CN1811430A - Singlet oxygen europium coordination compound fluorescent probe and application thereof - Google Patents

Abstract

The present invention relates to a novel singlet oxygen europium fluorescent probe and its application. It uses trivalent europium ion Eu ³⁺ to match with a functional group-containing 2,2':6'2"-biterpyridine skeleton structure. A complex formed by a ligand, wherein the ligand structural formula is as above, R=H, C _n H _2n+1 , C ₆ H ₅ . This type of complex can interact with singlet oxygen through its functional group to make The fluorescence intensity of the complex changes greatly, and then it is used for the fluorescence determination of singlet oxygen.

Description

A kind of fluorescent probe of singlet oxygen europium complex and its application

technical field

The present invention relates to the measurement technology of singlet oxygen ( ¹ O ₂ ) in solution, specifically a novel ¹ O ₂ europium complex fluorescent probe (that is, a novel singlet oxygen fluorescent probe based on europium complex) and its application.

Background technique

Singlet oxygen ( ¹ O ₂ ) is an excited state of oxygen molecule. It is not only a very useful oxidant in organic synthesis, but also has very important physiological activities. In organic synthesis, ¹ O ₂ makes it extremely easy to introduce oxygen into highly stereospecific organic compounds. ¹ O ₂ has the following important reactions: First, ¹ O ₂ can react with monoolefins to generate dioxanes or hydrocarbon peroxides. The former usually decomposes into corresponding carbonyl compounds and emits light at room temperature, while the latter generates unsaturated ketones, allyl alcohol or hydroxyl compounds under corresponding conditions. Secondly, ¹ O ₂ can react with active double bonds with electron-donating heteroatoms such as N, O, or S to generate quaternary 1,2-dioxo rings, followed by splitting into compounds with dialdehyde structures. At the same time, ¹ O ₂ can undergo Diels-Alder addition reaction with chain, ring, aromatic and heterocyclic aromatic conjugated π bonds to generate internal oxides. In addition, ¹ O ₂ can also have complex chemical and physical interactions with carotenoids, amines, phenols and heterocyclic compounds, making ¹ O ₂ highly reactive in biological systems . In the living system, ¹ O ₂ is a common active oxygen in organisms. It is active and electrophilic, and can react with various biomolecules such as DNA, protein and lipids, and plays a very important physiological role. effect. ¹ O ₂ is produced as a reaction precursor in almost all biological photooxidative systems, and is the main substance responsible for photosensitization damage of biological cells. It can destroy the cell membrane of red blood cells in the blood and cause hemolysis; it can also interact with amino acids to inhibit the activity of hydroxyl dehydrogenase and insulin. In the immune system, ¹ O ₂ is the main bactericide for leukocytes to phagocytose and dissolve invading organisms.

