CN102732246A - Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application - Google Patents
Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application Download PDFInfo
- Publication number
- CN102732246A CN102732246A CN2012101977185A CN201210197718A CN102732246A CN 102732246 A CN102732246 A CN 102732246A CN 2012101977185 A CN2012101977185 A CN 2012101977185A CN 201210197718 A CN201210197718 A CN 201210197718A CN 102732246 A CN102732246 A CN 102732246A
- Authority
- CN
- China
- Prior art keywords
- singlet oxygen
- mtdta
- cell
- fluorescence
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to a europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application. The probe has a structure as the following. The probe can directly enter a cell through a cell co-culture method, and can be used for time-resolved fluorescence assay of intracellular singlet oxygen and other biochemical determination. The probe can also be used for fluorescence determination or time-resolved fluorescence determination of singlet oxygen in various chemical, photochemical and biochemical systems.
Description
Technical field
The present invention relates to a kind of singlet oxygen and measure with fluorescent probe and application thereof specifically a kind of europium coordination compound fluorescent probe and application thereof with permeability of cell membrane.
Background technology
Singlet oxygen is a kind of unsettled oxygen molecule that is in high-energy excited state; Recent two decades comes, and comes the cancer optical dynamic therapy technology (photodynamic therapy is called for short PDT) of kill tumor cell in clinical medicine, to obtain increasing application based on the strong oxidizing property matter of singlet oxygen; This technology uses derivatives of porphyrin such as photosensitizers such as porporino, hematoporphyrin monomethyl ether as medicine (document 1:W.M.Sharman usually; C.M.Allen, J.E.Lier, Drug.Discov.Today.1999; 4,507; Document 2:T.J.Dougherty, J.Clin.Laser.Med.Surg.2002,20,3; Document 3:X.Ding, Q.Xu, F.Liu, P.Zhou; Y.Gu, J.Zeng, J.An, W.Dai; X.Li, CancerLett.2004,216,43); After photosensitizer was enriched in the tumour cell, through tumor locus is carried out illumination, photosensitizer can be given transmission ofenergy oxygen molecule and generate singlet oxygen after absorbing luminous energy, and then utilizes the oxygenizement of singlet oxygen to reach the purpose that makes apoptosis of tumor cells or necrosis.In recent years, 5-aminolevulinic acid and verivate thereof had also obtained many application (document 4:A.Casas, H.Fukuda, G.D.Venosa, A.B.Batlle, J.Cancer2001,85,279 as the PDT medicine in the cancer clinical treatment; Document 5:J.M.Gauffier, K.Berg, Q.Peng, H.Anholt; P.K.Selbo, L.Ma, J.Moan; CancerRes.1997,57,1481); The 5-aminolevulinic acid itself is not a kind of photosensitizer, but it can be metabolized to protoheme precursor protoporphyrin IX after getting into cell, thereby becomes a kind of photosensitizer that under illumination, can produce singlet oxygen.
Because singlet oxygen has important effect at aspects such as cancer PDT medicine performance evaluation and treatment effectiveness evaluations, the determination techniques research of singlet oxygen has caused increasing concern in the active somatic cell.The detection that can be used for singlet oxygen in the active somatic cell at present mainly contains following several method: (1) utilizes singlet oxygen self cancellation process to produce detection method (the document 6:S.Oelckers of phosphorescence; T.Ziegler; I.Michler, B.R der, J.Photochem.Photobiol.B:Biol.1999; 53,121; Document 7:L.K.Andersen, P.R.Ogilby, Photochem.Photobiol.2001,73,489; Document 8:L.K.Andersen, Z.Gao, P.R.Ogilby, L.Poulsen, I.Zebger, J.Phys.Chem.A2002,106,8488).This method specificity is very high, but a little less than the detecting signal, at the bottom of the sensitivity, can't be used for the detection of lower concentration singlet oxygen.(2) utilization has the detection method (document 9:K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, J.Am.Chem.Soc.2001,123,2530) of the fluoresceins fluorescent probe of anthracene nucleus.This method is highly sensitive, but because photosensitizer medicine itself just has very strong fluorescence, this method can not be used for the photosensitizer medicine coexistence fluorometric assay of the interior singlet oxygen of cell down.(3) the europium coordination compound fluorescent probe binding time that utilizes that we set up is differentiated the detection method (document 10:B.Song, G.Wang, M.Tan, J.Yuan, J.Am.Chem.Soc.2006,128,13442) of fluorometric assay technology.This method has been utilized the long lifetime fluorescent characteristic of europium complex, adopts the time-resolved fluorometry technology to eliminate the background fluorescence of photosensitizer medicine, and then can realize the real-time in-situ mensuration of singlet oxygen in the active somatic cell.But can not get in the cell through cytolemma during owing to probe and co-culture of cells, make it in cancer PDT research, be widely used than difficulty.
