CN1811429A - Terbium coordination compound singlet oxygen fluorescent probe and application thereof - Google Patents
Terbium coordination compound singlet oxygen fluorescent probe and application thereof Download PDFInfo
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Abstract
The present invention relates to a determination technique of singlet oxygen (1O2) in solution, in the concrete, it relates to a new type terbium coordination compound singlet oxygen fluorescent probe and its application. Said coordination compound is formed by using trivalent terbium ion Tb3+ and ligand containing anthracene and its derivative substituted 2,6-dipyrazolylpyridine skeleton structure, which can be reacted with singlet oxygen molecule characteristics. Besides, said invention also provides the structure formula of said ligand. Said invention has good selectivity for singlet oxygen molecule, and can be used for quantitatively detecting singlet oxygen.
Description
Technical field
The present invention relates to singlet oxygen in the solution (
1O
2) determination techniques, specifically a kind of new terbium complex singlet oxygen fluorescence probe and application thereof.
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, chemiluminescence, biosome deterioration by oxidation or even photocarcinogenesis as light degradation, pollutant, is all playing the part of crucial role.The application of singlet oxygen aspect organic synthesis had many reports, but its physiology oxidation in living things system more and more receives researcher's concern, it is containing the significant impact to biology and life system, various biomolecule comprise that membrane lipid, protein, amino acid, nucleotide, carbohydrates 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).
Just because of the outstanding critical role of singlet oxygen in photochemistry and photo bio process, people just deepen continuously to its research, and the detection method of several singlet oxygens of having reported is as follows:
1) phosphorimetry.Phosphoresce owing to two singlet oxygen moleculars can release energy with the form of light when singlet transits to ground state simultaneously, this character can be used to detect singlet oxygen (document 1:A.A.Jr.Krasnovsky, Biofzika 1976,21,748).This method has become one of standard method that detects singlet oxygen, is used to measure growing amount, the life-span of singlet oxygen and the constant that deactivates.Because the vital role of singlet oxygen in numerous optical-biological reaction processes developed the singlet oxygen detection method that can be adapted to the living things system environment so press for, phosphorimetry can't be used for low concentration because its sensitivity is low
1O
2Detection by quantitative.
2) chemical agent for capturing method.9,10-diphenylanthrancene (DPA) is the most frequently used compound, itself and singlet oxygen reaction back generate stable inner oxide, can change amount (document 2:M.J.Stenbeck, A.U.Khan, the M.J.Karnovsy that measures singlet oxygen in the absorbance of 355nm by measuring DPA, J.Biol.Chem., 1992,267,13425).This method has been used to measure singlet oxygen (document 3:M.J.Stenbeck, A.U.Khan, the M.J.Kamovsy that produces in the phagocyte, J.Biol.Chem., 1993,268,15649), but because this method is based on the detection method of absorbance log, so sensitivity is lower.
3) organic fluorescence sonde method.Recently, utilizing fluorescence signal is that the organic fluorescence molecular probe method that detects means is developed, and this probe comprises two classes: the no fluorescence of (1) probe itself, significantly strengthen (document 4:N.Umezawa with singlet oxygen reaction back fluorescence, K.Tanaka, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Angew.Chem.Int.Ed.Engl., 1999,38,2899.Document 5:K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, J.Am.Chem.Soc., 2001,123,2530).This class fluorescence probe mainly is the fluorescein molecule that anthracene and derivant thereof replace, and this method is short, highly sensitive detection time, but is not suitable for low pH value system and real-time the detection.(2) fluorescence significantly strengthens (document 6:A.A.Krasnovskii, C.Schweitzer, H.Leismann, C.Tanielian, E.A.Luk ' yanets, Quantum Electron., 2000,30,445-448 after the NE BY ENERGY TRANSFER of reception singlet oxygen.Document 7:A.A.Krasnovskii, M.E.Bashtanov, N.N.Drczdova, O.A.Yuzhakova, E.A.Luk ' yanets, Quantum Electron., 2002,32,83-86).This class probe mainly contains phthalocyanine dye (phthalocyanines) and porphyrazine (porphyrazine) derivant etc., can send fluorescence and be used for detecting behind the energy that receives singlet oxygen near 700nm.Use the assay method of organic fluorescence probe mainly to exist fluorometric assay to be subjected to the problem of the interference of light at random and sample background fluorescence easily.