Because ¹ O ₂ plays such an important role, the detection of ¹ O ₂ , especially ¹ O ₂ in biological systems, has attracted more and more attention. From the 1970s to the present, there are mainly the following methods for the detection of ¹ O ₂ : (1) Use the phosphorescence generated by ¹ O ₂ self-quenching ¹ Δ _g → ³ ∑ _g - to detect ¹ O ₂ (Ref. 1: AA, Jr. Krasnovsky, Biofzika, 1976, 21, 748). This method has high specificity, but its sensitivity is low and the detection signal is weak, so it cannot be used for the detection of low concentration ¹ O ₂ . At present, with the improvement of the performance of detection instruments, this method has been used in the research of the production, physiological effects and spatial distribution of ¹ O ₂ in some biological systems (document 2: C.Kiryu, M.Makiuchi, J.Miyazaki, T.Fujinaga, K.Kakinuma, FEBS Lett.1999, 443, 154; Literature 3: S.Oelckers, T.Ziegler, I.Michler, BRder, J.Photochem.Photobiol.B: Biol.1999, 53, 121; Literature 4: LKAndersen, PROgilby, Photochem.Photobiol.2001, 73, 489; Literature 5: LKAndersen, Z.Gao, PROgilby, L.Poulsen, I.Zebger, J.Phys.Chem.A 2002, 106, 8488 ). (2) Using ¹ O ₂ to specifically react with the fluorescein probe molecule with anthracycline, the probe changes from the original non-fluorescent molecule to a strong fluorescent molecule, so that it can be used for the detection of ¹ O ₂ ( Literature 6: N.Umezawa, K.Tanaka, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Angew.Chem.Int.Ed.Engl.1999, 38, 2899; Literature 7: K.Tanaka, T. Miura, N. Umezawa, Y. Urano, K. Kikuchi, T. Higuchi, T. Nagano, J. Am. Chem. Soc. 2001, 123, 2530). This method has short detection time and high sensitivity, but it is not suitable for low pH environment and real-time detection. (3) Utilize the characteristic reaction of 9,10-diphenylanthracene (DPA) and ¹ O ₂ to generate stable internal oxides, and measure ¹ O ₂ by measuring the change of DPA absorption spectrum (Document 8: MJStenbeck, AUKhan, MJ Kamovsy, J. Biol. Chem. 1992, 267, 13425; Document 9: MJ Stenbeck, AU Khan, MJ Kamovsy, J. Biol. Chem. 1993, 268, 15649). This method has been used to measure ¹ O ₂ produced in phagocytes, but the sensitivity is low because the detection is based on absorption spectra. (4) Using the energy transfer between ¹ O ₂ and the probe molecule, the probe molecule is excited to emit strong delayed fluorescence, which is then used for the detection of ¹ O ₂ (document 10: AAJr.Krasnovsky, C.Schweitzer, H.Lesmann , C. Tanielian, EALuk'yanets, Quantum Electron. 2000, 30, 445; Literature 11: AAJr. Krasnovsky, ME Bashtanov, NNDrozdova, OA Yuzhakova, EALuk'yanets, Quantum Electron. 2002, 32, 83). This method is applicable to a variety of systems, and the quantum yield of luminescence is 3-5 times that of ¹ O ₂ phosphorescence. However, the probe used in the detection is also sensitized to the generation of ¹ O ₂ , which leads to deviations in the measurement results. (5) A chemiluminescent method for detecting ¹ O ₂ based on photoinduced electron transfer process (Document 12: XHLi, GXZhang, HMMa, DQZhang, J.Li, DBZhu, J.Am.Chem.Soc.2004, 126, 11543 ). This method has good selectivity, high sensitivity, and rapid detection, but the probe has poor water solubility, which is not conducive to the determination of ¹ O ₂ in biological systems.

In recent years, time-resolved fluorescence assays using rare earth fluorescent complexes as probes have been widely used in various assays such as immunoassay, DNA hybridization analysis, and fluorescence microbiological imaging analysis. Rare earth fluorescent complexes have the characteristics of long fluorescence lifetime, large Stokes shift, and sharp fluorescence peak. By using time-resolved fluorescence measurement method, the influence of various scattered light and sample background fluorescence on fluorescence measurement can be effectively removed, thereby greatly improving the measurement efficiency. sensitivity.

Contents of the invention

The purpose of the present invention is to apply the time-resolved fluorescence measurement method to the detection of ¹ O ₂ , and provide a novel ¹ O ₂ europium complex fluorescent probe with high sensitivity, good selectivity, good water solubility and wide application range.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A fluorescent probe formed by a rare earth ion and a ligand containing an anthracycline-substituted 2,2':6',2"-biteropyridine skeleton structure, the ligand structural formula is:

Wherein R is hydrogen (H), alkyl (CnH _2n+1 ) or phenyl (C ₆ H ₅ ); wherein n=1-6 is preferred.

The fluorescent probe of the present invention can be used for the determination of ¹ O ₂ in various environments. In various biological and non-biological environments, ^{the 1} O ₂ produced in the probe capture system is used to make the fluorescence intensity of the probe extremely high Then, the concentration of ¹ O ₂ (production and production amount) was determined by measuring the change of fluorescence intensity before and after the probe interacted with ¹ O ₂ .

The fluorescence assay mentioned therein includes time-resolved fluorescence assay and time-resolved fluorescence microscope assay in addition to conventional fluorescence assay.

The europium complex fluorescent probe of the present invention can be prepared as a ¹ O ₂ detection reagent and kit for the singlet oxygen europium complex fluorescent probe.

The fluorescent probe of the present invention has the following advantages:

1. The novel ¹ O ₂ fluorescent probe of the present invention has good water solubility and is suitable for the determination of ¹ O ₂ in biological systems.

2. The novel ¹ O ₂ fluorescent probe of the present invention has high stability, can be stored and used for a long time, and is suitable for various environments such as weak acidity, neutrality and alkalinity.

3. The novel ¹ O ₂ fluorescent probe of the present invention has extremely high sensitivity, and its lowest detection limit is 28 times lower than that of the reported chemiluminescence method.

4. The novel ¹ O ₂ fluorescent probe of the present invention has good selectivity to ¹ O ₂ , and the fluorescent signal hardly changes when interacting with other reactive oxygen species.