The present invention is on the basis of our above-mentioned europium complex singlet oxygen fluorescence probe development; Improve through structure and to prepare a kind of permeability of cell membrane that has title complex; Can get into intracellular europium coordination compound fluorescent probe through cytolemma with co-culture of cells, utilize this fluorescent probe binding time to differentiate the fluorometric assay technology and set up a kind of PDT of being used for medicine (hematoporphyrin monomethyl ether and 5-aminolevulinic acid) produces singlet oxygen in cell real-time in-situ fluorometric assay technology.
Summary of the invention
The invention provides a kind of highly sensitive, selectivity and good water solubility, applied widely, have permeability of cell membrane, can be used for the singlet oxygen that PDT medicine illumination in the active somatic cell produces is down carried out fluorimetric europium coordination compound fluorescent probe of real-time in-situ and application.
Technical scheme of the present invention is following:
A kind of europium complex singlet oxygen fluorescence probe with permeability of cell membrane; Be that to replace the neutral compound (being called for short [MTDTA-Eu]) that the macrocyclic ligand of ter cycloheptapyridine skeleton structure forms with trivalent europium ion and a kind of 9-of containing methyl anthracene be fluorescent probe, the structural formula of wherein said title complex [MTDTA-Eu] is:
The application process of singlet oxygen fluorescence probe is in the said active somatic cell based on europium complex [MTDTA-Eu]: the cell of described fluorescent probe [MTDTA-Eu] and PDT medicine and co-culture of cells being prepared [MTDTA-Eu] and the common mark of PDT medicine; Utilize [MTDTA-Eu] to catch the singlet oxygen that produces in the system under the illumination then; Make the fluorescence intensity of probe significantly strengthen, and then confirm through time-resolved fluorometry to distribute in production process and the cell of singlet oxygen.Said time-resolved fluorometry comprises time resolution fluorescence spectral assay method and time resolution fluorescent microscopic imaging assay method.
Cause its fluorescence significantly to strengthen through the singlet oxygen in the title complex and the aqueous solution is reacted, and then be used for the fluorometric assay of aqueous solution singlet oxygen.
Fluorescent probe of the present invention has following advantage:
1. have well water-solublely and stable, can prolonged preservation use.
2. be applicable to the mensuration of singlet oxygen in the multiple environment such as slightly acidic, neutrality and alkalescence.
3. have good permeability of cell membrane, can get in the active somatic cell with co-culture of cells, to be used for the mensuration of singlet oxygen in the cell.
4. have high singlet oxygen and measure sensitivity, its lowest detection lower limit can reach the nanomolar concentration level.
5. singlet oxygen there is good selectivity, with other reactive oxygen species effect fluorescent signal no change almost.
With the singlet oxygen effect after fluorescence radiation quantum yield and fluorescence lifetime all significantly strengthen, can be used for the time-resolved fluorometry of hundreds of microsecond levels.
Description of drawings
Fig. 1 is the synthetic route chart of probe [MTDTA-Eu].
Fig. 2 is the fluorescence spectrum figure of reaction product [EP-MTDTA-Eu] (dotted line, 5.0 μ mol/L) in 0.05 mol/L borate buffer solution of pH value 9.1 of [MTDTA-Eu] (solid line, 5.0 μ mol/L) and itself and singlet oxygen.
Fig. 3 is fluorescence intensity and the fluorescence lifetime of [EP-MTDTA-Eu] (5.0 μ mol/L) in 0.05 mol/L Tris-HCl buffered soln of different pH values.Among the figure: fluorescence intensity ■; Fluorescence lifetime.
Fig. 4 is that [MTDTA-Eu] relatively schemes with the fluorescence intensity of various reactive oxygen species reaction product.
Fig. 5 is that singlet oxygen produces system Na under [MTDTA-Eu] and the alkaline condition
2MoO
4-H
2O
2Fix N a during reaction
2MoO
4Concentration changes H
2O
2Fluorescence spectrum figure under the concentration situation.
Fig. 6 is that singlet oxygen produces system H under [MTDTA-Eu] and the neutrallty condition
2O
2In-NaOCl when reaction, be H fixedly
2O
2Concentration changes the fluorescence spectrum figure under the NaOCl concentration situation.