4) chemiluminescence probe method.This probe is a kind of singlet oxygen chemiluminescence probe based on the photoinduction electron transport mechanism (document 8:X.H.Li, G.X.Zhang, H.M.Ma, D.Q.Zhang, J.Li, D.B.Zhu, J.Am.Chem.Soc.2004,126,11543).This method selectivity is good, highly sensitive, detection speed is fast, but the probe poorly water-soluble is unfavorable in the living things system
1O
2Mensuration, can not be used for the real-time follow-up that singlet oxygen produces system in addition and detect.
Because the life-span of singlet oxygen itself is very short, the high sensitivity quantitation to it detects the challenging task that remains at present.Owing to the time-resolved fluorescence detection technique that the long-life fluorescent characteristics based on the rare-earth fluorescent complex grows up can be eliminated short-life light at random and the interference of sample background fluorescence to measuring very effectively, measure sensitivity and greatly improve, be subjected to extensive concern (document 9:J.Yuan at the high-sensitivity biological detection range in recent years, K.Matsumoto and H.Kimura, Anal.Chem., 1998,70,596-601. document 10:K.Matsumoto, J.Yuan, United States Patent, 1999, Patent No.58592973.Document 11:I.Hemmil , V.-M.Mukkala, Crit.Rev.Clin.Lab.Sci., 2001,38,441-519.Document 12:K.Matsumoto, J.Yuan, Metal Ions in Biological Systems, Marcel Dekker, Inc., New York and Basel, 2003, Vol.40, pp.191-232).But the singlet oxygen fluorescence probe and the time-resolved fluorescence detection technique thereof that are based on rare earth compounding yet there are no report.
Summary of the invention
For the high sensitivity quantitation that solves singlet oxygen detects problem, the purpose of this invention is to provide the high-sensitivity detection that a kind of new terbium complex fluorescence probe is used for singlet oxygen.
To achieve these goals, the technical solution used in the present invention is as follows:
With trivalent terbium ion Tb
3+With can with the anthracene of singlet oxygen molecular characteristic reaction and derivant thereof replace 2, the complex that 6-two pyrazolyl pyridine skeleton structure class ligands form is a probe molecule, with the terbium coordination compound that forms hyperfluorescence and have long fluorescence lifetime after the singlet oxygen reaction, and then be used for fluorometric assay or time-resolved fluorometry.Described ligand structure formula is specially:
In the formula wherein R be hydrogen (H), alkyl (C
nH
2n+1) or phenyl (C
6H
5); Wherein best n=1～6.
Described terbium coordination compound probe is used for the detection of singlet oxygen, in all kinds of biologies and abiotic environment, utilizes terbium coordination compound to catch in the system
1O
2, make the fluorescence intensity of complex significantly strengthen, determine the generation and the growing amount of singlet oxygen then by fluorometry;
It is as follows that probe molecule of the present invention is used for the high-sensitivity detecting method of singlet oxygen: probe (almost not having fluorescence) is joined system to be measured, after the anthryl in the complex is partly caught the singlet oxygen that produces in the system, the fluorescence intensity of probe will increase tens of times, the fluorescence intensity of probe behind the assaying reaction can detect the growing amount of singlet oxygen.
Wherein said fluorometry also comprises time-resolved fluorometry and time resolution fluorescent microscope determination method except the fluorometry of routine.
Terbium coordination compound fluorescence probe of the present invention can be prepared into singlet oxygen terbium coordination compound fluorescence probe
1O
2Detectable and kit.
