitization reagent TMPyP
1O
2Measurement result.
Fig. 8 is TMPyP and MTTA-Eu
3+Produce in 100 watts of mercury lamps (using wavelength 450-490nm) irradiation process with the Hela cell of tense marker
1O
2Real-time time differentiate the fluorescence imaging measurement result.
Fig. 9 is 3 TMPyP and MTTA-Eu among Fig. 8
3+Dependence curve with the Hela cell of tense marker and white space fluorescence intensity and irradiation time in 100 watts of mercury lamp irradiation process.
Embodiment
The invention will be further described below by embodiment.
Embodiment 1. ligands [4 '-(10-methyl-9-anthryl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] tetraacethyl (be called for short MTTA) synthetic.
Synthetic route as shown in Figure 1, the matrix operating process is as follows.
(1) (E)-3-(10-methyl-9-anthryl)-1-(2 '-pyridyl)-2-acrylketone (compound 1) synthetic
After mixing 11.01 gram 10-methyl-9-anthracene aldehyde (50mmol) and 2.81 gram KOH (50mmol) in the mixing solutions that is dissolved in 150ml methyl alcohol and 30ml water composition; slowly drip 6.06 gram 2-acetylpyridine (50mmol) in reaction system, stirred 24 hours under the room temperature.Filter collecting precipitation, dry that target compound 13.19 restrains productive rate: 81.6% with ethanol thorough washing final vacuum.
1HNMR (CDCl
3) measurement result: δ=3.15 (s, 3H); 7.48-7.57 (m, 5H); 7.92 (m, 1H); 8.23 (d, 1H); 8.30 (d, 1H); 8.34-8.40 (m, 4H); 8.69 (d, 1H); 8.94 (d, 1H).
(2) 4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 2) synthetic
In the 100ml dry tetrahydrofuran, add 2.8 gram potassium tert.-butoxide (26mmol) and 2.0ml 2-acetylpyridine (16.8mmol), stir under the room temperature after 2 hours, add 4 and digest compound 1 (12.8mmol).Stir under the room temperature after 14 hours, add 20 gram exsiccant ammonium acetate and 100ml dehydrated alcohols.Reaction solution refluxes and stirred 4 hours.Remove solvent under reduced pressure, thick product separates with silica gel column chromatography, makees eluent with 2: 1 petroleum ether-ethyl acetate, removes solvent under reduced pressure, and product washs with acetonitrile, and vacuum-drying gets target compound 1.58 grams, productive rate: 29.1%.
1H NMR (CDCl
3) measurement result: δ=3.20 (s, 3H); 7.33-7.37 (m, 4H); 7.52 (t, 2H); 7.69 (d, 2H); 7.92 (m, 2H); 8.38 (d, 2H); 8.59 (s, 2H); 8.62 (d, 2H), 8.82 (d, 2H).
(3) 6,6 "-dinitrile-4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 3) synthetic
Digest compound 2 (10.0mmol) and 6.9 gram m-chloro-benzoic acid peroxides (40.0mmol) are dissolved in the 200ml methylene dichloride with 4.2, stirred under the room temperature 24 hours.The organic phase Na of 150ml 10%
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 200ml CH
2Cl
2In, add 7.94 gram Me
3SiCN (80.0mmol) stirs after 1 hour under the room temperature, slowly is added dropwise to 7.03 gram Benzoyl chlorides (50.0mmol), stirs 24 hours under the room temperature.Remove solvent under reduced pressure, add 200ml10%K
2CO
3Solution stirred 2 hours under the room temperature.Filter collecting precipitation, with acetonitrile washing final vacuum drying.Get target compound 3.10 grams, productive rate: 65.6%.
1H NMR (CDCl
3) measurement result: δ=3.23 (s, 3H); 7.39 (t, 2H); 7.55 (t, 2H); 7.62 (d, 2H); 7.75 (d, 2H); 8.05-8.09 (m, 2H); 8.42 (d, 2H); 8.72 (s, 2H); 8.99 (d, 2H).
(4) 4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dioctyl phthalate dimethyl ester (compound 4) synthetic
Digest compound 3 (5.4mmol) with 2.57 and be dissolved in the mixing solutions that the 16ml vitriol oil, 32ml Glacial acetic acid and 8ml water is made into, 90 ℃ were stirred 12 hours down.Reaction solution filters collecting precipitation to going in the 400ml frozen water, washing final vacuum drying.
With 6.04 gram SOCl
2(50.8mmol) under ice bath, slowly join in the 200ml exsiccant methyl alcohol, stir after 30 minutes, add the said hydrolyzed product, stirring and refluxing 24 hours.Remove solvent under reduced pressure, add saturated NaHCO
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 1.96 grams, productive rate: 67.2%.