Fig. 7 be [MTDTA-Eu] with solutions of weak acidity under singlet oxygen fixing horseradish peroxidase concentration when producing system horseradish peroxidase-indole-3-acetic acid reaction, the fluorescence response kinetic curve figure under the change indole-3-acetic acid concentration situation.
Fig. 8 differentiates fluorescence imaging (figure right part) mensuration result's (scale: 10 microns) the steady-state fluorescence imaging (figure left part) and the time of the HeLa cell of hematoporphyrin monomethyl ether (figure top) and [MTDTA-Eu] (figure bottom) difference mark.
To be hematoporphyrin monomethyl ether measure figure as a result with the time resolved fluorescence imaging during different irradiation time in 100 watts of mercury lamp irradiation process of the HeLa cell of [MTDTA-Eu] common mark to Fig. 9.
Figure 10 be among Fig. 9 the border circular areas fluorescence intensity with the variation diagram of irradiation time.
To be the 5-aminolevulinic acid measure figure as a result with the time resolved fluorescence imaging during different irradiation time in 100 watts of mercury lamp irradiation process of the HeLa cell of [MTDTA-Eu] common mark to Figure 11.
Figure 12 be among Figure 11 the elliptical region fluorescence intensity with the variation diagram of irradiation time.
Embodiment
Through embodiment the present invention is described further below.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: probe complexes [MTDTA-Eu] synthetic and with the reaction of singlet oxygen
Synthetic route is as shown in Figure 1, and the matrix operating process is following.
(1) compound 3 is synthetic
At first adopt document 10 methods and document 11 methods to synthesize compound 1 and compound 2 (document 11:C.Galaup, J.M.Couchet, S.Bedel, P.Tisnes, C.Picard, J.Org.Chem.2005,70,2274) respectively.300mg compound 1 (0.49mmol), 218mg compound 2 (0.49mmol) and 519mg salt of wormwood (4.9mmol) are joined in the mixed solvent of dry acetonitrile of 120mL and 100mL dry tetrahydrofuran (THF), stir refluxed reaction 24 hours.Remove by filter insolubles, the pressure reducing and steaming solvent.Resultant is that eluent separates with silicagel column with ETHYLE ACETATE-triethyl ammonia-methyl alcohol of 40:8:5 (v/v/v) earlier, and the chloroform-methanol with 25:1 (v/v) is that eluent separates with alumina column again, obtains the yellow oily compound 3 of 120mg, productive rate 27.4%.
1HNMR (400MHz, CDCl
3) mensuration result: δ=1.18 (s, 9H); 1.30 (s, 18H); 2.82 (s, 4H); 2.98 (d, J=8.0Hz, 4H); 3.23-3.26 (m, 9H); 4.15 (s, 4H); 7.39-7.42 (m, 4H); 7.56-7.63 (m, 4H); 7.85 (d, J=4.0Hz, 4H); 8.08 (s, 2H); 8.46 (d, J=8.0Hz, 2H).Mass spectrum (ESI-MS) is measured result: m/z=893.5 [M+H]
-
(2) macrocyclic ligands MTDTA's is synthetic
200mg compound 3 (0.28 mmol) is dissolved in the 1:1 CH of 30mL
2Cl
2-CF
3Among the COOH, stirring reaction is 20 hours under the room temperature.Behind the pressure reducing and steaming solvent resultant is dissolved in the methyl alcohol of 5mL, the ether that adds 50mL is then separated out product.Filter collecting precipitation, after the vacuum-drying product is joined in the dry acetonitrile of 10mL again, stirred refluxed 20 minutes.。Filter collecting precipitation, obtain the yellow powder shape target compound MTDTA of 150mg after the vacuum-drying, productive rate 73.9%.
1HNMR (400MHz, DMSO-d
6) mensuration result: δ=2.76 (d, J=8.0Hz, 4H); 2.91 (d, J=12.0Hz, 4H); 3.21-3.45 (m, 9H); 4.02 (s, 4H); 7.45 (d, J=7.6Hz, 2H); 7.49-7.53 (m, 2H); 7.61-7.65 (m, 4H); 7.91 (t, J=8.0Hz, 2H); 8.26 (d, J=6.8Hz, 2H); 8.45 (s, 2H); 8.53 (d, J=8.8Hz, 2H).