Description of drawings

Fig. 1 is the structural formula of europium complex ¹ O ₂ fluorescent probes;

Fig. 2 is the synthetic route of ligand ATTA;

Figure 3 is a synthetic route for ligands containing anthracycline-substituted 2,2':6',2"-biterpyridine skeleton structure;

Fig. 4 is the fluorescence spectrum of ATTA-Eu ³⁺ (solid line, 1.0 μ mol/L) and EP-ATTA-Eu ³⁺ (dotted line, 1.0 μ mol/L) in the 0.05mol/L boric acid buffer solution of pH value 9.1;

Figure 5 is the fluorescence intensity (●) and fluorescence lifetime (○) of EP-ATTA-Eu ³⁺ (1.0 μmol/L) in 0.05mol/L Tris-HCl buffer solution of different pH values;

Fig. 6 is the photostability experiment result of EP-ATTA-Eu ³⁺ (■) and fluorescein (□) in the 0.05mol/L boric acid buffer solution of pH value 9.1;

Fig. 7 is a working curve for detecting quantitatively produced ¹ O ₂ in the Na ₂ MoO ₄ /H ₂ O ₂ system using ATTA-Eu ³⁺ .

Detailed ways

Below by embodiment the present invention will be further described.

Example 1. Synthesis of ligand [4'-(9-anthracenyl)-2,2':6'2"-biteropyridine-6,6"-dimethylamine]tetraacetic acid (ATTA for short).

The synthetic route is shown in Figure 2, and the matrix operation process is as follows.

(1) Synthesis of (E)-3-(9-anthracenyl)-1-(2'-pyridyl)-2-propenone (compound 1)

10.31 grams of 9-anthral (50mmol) and 2.81 grams of KOH (50mmol) were mixed and dissolved in a mixed solution composed of 200ml of methanol and 40ml of water; after stirring for 30 minutes, 6.06 grams of 2-acetylpyridine (50mmol) was slowly added dropwise to The reaction system was stirred at room temperature for 24 hours; the precipitate was collected by filtration, and the crude product was recrystallized from ethanol; 12.77 g of the target compound was obtained, with a yield of 82.6%. ¹ H NMR (CDCl ₃ ) measurement results: δ=8.48-8.54 (m, 5H); 7.92 (m, 1H); 8.03 (d, J=8.4Hz, 2H); 8.27-8.31 (m, 2H); 8.37 (d, J = 8.0 Hz, 2H); 8.48 (s, 1H); 8.70 (d, J = 4.4 Hz, 1H); 8.93 (d, J = 16.0 Hz, 1H).

(2) Synthesis of 4'-(9-anthracenyl)-2,2':6',2"-biterpyridine (compound 2)

In 500ml of dry methanol, add 15.47 grams of compound 1, 16.46 grams of [2-(2'-pyridyl)-2-oxoethyl]pyridinium iodide (50mmol) and 23.12 grams of dry ammonium acetate, and the reaction solution was refluxed and stirred for 24 hour; the solvent was evaporated under reduced pressure, and the residue was extracted with 400ml chloroform; after the chloroform was evaporated, the crude product was separated by silica gel column chromatography, and the eluent was collected with 2:1 petroleum ether-ethyl acetate. Components: The product was washed with acetonitrile and dried in vacuo to obtain 5.48 g of the target compound, yield: 26.8%. ¹ H NMR (CDCl ₃ ) measurement results: δ=7.26-7.37(m, 4H); 7.47(t, J=7.6Hz, 2H); 7.71(d, J=8.8Hz, 2H); 7.92(m, 2H ); 8.07(d, J=8.8Hz, 2H); 8.55(s, 1H); 8.61(s, 2H); 8.63(d, J=4.0Hz, 2H); 8.79(d, J=8.0Hz, 2H ). Elemental analysis results, calculated values: C 85.06%, H 4.68%, N 10.26%; measured values: C 84.66%, H 4.63%, N 9.90%.

(3) Synthesis of 6,6"-dicyano-4'-(9-anthracenyl)-2,2':6',2"-biterpyridine (compound 3)

3.3 grams of compound ₂ (8.0mmol) and 6.9 grams of 80% m-chloroperoxybenzoic acid (32.0mmol) were dissolved in 160ml of dichloromethane and stirred at room temperature for 24 hours _; The solution was washed twice, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and dried under vacuum; the product obtained above and 6.34 g of Me ₃ SiCN (64.0 mmol) were dissolved in 200 ml of CH ₂ Cl ₂ and stirred at room temperature for 1 hour Finally, slowly add 5.62 g of benzoyl chloride (40.0 mmol) dropwise, and stir at room temperature for 24 hours; evaporate the solvent under reduced pressure, add 200ml of 10% K ₂ CO ₃ solution, and stir for 1 hour at room temperature; Washing with acetonitrile; 2.34 g of the target compound was obtained, yield: 63.6%. ¹ H NMR (CDCl ₃ ) measurement results: δ=7.40(t, J=7.2Hz, 2H); 7.50(t, J=8.0Hz, 2H); 7.63(d, J=8.8Hz, 2H); 7.75( d, J = 7.2 Hz, 2H); 8.05-8.12 (m, 4H); 8.61 (s, 1H); 8.72 (s, 2H); 8.98 (d, J = 8.0 Hz, 2H).