Singlet oxygen fluorescence probe of the present invention has following advantage:
(1) highly sensitive.Because catching the terbium fluorescence complex that generates behind the singlet oxygen, the probe molecule of being invented has long fluorescence lifetime, utilize the time-resolved fluorometry technology can eliminate background fluorescence and the interference of light at random effectively, thereby can obtain very high detection sensitivity measuring.New terbium complex fluoresces before reacting with singlet oxygen hardly, can send strong terbium ion long-life fluorescence after reacting with singlet oxygen, and fluorescence lifetime is 2.76 milliseconds; Utilize the enhancing of fluorescence signal can realize detection by quantitative to singlet oxygen, lowest detectable limit can reach 1.1 * 10
8Mol/L.
(2) good water solubility.The terbium coordination compound probe of being invented is very soluble in water, can in various buffer solution, use, overcome the water-insoluble fluorescence probe and need add that organic solvent helps dissolving and to the interference that mensuration is brought, the real-time in-situ that is highly suitable for singlet oxygen in the various living things systems detects.
(3) selectivity is good.Selectivity experimental result to various active oxygen species (comprising hydroxyl radical free radical, superoxide anion, hydrogen peroxide and singlet oxygen) shows that the new terbium complex probe has very high selectivity to singlet oxygen molecular.
(4) fast light bleaching power is strong.Compare with organic fluorescent compounds fluorescein (common organic fluorescent dye), the new terbium complex probe is caught the fluorescence complex that generates behind the singlet oxygen and is had higher anti-photobleaching performance (fast light bleach stability), be applicable to need longer exposure time living things system singlet oxygen generative process in-site detecting.
(5) the fluorescigenic Stokes displacement of probe molecule institute of the present invention is big, and glow peak is shaped as the sharp-pointed shape glow peak of trivalent terbium ion feature, measures the not interference of stimulated luminescence.
Description of drawings
Fig. 1 is a ligand PATA synthetic route;
Fig. 2 is EP-PATA-Tb
3+Mass spectrogram [M-H]
-(mass spectroscopy result);
Fig. 3 is PATA-Tb
3+Catch the time resolution fluorescence spectral figure of (a) back (b) before the singlet oxygen;
Fig. 4 be in the 0.05mol/L borate buffer solution pH value to EP-PATA-Tb
3+The influence of (1.0 μ mol/L) fluorescence intensity (■) and fluorescence lifetime (zero);
Fig. 5 is EP-PATA-Tb in aqueous solution
3+(■) and the light stability experimental result of fluorescein (zero);
Fig. 6 is that the working curve of time-resolved fluorescence quantitative measurement singlet oxygen is (at the 0.1mol/L of pH value 10.5 NaHCO
3Detect in the buffer solution, use Na
2MoO
4Concentration is 10mmol/L, PATA-Tb
3+Concentration is 0.5 μ mol/L).
Embodiment
The invention will be further described below by embodiment.
Ligand PATA synthetic route as shown in Figure 1, specific operation process is as follows:
Synthesizing of (1) 2,6-two bromo-4-(9 '-anthryl) pyridines (compound 1)
Add 2.52 gram 4-amino-2 in 150ml acetate, 50ml methylene chloride, 6-dibromo pyridine (10.0mmol) and 2.14 gram anthracenes (12.0mmol) are added dropwise to 2.0 gram nitrosyl isopentyl ester under 0 ℃ of stirring; Stirring reaction added saturated Na after 48 hours under the reactant liquor room temperature
2CO
3The solution neutralization uses the chloroform of 4 * 60ml to extract product out then; Chloroformic solution with anhydrous sodium sulfate drying after, the pressure reducing and steaming solvent; Product separates with silica gel column chromatography, with methylene chloride-sherwood oil (v/v, 1: 2) wash out, boil off solvent after, product ethyl alcohol recrystallization, vacuum drying; Get target compound 1.24 grams (yield 30.02%).
1HNMR measurement result (400MHz, CDCl
3): δ 8.56 (s, 1H), 8.06 (d, J, 8.4Hz, 2H), 7.57 (s, 2H), 7.54-7.46 (m, 6H).