13CNMR (100MHz, DMSO-d
6) mensuration result: δ=14.53,50.22,50.41,54.50,58.02,122.62,125.13,125.54,125.62,125.94,126.55,126.84,129.19,129.63,132.09,138.91,150.35,155.77,156.66,157.95,172.60.Determination of elemental analysis result: by molecular formula C
42H
40N
6O
63.5H
2O (MTDTA3.5H
2O) calculated value (%), C=64.03, H=6.01, N=10.67; Measured value (%), C=63.95, H=5.82, N=10.73.Mass spectrum (ESI-MS) is measured result: m/z=747.5 [M+Na]
+
(3) probe complexes [MTDTA-Eu] is synthetic
With the MTDTA (0.038mmol) of 30mg and the EuCl of 14.0mg
36H
2O (0.038mmol) adds in the solution of 8.0mL methyl alcohol and 3.0mL triethyl ammonia, stirs refluxed after 2 hours, the pressure reducing and steaming solvent.Add the 5.0mL ether in the resultant, centrifugal collecting precipitation and with washing with acetone three times obtains the probe complexes [MTDTA-Eu] of 19.3mg, productive rate 58.1% after the vacuum-drying.Mass spectrum (ESI-MS) is measured result: m/z=897.4 [M+Na]
+
(4) reaction of probe complexes [MTDTA-Eu] and singlet oxygen
With the MTDTA (0.025mmol) of 19.7mg and the EuCl of 9.2mg
36H
2It is in 10.5 the 0.1mol/L carbonate buffer solution that O (0.025mmol) joins 10mL pH value, stirs after 2 hours the Na of adding 12.1mg in the solution under the room temperature
2MoO
42H
2The 30%H of O (0.05mmol) and 200 microlitres
2O
2After solution at room temperature continues to stir 30 minutes, add the 30%H of 200 microlitres again
2O
2, solution continues to be stirred to fluorescence no longer strengthen till, the standardized solution that this solution is used as [MTDTA-Eu] and singlet oxygen reaction product [EP-MTDTA-Eu] is used for the photoluminescent property of [EP-MTDTA-Eu] and measures after dilution.Mass spectrum (ESI-MS) is measured result: m/z=929.2 [M+Na]
+
Embodiment 2: probe [MTDTA-Eu] and measure with the photoluminescent property of singlet oxygen reaction product [EP-MTDTA-Eu]
(1) spectrum and photoluminescent property
Use 0.05 mol/L sodium borate buffer solution of pH value 9.1 to measure [MTDTA-Eu] and [EP-MTDTA-Eu] thereof uv-vis spectra, fluorescence spectrum, molar extinction coefficient (ε), fluorescent quantum yield (φ) and fluorescence lifetime (τ) in this solvent as solvent, the gained result sees table 1.
Table 1. MTTA-Eu
3+And EP-MTTA-Eu
3+Absorption in sodium borate buffer solution and photoluminescent property
Visible by table 1 result, probe [MTDTA-Eu] and singlet oxygen reaction generate [EP-MTDTA-Eu] back fluorescence radiation quantum yield and have been increased to 13.4% from 0.88%, and fluorescence lifetime has been increased to 0.96ms from 0.31ms, all significantly strengthens.Show that probe [MTDTA-Eu] has significant fluorescent signal response to singlet oxygen, the fluorescence spectrum of two kinds of compounds is as shown in Figure 2.
(2) the pH value of solution value is to the influence of [EP-MTDTA-Eu] photoluminescent property
[EP-MTDTA-Eu] become the solution of 5.0 μ mol/L with the 0.05mol/L Tris-HCl buffer preparation of different pH values, measured its fluorescence intensity and fluorescence lifetime under different pH values, the result sees Fig. 3.It is thus clear that the pH value greater than 3 scope in, the fluorescence intensity of [EP-MTDTA-Eu] and fluorescence lifetime receive the influence of pH value little, show that this probe all can use in slightly acidic, neutrality and weakly alkaline environment.
(3) probe [MTDTA-Eu] is to the specificity of singlet oxygen fluorescence response
Owing in PDT treatment illumination process, except generating the principal product singlet oxygen, also possibly be attended by the generation of other reactive oxygen species, therefore through experiment measured respectively probe [MTDTA-Eu] to reactive oxygen species peroxo-nitrous acid with (ONOO
-), hydroxyl radical free radical (
.OH), ultra-oxygen anion free radical (O
2 .), H
2O
2And singlet oxygen (
1O
2) the fluorescence response situation, the gained result is as shown in Figure 4.It is thus clear that [MTDTA-Eu] is to ONOO
-,
.OH, O
2 .And H
2O
2Basically do not have fluorescence response, show that [MTDTA-Eu] can not react with these reactive oxygen species.And after working as [MTDTA-Eu] and singlet oxygen reaction, owing to formed the inner oxide [EP-MTDTA-Eu] of hyperfluorescence property, and then cause the fluorescence intensity of probe significantly to strengthen.This result shows that probe [MTDTA-Eu] has excellent specificity to the fluorescence response of singlet oxygen.