(4) Synthesis of 4'-(9-anthracenyl)-2,2':6',2"-biterpyridine-6,6"-dicarboxylic acid dimethyl ester (compound 4)

Dissolve 1.89 g of compound 3 (4.1 mmol) in a mixed solution of 12 ml of concentrated sulfuric acid, 24 ml of glacial acetic acid, and 6 ml of water, and stir for 12 hours at 85-90° C.; the reaction solution is poured into 500 ml of ice water, and the precipitate is collected by filtration and vacuum dry;

Slowly add 4.11 grams of SOCl ₂ (34.6 mmol) into 125 ml of dry methanol under ice bath, stir for 20 minutes, add the above-mentioned hydrolyzate, stir and reflux for 24 hours; evaporate the solvent under reduced pressure, add 100 ml of 10% Na ₂ In CO ₃ solution, the precipitate was collected by filtration and dried in vacuo; the crude product was separated by silica gel column chromatography, eluted with 95:5 chloroform-methanol, and the first fraction was collected; 1.56 g of the target compound was obtained, yield: 72.4% . ¹ H NMR (CDCl ₃ ) measurement results: δ=3.90(s, 6H); 7.36(t, J=8.8Hz, 2H); 7.50(t, J=7.2Hz, 2H); 7.66(d, J=8.8 Hz, 2H); 8.06-8.12(m, 4H); 8.19(d, J=8.0Hz, 2H); 8.61(s, 1H); 8.73(s, 2H); 8.99(d, J=7.2Hz, 2H ).

(5) Synthesis of 6,6"-dimethylol 4'-(9-anthracenyl)-2,2':6',2"-biterpyridine (compound 5)

Dissolve 1.52 g of compound 4 (2.9 mmol) in 48 ml of absolute ethanol, and stir to form a suspension; add 0.44 g of NaBH ₄ (11.6 mmol) and stir at room temperature for 2 hours, then reflux and stir for 8 hours; evaporate the solvent under reduced pressure, Add 8ml of saturated NaHCO ₃ solution and heat to boiling; add 60ml of water after cooling, collect the precipitate by filtration, wash the crude product with water and acetonitrile, and dry in vacuo; 1.02g of the target compound is obtained, yield: 74.9%. ¹ H NMR (CDCl ₃ ) measurement results: δ=4.74(s, 4H); 7.25(d, J=7.8Hz, 2H); 7.36(t, J=7.8Hz, 2H); 7.48(t, J=7.8 Hz, 2H); 7.67(d, J=8.0Hz, 2H); 7.90(t, J=7.8Hz, 2H); 8.09(d, J=8.8Hz, 2H); 8.58(s, 1H); 8.60( s, 2H); 8.68 (d, J = 8.0 Hz, 2H).

(6) Synthesis of 6,6"-dibromomethyl-4'-(9-anthracenyl)-2,2':6',2"-biterpyridine (compound 6)

Add 1.21 grams of PBr ₃ (4.5 mmol) to 40 ml of anhydrous DMF, stir at room temperature for 15 minutes, add 0.84 grams of compound 5 (1.8 mmol), and continue stirring for 24 hours; after the reaction, add saturated NaHCO ₃ solution for neutralization, and collect by filtration Precipitate, the crude product was washed with water and acetonitrile, and dried in vacuo; 0.83 g of the target compound was obtained, yield: 77.9%. ¹ H NMR (CDCl ₃ ) measurement results: δ=4.52(s, 4H); 7.38(t, J=7.8Hz, 2H); 7.49(m, 4H); 7.70(d, J=7.8Hz, 2H); 7.92(t, J=7.8Hz, 2H); 8.10(d, J=8.0Hz, 2H); 8.59(s, 1H); 8.65(s, 2H); 8.70(d, J=8.0Hz, 2H) .