Synthesizing of (2) 2,6-two (3 '-carboxylate methyl ester base-1 '-pyrazolyl)-4-(9 '-anthryl) pyridine (compound 2)
In the 60ml dry tetrahydrofuran, add 7.14 gram 3-carboxylate methyl ester base pyrazoles (56.26mmol), the metallic potassium that adds 2.23 gram fritters after the stirring and dissolving, solution adds the compound 1 (12.59mmol) of 5.2 grams after 60 ℃ of following stirring reactions are intact to the metallic potassium total overall reaction; The reactant liquor continuous backflow stirred after 9 days, the pressure reducing and steaming solvent, and product is with 6 * 150ml chloroform extracting solvend; The pressure reducing and steaming solvent, product separates with silica gel column chromatography, washes out with methylene chloride-sherwood oil (v/v, 1: 1); Boil off product toluene recrystallization behind the solvent, vacuum drying; Get target compound 2.04 grams (yield 32.2%).
1HNMR measurement result (400MHz CDCl
3): δ 8.75 (d, J, 2.8Hz, 2H), 8.58 (s, 1H), 8.22 (s, 2H), 8.07 (d, J, 8.4Hz, 2H), 7.58 (d, J, 8.4Hz, 2H), 7.49 (t, J, 8.0Hz, 2H), 7.38 (t, J, 7.2Hz, 2H), 7.08 (d, J, 2.8Hz, 2H), 3.90 (s, 6H).
Synthesizing of (3) 2,6-two (3 '-methylol 1 '-pyrazolyl)-4-(9 '-anthryl) pyridine (compound 3)
In the 160ml dry tetrahydrofuran, add 0.63 gram LiAlH
4, add 1.6 then and digest compound 2 (3.2mmol), stir after 4 hours under the reactant liquor room temperature, slowly splash into 0.55ml water, 15% NaOH of 0.55ml and 2.2ml water; Stir after 30 minutes under the room temperature, remove by filter precipitation, the solvend with in 3 * 100ml tetrahydrofuran extracting precipitation merges tetrahydrofuran solution; Behind the pressure reducing and steaming solvent, product fully washs with acetonitrile, and vacuum drying gets target compound 0.90 gram (yield 63.2%).
1H NMR measurement result (400MHz, DMSO-d
6): δ 9.03 (d, J, 2.4Hz, 2H), 8.81 (s, 1H), 8.22 (d, J, 8.4Hz, 2H), 7.73 (s, 2H), 7.71 (d, J, 8.4Hz, 2H), 7.59 (t, J, 8.0Hz, 2H), 7.50 (t, J, 8.0Hz, 2H), 6.63 (d, J, 2.4Hz, 2H), 4.46 (s, 4H).
Synthesizing of (4) 2,6-two (3 '-bromomethyl-1 '-pyrazolyl)-4-(9 '-anthryl) pyridine (compound 4)
At the anhydrous N of 70ml, add 0.9 in the dinethylformamide and digest compound 3 (2.0mmol), stir the 2.0 gram PBr of adding down
3, the reactant liquor stirring at room added 10% sodium carbonate liquor of 80ml after 24 hours, and fully collecting precipitation is filtered in the stirring back, and vacuum drying after the precipitation water fully washs gets target compound 1.11 grams (yield 96.4%).
1H NMR measurement result (400MHz, CDCl
3): δ 8.65 (d, J, 2.4Hz, 2H), 8.57 (s, 1H), 8.07 (d, J, 8.4Hz, 2H), 7.97 (s, 2H), 7.67 (d, J, 8.4Hz, 2H), 7.48 (t, J, 8.0Hz, 2H), 7.41 (t, J, 8.0Hz, 2H), 6.60 (d, J, 2.4Hz, 2H), 4.48 (s, 4H).