Embodiment 3: use probe [MTDTA-Eu] quantitatively determined Na
2MoO
4-H
2O
2The singlet oxygen that system produces
In the 0.1mol/L of pH value 10.5 yellow soda ash buffered soln, add [MTDTA-Eu] (10 μ mol/L), Na respectively
2MoO
4(1.0mmol/L) with the H of a series of concentration
2O
2, the reaction of room temperature held was measured the time resolution fluorescence spectral of each reaction solution after 4 hours, and the gained result is as shown in Figure 5.It is thus clear that along with H
2O
2The increase of concentration (being singlet oxygen concentration), the fluorescence intensity of probe also increase gradually, show that [MTDTA-Eu] can be used for the singlet oxygen that generates in the quantitatively determined solution.Adopt 96 microwell plates on Perkin-Elmer Victor 1420 time-resolved fluorometry appearance, utilization [MTDTA-Eu] to be detected Na
2MoO
4-H
2O
2The minimum detectability that produces singlet oxygen in the system is measured, and the minimum detectability that gets present method mensuration singlet oxygen is 1.3nmol/L, hangs down about 3 times than document 10 methods that we have reported, shows use [MTDTA-Eu] quantitatively determined Na
2MoO
4-H
2O
2The singlet oxygen that system produces has very high sensitivity.
Embodiment 4: use probe [MTDTA-Eu] quantitatively determined H
2O
2The singlet oxygen that-NaOCl system produces adds [MTDTA-Eu] (10 μ mol/L), H respectively in the 0.05mol/LTris-HCl of pH value 7.4 buffered soln
2O
2(50mmol/L) with the NaOCl of a series of concentration, the reaction of room temperature held was measured the time resolution fluorescence spectral of each reaction solution after 2 hours, and the gained result is as shown in Figure 6.It is thus clear that along with the increase of NaOCl concentration (being singlet oxygen concentration), the fluorescence intensity of probe also increases gradually, shows that [MTDTA-Eu] can be used for the singlet oxygen that generates in the quantitatively determined solution.Adopt 96 microwell plates on Perkin-Elmer Victor 1420 time-resolved fluorometry appearance, utilization [MTDTA-Eu] to be detected H
2O
2The minimum detectability that produces singlet oxygen in the-NaOCl system is measured, and the minimum detectability that gets present method mensuration singlet oxygen is 3.8nmol/L, shows use [MTDTA-Eu] quantitatively determined H
2O
2The singlet oxygen that-NaOCl system produces also has very high sensitivity.
Embodiment 5: probe [MTDTA-Eu] is to the fluorescence response kinetic determination of the singlet oxygen of horseradish peroxidase-indole-3-acetic acid system generation
Because horseradish peroxidase-indole-3-acetic acid system can produce singlet oxygen (document 12:M.P.De Mello, S.M.De Toledo, M.Haun apace under solutions of weak acidity; G.Cilento; N.Duran, Biochemistry1980,19; 5270), present embodiment utilizes this system to investigate the kinetics of probe [MTDTA-Eu] to the singlet oxygen fluorescence response.At first in the 0.05mol/L of pH value 4.0 acetic acid-sodium acetate buffer solution, add [MTDTA-Eu] (5 μ mol/L) and horseradish peroxidase (0.5 μ mol/L) respectively; The indole-3-acetic acid that adds different concns then respectively; Write down each solution simultaneously in the relation of 608nm fluorescence intensity variation with the reaction times, the gained result is as shown in Figure 7.It is thus clear that the increase of each solution fluorescence intensity all can reach peak in 2 minutes, show that probe [MTDTA-Eu] has fluorescence response fast to singlet oxygen, is very beneficial for the fluorometric assay of short life singlet oxygen.
Embodiment 6: the time resolved fluorescence imaging that singlet oxygen generates in the cell under the preparation of probe [MTDTA-Eu] and the common mark HeLa of hematoporphyrin monomethyl ether cell and the illumination is measured
The grade that will contain 0.5mmol/L [MTDTA-Eu] is oozed salts solution (aqueous solution that contains 140mmol/LNaCl, 10mmol/L glucose and 3.5mmol/LKCl) and is joined in the HeLa cell of cultivation, at 5%CO
237 ° of C cultivated down after 3.5 hours in the incubator, and cell oozes the salts solution thorough washing with waiting.The grade that just contains 10 μ mol/L hematoporphyrin monomethyl ethers is then oozed salts solution and is joined in the HeLa cell, at 5%CO
237 ° of C cultivated 0.5 hour down in the incubator.After cell oozes the salts solution thorough washing with grade; Mercury lamp irradiation (irradiates light wavelength 450-490 nm) with 100 watts; Differentiate fluorescent microscope with the time simultaneously the cell in the irradiation process is carried out time resolved fluorescence imaging mensuration, thereby the singlet oxygen that in cell, produces in the irradiation process is carried out real-time monitoring and mensuration.