1H NMR (CDCl
3) measurement result: δ=3.23 (s, 3H); 3.88 (s, 6H); 7.35 (t, 2H); 7.54 (t, 2H); 7.67 (d, 2H); 8.07 (t, 2H); 8.18 (d, 2H); 8.41 (d, 2H); 8.72 (s, 2H); 8.99 (d, 2H).
(5) 6,6 "-dihydroxymethyl-4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 5) synthetic
Digest compound 4 (2.9mmol) with 1.58 and be dissolved in the 48ml dehydrated alcohol, stir and form suspension liquid.Add 0.44 gram NaBH
4(11.6mmol) stirred 2 hours under the room temperature, reflux again and stirred 6 hours.Remove solvent under reduced pressure, add the saturated NaHCO of 8ml
3Solution is heated to and boils.The cooling back adds 60ml 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 0.98 gram, productive rate: 68.9%.
1H NMR (CDCl
3) measurement result: δ=3.23 (s, 3H); 4.75 (s, 4H); 7.24 (d, 2H); 7.36 (t, 2H); 7.54 (t, 2H); 7.67 (d, 2H); 7.92 (t, 2H); 8.41 (d, 2H); 8.59 (s, 2H); 8.71 (d, 2H).
(6) 6,6 "-two brooethyls-4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine (compound 6) synthetic
In the 70ml dry DMF, add 1.90 gram PBr
3(7.0mmol), stir under the room temperature and add 1.36 after 15 minutes and digest compound 5 (2.8mmol), 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 1.30 grams, productive rate: 75.9%.
1H NMR (CDCl
3) measurement result: δ=3.23 (s, 3H); 4.51 (s, 4H); 7.38 (t, 2H); 7.49-7.56 (m, 4H); 7.70 (d, 2H); 7.91 (t, 2H); 8.41 (d, 2H); 8.64 (s, 2H); 8.70 (d, 2H).
(7) 4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine tetraacethyl ethyl ester (compound 7) synthetic
Digest compound 6 (1.7mmol), 0.71 gram ethyl diacetate base amine (3.7mmol) and 2.36 gram anhydrous K with 1.04
2CO
3(17mmo1) in the mixing solutions that adding 120ml exsiccant acetonitrile and 36ml 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 80ml chloroform, with the saturated 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 petroleum ether-ethyl acetate-triethylamine of 15: 5: 2, removes a small amount of petroleum ether of product behind the solvent under reduced pressure, vacuum-drying.Get target compound 1.02 grams, productive rate: 72.6%.
1H NMR (CDCl
3) measurement result: δ=1.07 (t, 12H); 3.23 (s, 3H); 3.58 (s, 8H); 3.99 (q, 8H); 4.03 (s, 4H); 7.36 (t, 2H); 7.53 (t, 2H); 7.65 (d, 2H); 7.72 (d, 2H); 7.91 (t, 2H); 8.39 (d, 2H); 8.55 (s, 3H); 8.67 (d, 2H).
(8) [4 '-(10-methyl-9-anthryl)-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] tetraacethyl (MTTA) synthetic
Digest compound 7 (1.44mmol) with 1.19 and be dissolved in the mixing solutions that 48ml ethanol and 4ml water is made into, add 1.84 gram KOH (33mmol) again, reflux and stirred 2 hours.After removing solvent under reduced pressure, product is dissolved in the 16ml water, with about the hydrochloric acid adjust pH to 2 of 1mol/L, stirs under the room temperature and spends the night.Filter collecting precipitation, wash with water.After the vacuum-drying product is added in the 50ml acetonitrile, stirring and refluxing 30 minutes is filtered collecting precipitation, gets target compound 0.84 gram after the vacuum-drying, productive rate: 76.8%.