(5) N, N, N
1, N
1-[2,6-two (3 '-amine methyl isophthalic acid '-pyrazolyl)-4-(9 '-anthryl) pyridine] tetraacetic acid tetraethyl ester (compound 5) synthetic
Add 1.11 and digest compound 4 (1.9mmol) in dry acetonitrile of 140ml and 40ml dry tetrahydrofuran, the ethyl diacetate base amine (4.30mmol) of 810mg is dissolved in the solution and the 2.72 gram K of the dry acetonitrile of 10ml
2CO
3(20mmol), reactant liquor refluxes and stirs after 24 hours, removes by filter insolubles; The pressure reducing and steaming solvent is dissolved in product in the 100ml chloroform, chloroformic solution with 2 * 50ml water washing after, use anhydrous sodium sulfate drying; Filter back pressure reducing and steaming solvent, vacuum drying; Gained grease separates with silica gel column chromatography, with ethyl acetate-sherwood oil (2: 3, v/v) wash out, boil off solvent after, vacuum drying; Get target compound 1.17 grams (yield 76.2%).
1H NMR measurement result (400MHz, CDCl
3): δ 8.64 (d, J, 2.4Hz, 2H), 8.55 (s, 1H), 8.06 (d, J, 8.4Hz, 2H), 7.91 (s, 2H), 7.69 (d, J, 8.4Hz, 2H), 7.48 (t, J, 6.4Hz, 2H), 7.39 (t, J, 6.8Hz, 2H), 6.59 (d, J, 2.4Hz, 2H), 4.10 (q, J, 7.2Hz, 8H), 3.98 (s, 4H), 3.57 (s, 8H), 1.16 (t, J, 7.2Hz, 12H).
(6) N, N, N
1, N
1-[2,6-two (3 '-amine methyl isophthalic acid '-pyrazolyl)-4-(9 '-anthryl) pyridine] tetraacethyls (being called for short PATA) synthetic
The compound 5 (1.4mmol) of 1.11g is joined in the 85ml ethanol, add 2.2 gram potassium hydroxide and 7ml water then, reactant liquor refluxes and stirred 2 hours; The pressure reducing and steaming solvent is dissolved in product in the 50ml water, removes by filter micro-insolubles, and very long trifluoroacetic acid aqueous solution to the pH value of solution that splashed into 1: 1 is about till 1 in the solution under stirring; Stir under the solution room temperature after 3 hours, filter collecting precipitation, fully wash vacuum drying with the trifluoroacetic acid aqueous solution of 100 times of dilutions; Get target compound 922mg (yield 97.0%).Results of elemental analyses (%) is pressed C
35H
38N
7O
11.5S (PATA3.5H2O) calculated value (%): C, 56.75; H, 5.17; N, 13.24, measured value (%): C, 56.71; H, 5.16; N, 13.30.
1H NMR measurement result (400MHz DMSO-d
6): δ 9.04 (d, J, 2.4Hz, 2H), 8.81 (s, 1H), 8.22 (d, J, 8.4Hz, 2H), 7.72 (d, J, 8.4Hz, 2H), 7.71 (s, 2H), 7.58 (t, J, 8.0Hz, 2H), 7.50 (t, J, 8.0Hz, 2H), 6.62 (d, J, 2.4Hz, 2H), 3.88 (s, 4H), 3.43 (s, 8H).The mass spectroscopy result (ESI-MS, m/z): 676[M
--H].
Embodiment 2.PATA-Tb
3+The reaction of complex and singlet oxygen
PATA and TbCl with equimolar amounts
3Be dissolved in the NaHCO of 0.1mol/L
3In the aqueous solution, promptly obtain PATA-Tb
3+The solution of complex; Adopt Na
2MoO
4Catalysis H under weak basic condition
2O
2The singlet oxygen that produces has been measured PATA-Tb as the singlet oxygen source
3+The reaction product of complex and singlet oxygen.Be specially in the 0.1mol/L of pH value 10.5 carbonate buffer solution and add PATA-Tb
3+Complex, Na
2MoO
4And certain amount of H
2O
2, reactant liquor is measured after 20 hours 25 ℃ of reactions.Fig. 2 is PATA-Tb
3+Mass spectrum (ESI-MS) measurement result with singlet oxygen reaction afterproduct shows that product is PATA-Tb
3+Inner oxide (be called for short EP-PATA-Tb
3+).