For the fluorescence imaging that above-mentioned experiment is described is that probe is produced with singlet oxygen reaction back in the HeLa cell; Present embodiment at first to hematoporphyrin monomethyl ether and [MTDTA-Eu] respectively the HeLa cell of mark carried out that the steady-state fluorescence imaging is measured and the time differentiates fluorescence imaging and measures, the gained result is as shown in Figure 8.It is thus clear that the HeLa cell of hematoporphyrin monomethyl ether mark can send the red fluorescence that intensive comes from hematoporphyrin monomethyl ether under the steady-state fluorescence imaging pattern, then completely dissolve of red fluorescence under the time resolved fluorescence imaging pattern.The HeLa cell of [MTDTA-Eu] mark is not all having fluorescence to send under the steady-state fluorescence imaging pattern and under the time resolution fluorescence imaging pattern simultaneously.The above results shows that the singlet oxygen that can adopt the time resolved fluorescence imaging pattern that the HeLa cell of [MTDTA-Eu] and the common mark of hematoporphyrin monomethyl ether is produced carries out fluorescence imaging and measures in the illumination process.
The HeLa cell that Fig. 9 has provided [MTDTA-Eu] and the common mark of hematoporphyrin monomethyl ether is in 100 watts of mercury lamps irradiations time resolved fluorescence imaging mensuration result during different irradiation time down, and Figure 10 has provided the variation of a cell internal fixing border circular areas fluorescence intensity with irradiation time.It is thus clear that along with the increase of irradiation time, the fluorescence intensity that cell sends also obviously strengthens, show that the singlet oxygen amount that generates in the increase cell along with irradiation time also increases gradually.In addition; The distribution situation of fluorescence intensity can be found out in the cell; With the hematoporphyrin monomethyl ether is that the singlet oxygen that the photosensitization medicine produces in cell mainly is distributed in the cytoplasm; In nucleus, then seldom, explain that hematoporphyrin monomethyl ether mainly occurs in (as to mitochondrial destruction) in the cytoplasm as the effect of PDT medicine, the karyomit(e) effect in this medicine pair cell nuclear is less.
Embodiment 7: the time resolved fluorescence imaging that singlet oxygen generates in the cell under the preparation of probe [MTDTA-Eu] and 5-aminolevulinic acid mark HeLa cell and the illumination is measured
With contain simultaneously 0.5mmol/L [MTDTA-Eu] and 4.0 μ mol/L5-aminolevulinic acids etc. ooze in the HeLa cell that salts solution joins cultivation, at 5%CO
237 ° of C cultivated 3.5 hours down in the incubator.After cell oozes the salts solution thorough washing with grade; Mercury lamp irradiation (irradiates light wavelength 450-490 nm) with 100 watts; Differentiate fluorescent microscope with the time simultaneously the cell in the irradiation process is carried out time resolved fluorescence imaging mensuration, thereby the singlet oxygen that in cell, produces in the irradiation process is carried out real-time monitoring and mensuration.
The HeLa cell that Figure 11 has provided [MTDTA-Eu] and the common mark of 5-aminolevulinic acid is in 100 watts of mercury lamps irradiations time resolved fluorescence imaging mensuration result during different irradiation time down, and Figure 12 has provided the variation of a cell internal fixing elliptical region fluorescence intensity with irradiation time.Similar with the result of embodiment 6, along with the increase of irradiation time, the fluorescence intensity that cell sends also obviously strengthens, and shows that the singlet oxygen amount that generates in the increase cell along with irradiation time also increases gradually.But the distribution situation of fluorescence intensity is seen in the cell; With different as the effect of PDT medicine with hematoporphyrin monomethyl ether; Both be distributed in the cytoplasm with the 5-aminolevulinic acid as the singlet oxygen that the photosensitization medicine produces in cell; Also be distributed in the nucleus, explain the 5-aminolevulinic acid can occur in the cytoplasm simultaneously as the effect of PDT medicine and nucleus in.