1H NMR (DMSO-d
6) measurement result: δ=3.21 (s, 3H); 3.45 (s, 8H); 3.96 (s, 4H); 7.46 (t, 2H); 7.60-7.62 (m, 4H); 7.68 (d, 2H); 8.08 (t, 2H); 8.41 (s, 2H); 8.51 (d, 2H); 8.70 (d, 2H).Results of elemental analyses is pressed C
40H
35N
5O
83H
2O calculated value: C62.57%, H5.38%, N9.12%; Measured value: C62.35%, H5.02%, N9.10%.MALDI-TOF-MS：m/z：714[M+H]
+。
(1) fluorescence spectrum, fluorescence intensity and fluorescence lifetime
0.05mol/L sodium borate buffer solution with pH value 9.1 is that solvent has been measured MTTA-Eu
3+Uv-vis spectra in this solvent, fluorescence spectrum, molar extinction coefficient (ε), fluorescent quantum yield (φ) and fluorescence lifetime (τ).EP-MTTA-Eu
3+By MTTA-Eu
3+With Na
2MoO
4/ H
2O
2At the 0.1mol/L of pH value 10.5 NaHCO
3Preparation (product is verified through the mass spectrum) in-NaOH buffered soln, dilute 100 times with the 0.05mol/L sodium borate buffer solution of pH value 9.1 again after, measure its photoluminescent property in this solvent.It is Perkin Elmer Lambda35 type spectrophotometer that uv-vis spectra 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 " the title complex of-dimethylamine tetraacethyl europium records (document 18:M.Latva, H.Takalo, V.M.Mukkala as standard substance, C.Matachescu, J.C.Rodriguez-Ubis, J.Kankare, J.Lumin., 1997,75,149-169), 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.MTTA-Eu
3+And EP-MTTA-Eu
3+Absorption in sodium borate buffer solution and photoluminescent property
| Compound | Maximum absorption wavelength (nm) | Molar extinction coefficient 335nm (cm -1M -1) | Maximum fluorescence emission wavelength (nm) | Fluorescence quantum yield (%) | Fluorescence lifetime (ms) |
| MTTA-Eu 3+EP-MTTA-Eu 3+ | 294,335 294,335 | 18100 16400 | 614 614 | 0.90 13.8 | 0.80 1.29 |
By table 1 as seen, probe MTTA-Eu
3+With
1O
2Very big variation, MTTA-Eu have taken place in its fluorescence quantum yield before and after the reaction
3+Fluorescence quantum yield has only 0.90%, and EP-MTTA-Eu
3+Fluorescence quantum yield reached 13.8%, show probe MTTA-Eu
3+With
1O
2Its fluorescence intensity of reaction back can increase by 15 times.The fluorescence spectrum of two kinds of compounds as shown in Figure 2.
(2) the pH value of solution value is to EP-MTTA-Eu
3+The influence of photoluminescent property
With EP-MTTA-Eu
3+With the 0.05mol/L Tris-HCl buffered soln dissolving of different pH values, measure its fluorescence intensity and fluorescence lifetime under different pH values, it the results are shown in Figure 3.As seen from the figure, the pH value greater than 3 scope in, EP-MTTA-Eu
3+Fluorescence intensity and fluorescence lifetime be subjected to the influence of pH value little, show that this probe all can use in slightly acidic, neutrality and weakly alkaline environment.
(3) probe MTTA-Eu
3+Right
1O
2Selectivity
Investigated active oxygen species ONOO respectively
-, OH, O
2 -, H
2O
2And
1O
2With MTTA-Eu
3+Response situation, same concentrations and reaction conditions (respond and all in the carbonate buffer solution of the pH of 0.05mol/L value 10.5, carry out under the room temperature, the reaction times is 4 hours, reaction density is: MTTA-Eu
3+: 100nmol/L;
1O
2: 10 μ mol/L H
2O
2+ 10mmol/L Na
2MoO
4OH:10 μ mol/L H
2O
2+ 10 μ mol/L (NH
4)
2Fe (SO
4)
2O
2 -: 10 μ mol/L KO
2H
2O
2: 10 μ mol/L H
2O
2) following 5 kinds of active oxygen species and MTTA-Eu
3+The fluorescent strength determining result of reaction product as shown in Figure 4.As seen from the figure, MTTA-Eu
3+With ONOO
-, OH, O
2 -And H
2O
2Do not cause MTTA-Eu after the reaction
3+Fluorescence intensity generation considerable change, show MTTA-Eu
3+Do not react with these active oxygen species.Work as MTTA-Eu
3+With
1O
2After the reaction, owing to formed the inner oxide EP-MTTA-Eu of hyperfluorescence
3+, cause the fluorescence intensity of probe significantly to strengthen.This result shows probe MTTA-Eu
3+Right
1O
2Has good selectivity.
Embodiment 3. uses MTTA-Eu
3+Quantitative assay Na
2MoO
4/ H
2O
2System produces
1O
2
At the 0.1mol/L of pH value 10.5 NaHCO
3Add MTTA-Eu in the-NaOH buffered soln respectively
3+(1.0 μ mol/L), Na
2MoO
4(1.0mmol/L) and the H of a series of concentration
2O
2(the H of per 2 molecules in this system
2O
2Produce 1 molecule
1O
2).Behind 37 ℃ of following placing responses reaction solution being carried out time resolution fluorescence spectral measures.Measuring with instrument is Perkin Elmer LS 50B spectrophotofluorometer.