Embodiment 3.PATA and rare earth ion (Eu
3+And Tb
3+) photoluminescent property of complex measures
1. fluorescence spectrum, fluorescence intensity and fluorescence lifetime
In aqueous solution PATA with can form stable fluorescigenic hardly complex rapidly after rare earth ion mixes, yet when with the singlet oxygen reaction after, fluorescence signal strengthens significantly.0.05mol/L borate buffer solution with pH value 9.1 is that solvent has been measured PATA-Tb
3+, PATA-Eu
3+And inner oxide EP-PATA-Tb
3+, EP-PATA-Eu
3+Uv-vis spectra in this solvent, fluorescence spectrum, molar absorptivity, fluorescence quantum yield and fluorescence lifetime; Uv-vis spectra day beautiful UV 7500 spectrophotometric determinations; Fluorescence Perkin Elmer LS 50B fluorescent spectrophotometer assay.Quantum yield is measured and is used 4 '-phenyl-2,2 ': 6 ', 2 "-ter cycloheptapyridine-6,6 "-complex of dimethylamine tetraacethyl and Eu3+ and Tb3+ records (document 13:M.Latva as reference material, H.Takalo, V.-M.Mukkala, C.Matachescu, J.C.Rodriguez-Ubis, J.Kankare J.Lumin., 1997,75,149-169).Measurement result is as shown in table 1.
Absorption and the photoluminescent property of several compounds of table 1. in the 0.05mol/L of pH value 9.1 borate buffer solution
| Compound | Maximum absorption wavelength (nm) | Molar absorptivity (cm -1M -1) | Maximum fluorescence wavelength (nm) | Fluorescence quantum yield (%) | Fluorescence lifetime (ms) |
| PATA-Tb 3+ EP-PATA-Tb 3+ PATA-Eu 3+ | 316 316 316 | 13600 18300 13100 | 543 543 620 | 0.46 10.5 0.05 | 2.000 2.765 0.246 |
| EP-PATA-Eu 3+ | 316 | 15300 | 620 | 1.87 | 1.296 |
By table 1 result as can be seen, PATA-Eu
3+And PATA-Tb
3+Be extremely weak fluorescence compound, and EP-PATA-Tb
3+Be hyperfluorescence complex, EP-PATA-Eu
3+Though the fluorescent quantum yield is than PATA-Eu
3+Strengthened manyfold, but still be the hypofluorescence compound, four kinds of compounds all have long fluorescence lifetime, with EP-PATA-Tb
3+Fluorescence lifetime the longest.Fig. 3 has provided PATA-Tb
3+Catch the time resolution fluorescence spectral figure of singlet oxygen front and back, show PATA-Tb
3+Significantly strengthen with singlet oxygen reaction back fluorescence signal, therefore can utilize this property testing singlet oxygen.
2. pH value of buffer solution is to the influence of complex fluorescent character
Borate buffer solution with the 0.05mol/L of different pH values is a solvent, has measured EP-PATA-Tb under the different pH values
3+Fluorescence intensity and fluorescence lifetime, it the results are shown in Figure 4.Measurement result shows that in the scope of pH value 9-3.0, the pH value is to EP-PATA-Tb
3+The fluorescence intensity and the influence of fluorescence lifetime little.
Embodiment 4.PATA-Tb
3+Selectivity to singlet oxygen
PATA-Tb
3+The complex probe is one of critical nature of desirable probe to the selectivity reaction of singlet oxygen molecular, so we have investigated PATA-Tb
3+With various reactive oxygen species (hydroxyl radical free radical OH, superoxide anion O
2 -, H
2O
2, singlet oxygen
1O
2) response situation.With PATA-Tb
3+With the reacted fluorescent value of singlet oxygen as 100, see Table 2 with the variation of various reactive oxygen species reaction back fluorescence intensity.From table 2 result as seen, PATA-Tb
3+With OH, O
2 -, H
2O
2The reaction back is with respect to pure PATA-Tb
3+The fluorescence intensity of solution increase is very little, and significantly strengthens with singlet oxygen reaction back fluorescence intensity, shows PATA-Tb
3+The complex probe has very high selectivity to singlet oxygen.