Claims (4)
1. europium complex singlet oxygen fluorescence probe with permeability of cell membrane; It is characterized in that: be that the formed title complex of big ring-type ligand that replaces the ter cycloheptapyridine skeleton structure with trivalent europium ion and a kind of 9-of containing methyl anthracene is a fluorescent probe, the structural formula of said trivalent europium complex is:
2. application rights requires 1 described europium complex singlet oxygen fluorescence probe; It is characterized in that: the method through with title complex and co-culture of cells changes title complex in the cell over to; Title complex and the reaction of intracellular singlet oxygen cause its fluorescence significantly to strengthen, and then are used for the fluorometric assay of singlet oxygen in the cell.
3. application rights requires 1 described europium complex singlet oxygen fluorescence probe, it is characterized in that: cause its fluorescence significantly to strengthen through the singlet oxygen in the title complex and the aqueous solution is reacted, and then be used for the fluorometric assay of aqueous solution singlet oxygen.
4. europium complex singlet oxygen fluorescence probe according to claim 1 is characterized in that, the europium complex singlet oxygen fluorescence probe is reagent or the test kit that contains it.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101977185A CN102732246A (en) | 2012-06-15 | 2012-06-15 | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101977185A CN102732246A (en) | 2012-06-15 | 2012-06-15 | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102732246A true CN102732246A (en) | 2012-10-17 |
Family
ID=46988560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101977185A Pending CN102732246A (en) | 2012-06-15 | 2012-06-15 | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102732246A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0288256A2 (en) * | 1987-04-20 | 1988-10-26 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Fluorescent labels |
| WO2003076938A1 (en) * | 2002-03-08 | 2003-09-18 | Mitsubishi Rayon Co., Ltd. | Novel fluorescent label compounds |
| CN1493647A (en) * | 2002-11-01 | 2004-05-05 | 中国科学院大连化学物理研究所 | Functional nano-rare earth fluorescent micro particle and its preparation and application |
| CN1811430A (en) * | 2005-01-26 | 2006-08-02 | 中国科学院大连化学物理研究所 | Singlet oxygen europium coordination compound fluorescent probe and application thereof |
| EP1785724A1 (en) * | 2004-08-17 | 2007-05-16 | Japan Science and Technology Agency | Method of labeling with use of rare earth fluorescent complex and relevant method of analysis and detection |
| CN1986550A (en) * | 2005-12-23 | 2007-06-27 | 中国科学院大连化学物理研究所 | Singlet oxygen fluorescence probe based on europium complex and its application |
| WO2007128873A1 (en) * | 2006-05-05 | 2007-11-15 | Wallac Oy | A method for the preparation of maleimido derivatives of biomolecule labeling reactants and conjugates derived thereof |
| WO2008025886A1 (en) * | 2006-09-01 | 2008-03-06 | Wallac Oy | Metal chelates and chelating agents containing triazolyl subunits |
| WO2010055207A1 (en) * | 2008-11-17 | 2010-05-20 | Wallac Oy | Chelating, chelating agents and conjugates deriver thereof |
-
2012
- 2012-06-15 CN CN2012101977185A patent/CN102732246A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0288256A2 (en) * | 1987-04-20 | 1988-10-26 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Fluorescent labels |
| WO2003076938A1 (en) * | 2002-03-08 | 2003-09-18 | Mitsubishi Rayon Co., Ltd. | Novel fluorescent label compounds |
| CN1493647A (en) * | 2002-11-01 | 2004-05-05 | 中国科学院大连化学物理研究所 | Functional nano-rare earth fluorescent micro particle and its preparation and application |
| EP1785724A1 (en) * | 2004-08-17 | 2007-05-16 | Japan Science and Technology Agency | Method of labeling with use of rare earth fluorescent complex and relevant method of analysis and detection |
| CN1811430A (en) * | 2005-01-26 | 2006-08-02 | 中国科学院大连化学物理研究所 | Singlet oxygen europium coordination compound fluorescent probe and application thereof |
| CN1986550A (en) * | 2005-12-23 | 2007-06-27 | 中国科学院大连化学物理研究所 | Singlet oxygen fluorescence probe based on europium complex and its application |
| WO2007128873A1 (en) * | 2006-05-05 | 2007-11-15 | Wallac Oy | A method for the preparation of maleimido derivatives of biomolecule labeling reactants and conjugates derived thereof |
| WO2008025886A1 (en) * | 2006-09-01 | 2008-03-06 | Wallac Oy | Metal chelates and chelating agents containing triazolyl subunits |