As shown in Figure 5, be accompanied by H
2O
2Concentration (promptly
1O
2Concentration) increase, the fluorescence intensity of probe also increase gradually, show MTTA-Eu
3+Can be used for generating in the quantitative assay solution
1O
2Concentration.Fig. 6 has provided use MTTA-Eu
3+Detect Na
2MoO
4/ H
2O
2Quantitatively produce in the system
1O
2Working curve.As shown in the figure,
1O
2Concentration and fluorescence intensity have good linear relationship, calculate minimum detectabilities with 3 times of background signal standard deviation, MTTA-Eu
3+Right
1O
2Minimum detectability be 3.8nmol/L, than low about 21 times of the minimum detectability of the chemiluminescence method of having reported, show and use MTTA-Eu
3+Quantitative assay
1O
2Has very high sensitivity.
To contain 10 μ mol/L MTTA-Eu
3+(its solution can produce under rayed for full name 5,10,15,20-four (N-methyl-4-pyridinium base) porphyrin four p-methyl benzenesulfonic acid salt to reach 10 μ M photosensitizer TMPyP
1O
2) the 0.05mol/L Tris-HCl solution of pH value 7.4 be placed on 100 watts tungsten lamp irradiation down, the fluorescence intensity of measuring probe under the different irradiation times changes, thereby investigates in the illumination process
1O
2The generation situation.As shown in Figure 7, along with the increase of light application time, the fluorescence intensity of probe obviously increases, and shows probe MTTA-Eu
3+But generate in the capture light photograph process
1O
2, and then can extrapolate in the illumination process
1O
2Growing amount.
To contain 0.4mmol/L MTTA-Eu
3+And the normal saline solution solution of 10 μ mol/L TMPyP joins in the Hela cell of cultivation, at 5%CO
2Cultivate after 5 hours down for 37 ℃ in the incubator, remove the MTTA-Eu that does not enter cell with normal saline solution solution thorough washing cell
3+And TMPyP, cell is added in the normal saline solution, use 100 watts mercury lamp irradiating cell (irradiates light wavelength 450-490nm) then, differentiate fluorescent microscope with the time and the cell in the irradiation process is carried out the time resolved fluorescence imaging measure, thereby in cell, producing in the irradiation process
1O
2Carry out real-time monitoring and mensuration.This is determined on the time resolved fluorescence microscope and carries out.
Fig. 8 has provided the MTTA-Eu under different irradiation times
3+And the time resolved fluorescence imaging measurement result of TMPyP labeled cell, as seen from the figure, being accompanied by the increase of irradiation time, the fluorescence intensity that cell sends also obviously strengthens, and shows MTTA-Eu
3+But in cell, generate in the capture light photograph process
1O
2Analyze by fluorescence intensity, find that the fluorescence intensity in cell centre zone will show MTTA-Eu apparently higher than the fringe region of cell individual cells
3+Reach TMPyP and mainly concentrate in the nucleus, this has the character of stronger keying action to conform to TMPyP and DNA.Fig. 9 is 3 dependence curves of selecting cell compartment and white space fluorescence intensity and irradiation time in 100 watts of mercury lamp irradiation process among Fig. 8, as seen uses MTTA-Eu
3+Probe can be to each single celled photoinduction
1O
2Real-time and quantitative monitoring and mensuration are carried out in formation, also show each single celled photoinduction
1O
2The formation ability has certain difference.
Claims (4)
1. the singlet oxygen fluorescence probe based on europium complex is characterized in that: be with trivalent europium ion Eu
3+With ligand [4 '-(10-methyl-9-anthryl)-2,2 ': 6 ' 2 "-ter cycloheptapyridine-6,6 "-dimethylamine] title complex that tetraacethyl forms, wherein said ligand structure formula is:
2. the application according to the described singlet oxygen fluorescence probe based on europium complex of claim 1 is characterized in that: in all kinds of biologies and abiotic environment, utilize described europium coordination compound fluorescent probe to catch in the system
1O
2, make the fluorescence intensity of probe significantly strengthen, determine by fluorometry then
1O
2Generation and growing amount.
3. according to the application of the described singlet oxygen fluorescence probe based on europium complex of claim 2, it is characterized in that: described fluorometry also comprises time-resolved fluorometry and time resolution fluorescent microscope imaging assay method except the fluorometry of routine.
4. mensuration test kit and related reagent based on the singlet oxygen fluorescence probe of the described europium complex of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732246A (en) * | 2012-06-15 | 2012-10-17 | 大连理工大学 | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application |
| CN110483539A (en) * | 2019-09-17 | 2019-11-22 | 临沂大学 | A kind of fluorescence probe and the preparation method and application thereof |
-
2005
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732246A (en) * | 2012-06-15 | 2012-10-17 | 大连理工大学 | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application |
| CN110483539A (en) * | 2019-09-17 | 2019-11-22 | 临沂大学 | A kind of fluorescence probe and the preparation method and application thereof |
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