Various reactive oxygen species of table 2. and PATA-Tb
3+Reacted relative intensity of fluorescence
| Compound | Pure PATA-Tb 3+ | With 1O 2After the reaction | After the OH reaction | With O 2 -After the reaction | With H 2O 2After the reaction |
| Relative intensity of fluorescence | 8.3 | 100 | 9.1 | 8.6 | 10.9 |
Embodiment 5.PATA-Tb
3+Light stability with the singlet oxygen reaction product
EP-PATA-Tb
3+See Fig. 5 with the photobleaching experimental result of fluorescein in the 0.05mol/L borate buffer solution.Per 5 minutes record first order fluorescence intensity in 60 minutes exposure times is as seen through EP-PATA-Tb after 60 minutes
3+Fluorescence intensity 61% when still keeping initial, and almost all decay of the fluorescence of fluorescein molecule have only kept for 2% when initial, show that the new terbium complex fluorescence probe has better light stability than fluoresceins probe, are applicable in the living things system more
1O
2The long-time fluorometric assay of generative process.
The detection sensitivity of embodiment 6. singlet oxygen fluorescence probes
With Na
2MoO
4/ H
2O
2The singlet oxygen that system produces at pH value 10.5 solution reactions uses PATA-Tb as standard singlet oxygen source
3+Probe has carried out quantitative measurement to the generation of singlet oxygen, and gained the results are shown in Figure 6.Measuring with instrument is Perkin ElmerVictor 1420 type multiple labeling calculating instruments, and condition determination is: excitation wavelength, 340nm; Detect wavelength, 545nm; Delay time, 0.2ms; The numeration window time, 0.4ms; Cycling time, 1.0ms.The concentration of fluorescence intensity and singlet oxygen is good linear relationship in measurement range as seen from Figure 6, shows PATA-Tb
3+Probe can be used for the quantitative measurement of singlet oxygen.3 times of the standard deviation of the fluorescence signal during with zero-dose (background) are calculated the minimum lower limit that detects, and minimum the detecting down that gets this law is limited to 1.1 * 10
8Mol/L.
Claims (5)
1. a terbium coordination compound singlet oxygen fluorescent probe is characterized in that: be with trivalent terbium ion Tb
3+With contain can with the anthracene of singlet oxygen molecular characteristic reaction and derivant thereof replace 2, the complex that 6-two pyrazolyl pyridine skeleton structure class ligands form; The structural formula of wherein said ligand is:
R＝H，C
nH
2n+1，C
6H
5
2. according to the described terbium coordination compound singlet oxygen fluorescent probe of claim 1, it is characterized in that: n=1～6 wherein.
3. the application of the described terbium coordination compound singlet oxygen fluorescent probe of claim 1 is characterized in that: be used for the detection of singlet oxygen, in all kinds of biologies and abiotic environment, utilize terbium coordination compound to catch in the system
1O
2, make the fluorescence intensity of complex significantly strengthen, determine the generation and the growing amount of singlet oxygen then by fluorometry.
4. according to the application of the described terbium coordination compound singlet oxygen fluorescent probe of claim 3, it is characterized in that: described fluorometry is conventional fluorometry, time-resolved fluorometry or time-resolved fluorescence measurement microscope method.
5. according to the application of the described terbium coordination compound singlet oxygen fluorescent probe of claim 3, it is characterized in that: be used to be prepared into the singlet oxygen detectable and the kit that contain the described terbium coordination compound fluorescence probe of claim 1.
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| WO2013054046A1 (en) | 2011-10-12 | 2013-04-18 | Centre National De La Recherche Scientifique | Luminescent probe for biological labelling and imaging, method for preparing same |
| CN107603600A (en) * | 2017-10-01 | 2018-01-19 | 桂林理工大学 | A kind of terbium coordination compound green luminescent material and synthetic method |
| CN108727277B (en) * | 2018-05-24 | 2021-03-30 | 陕西师范大学 | Singlet oxygen highly selective aggregation-induced chemiluminescence probe and preparation method and application thereof |
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