| WO2010055207A1 (en) * | 2008-11-17 | 2010-05-20 | Wallac Oy | Chelating, chelating agents and conjugates deriver thereof |
Non-Patent Citations (5)
| Title |
|---|
| BO SONG 等: "A Europium(III) Complex as an Efficient Singlet Oxygen Luminescence Probe", 《J.AM.CHEM.SOC.》, vol. 128, no. 41, 26 September 2006 (2006-09-26), pages 13442 - 13450 * |
| CHANTAL GALAUP 等: "Direct Access to Terpyridine-Containing Polyazamacrocycles as Photosensitizing Ligands for Eu(III) Luminescence in Aqueous Media", 《J.ORG.CHEM.》, vol. 70, no. 6, 16 February 2005 (2005-02-16), pages 2274 - 2284 * |
| CHANTAL GALAUP 等: "Novel terpyridine macrocyclic complexing agent and luminescence of its neutral Ln(III) complexes (Ln=Eu, Tb, Sm, Dy) in aqueous solution", 《TETRAHEDRON LETTERS》, vol. 42, no. 36, 3 September 2001 (2001-09-03), pages 6275 - 6278 * |
| 苑嗣纯等: "联三吡啶配体组装及其金属配合物性能", 《化学进展》, vol. 21, no. 10, 24 October 2009 (2009-10-24), pages 2132 - 2152 * |
| 袁景利 等: "稀土配合物时间分辨荧光探针的设计、合成与应用", 《第四届海峡两岸分析化学学术会议论文集》, 30 November 2006 (2006-11-30), pages 18 - 19 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dai et al. | A cell-membrane-permeable europium complex as an efficient luminescent probe for singlet oxygen | |
| Majumdar et al. | Cyclometalated Ir (III) complexes with styryl-BODIPY ligands showing near IR absorption/emission: preparation, study of photophysical properties and application as photodynamic/luminescence imaging materials | |
| Guo et al. | Recognition and sensing of various species using boronic acid derivatives | |
| Liu et al. | High-efficiency dynamic sensing of biothiols in cancer cells with a fluorescent β-cyclodextrin supramolecular assembly | |
| Yan et al. | NIR organic dyes based on phenazine-cyanine for photoacoustic imaging-guided photothermal therapy | |
| Wu et al. | Design of a β-diketonate–Eu 3+ complex-based time-gated luminescence probe for visualizing mitochondrial singlet oxygen | |
| Yang et al. | Diketopyrrolopyrrole-based multifunctional ratiometric fluorescent probe and γ-glutamyltranspeptidase-triggered activatable photosensitizer for tumor therapy | |
| Maruszewski et al. | Synthesis and spectroscopic properties of zeolite-entrapped bis-heteroleptic ruthenium (II) polypyridine complexes | |
| Ding et al. | From nonconjugation to conjugation: novel meso-OH substituted dipyrromethanes as fluorescence turn-on Zn 2+ probes | |
| CN109796483A (en) | A kind of water-soluble cationic photosensitizer and its preparation and application | |
| CN114805447B (en) | Iridium complex photosensitizer and preparation method and application thereof | |
| Guria et al. | A benzothiazole-conjugated hemicyanine dye as a ratiometric NIR fluorescent probe for the detection and imaging of peroxynitrite in living cells | |
| CN110372754B (en) | Novel metal iridium complex and preparation method and application thereof | |
| CN113480551B (en) | Targeted phenoxazine porphyrin, preparation method and application thereof as triplet photosensitizer | |
| Pan et al. | Near-infrared AIE-active phosphorescent iridium (III) complex for mitochondria-targeted photodynamic therapy | |
| CN112409365B (en) | 3-sulfopropane sulfydryl modified phthalocyanine, preparation method thereof and application thereof in pharmaceutical field | |
| CN111548373B (en) | Iridium complex azapyrrolidone photo-diagnosis and treatment reagent and preparation method and application thereof | |
| CN107286197B (en) | A kind of complex of iridium and its preparation method and application with phosphorescence ion-pair structure | |
| CN113512068A (en) | Double-ligand nitrosyl ruthenium complex and preparation method and application thereof | |
| CN113278036B (en) | Phenothiazine-containing iridium complex and preparation method and application thereof | |
| CN108358972B (en) | Phenanthroline ruthenium complex photosensitive dye and preparation method and application thereof | |
| CN102732246A (en) | Europium complex singlet oxygen fluorescent probe with cell membrane permeability and its application | |
| CN114409687B (en) | Photosensitive medicine capable of switching light treatment modes in tumor and preparation method and application thereof | |
| CN110642772A (en) | Near-infrared ratiometric fluorescent probe for detecting nitroreductase as well as preparation method and application thereof | |
| CN113603698B (en) | Phthalocyanine-perphenazine conjugate with type I photosensitive reaction and photothermal synergistic effect and application in pharmaceutical field |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121017 |