(7) Synthesis of 4'-(9-anthracenyl)-2,2':6',2"-tripyr-6,6"-dimethylaminetetraacetic acid ethyl ester (compound 7)

Add 0.60 g of compound 6 (1 mmol), 0.42 g of ethyl diacetate amine (2.2 mmol) and 1.38 g of anhydrous K ₂ CO ₃ (10 mmol) into a mixed solution prepared by 70 ml of dry acetonitrile and 21 ml of dry tetrahydrofuran, Stir and reflux for 24 hours; remove insoluble matter by filtration, and evaporate the solvent under reduced pressure; dissolve the resultant in 100ml of chloroform, and wash with the same volume of saturated NaHCO ₃ and water. The organic phase was dried with anhydrous sodium sulfate, evaporated to remove the solvent under reduced pressure, and then separated by silica gel column chromatography. The eluent was 2:1:0.3 petroleum ether-ethyl acetate-triethylamine, and the first component was collected; After distilling off the solvent under reduced pressure, the product was washed with a small amount of petroleum ether and dried in vacuo; 0.55 g of the target compound was obtained with a yield of 68.0%. ¹ H NMR (CDCl ₃ ) measurement results: δ=1.07(t, J=7.2Hz, 12H); 3.58(s, 8H); 3.97-4.02(m, 12H); 7.37(t, J=8.0Hz, 2H ); 7.48(t, J=7.2Hz, 2H); 7.64(d, J=8.0Hz, 2H); 7.72(d, J=7.2Hz, 2H); 7.90(t, J=8.0Hz, 2H); 7.72 (d, J=8.0 Hz, 2H); 8.57 (s, 3H); 7.72 (d, J=8.0 Hz, 2H).

(8) Synthesis of [4'-(9-anthracenyl)-2,2':6',2"-tripyridine-6,6"-dimethylamine]tetraacetic acid (ATTA)

Dissolve 1.39 g of compound 7 (1.7 mmol) in a mixed solution of 60 ml of ethanol and 10 ml of water, then add 2.02 g of KOH (36.0 mmol), and stir at reflux for 2 hours; after decompression to remove the solvent, the product is dissolved in 30 ml of water , adjusted the pH value to about 3 with 1:1 hydrochloric acid, and stirred at room temperature for 3 hours; the precipitate was collected by filtration, washed with water and acetonitrile; 0.81 g of the target compound was obtained, and the yield was 65.6%. ¹ H NMR (DMSO-d ₆ ) measurement results: δ=3.45(s, 8H); 3.96(s, 4H); 7.47(t, J=8.0Hz, 2H); 7.56-7.63(m, 4H); 7.80 (d, J=8.0Hz, 2H); 8.08(t, J=8.0Hz, 2H); 8.23(t, J=8.0Hz, 2H); 8.43(s, 2H); 8.69(d, J=8.0Hz , 2H); 8.82(s, 1H). Elemental analysis results, calculated by C ₃₉ H ₃₃ N ₅ O ₈ ·1.5H ₂ O: C 64.64%, H 4.99%, N 9.64%; measured values: C 64.52%, H 5.38%, N 9.30%. ESI-MS: m/z (%): 698 (100) M ^- -H].

Example 2. 10-methyl-9-anthracenyl and 10-phenyl-9-anthracenyl substituted [2,2':6',2"-biterpyridine-6,6"-dimethylamine] tetra Synthesis of acetic acid ligand

The synthetic route is shown in Figure 3, and the synthetic operation method is the same as in Example 1.

Example 3. The reaction of the complex of ligand ATTA and Eu ³⁺ (abbreviated as ATTA-Eu ³⁺ ) with ¹ O ₂

Dissolve 36mg ATTA (0.05mmol) and 18mg EuCl ₃ ·6H ₂ O (0.05mmol) in NaHCO ₃ -NaOH buffer solution with a pH value of 10.5, stir at room temperature for 2 hours, then add 1.2gNa ₂ MoO ₄ ·2H ₂ O (5mmol) and 500μl 30% H ₂ O ₂ , and stirred for another 30 minutes; observe the change in the fluorescence intensity of the reaction solution, and continue to add H ₂ O ₂ until the fluorescence intensity of the reaction solution does not change; use HCl to adjust the pH value of the reaction solution to 3 or so, the precipitate was collected by filtration, washed with water and dried in vacuum; the internal oxide formed by the reaction of ATTA-Eu ³⁺ and ¹ O ₂ (referred to as EP-ATTA-Eu ³⁺ ) was determined by ESI-MS: ESI-MS: m/z (%): 882(10) [M ^- -H].

Example 4. Determination of the fluorescence properties of the probe ATTA-Eu ³⁺ and the internal oxide EP-ATTA-Eu ³⁺ formed by combining with ¹ O ₂

(1) Fluorescence spectrum, fluorescence intensity and fluorescence lifetime

Using a 0.05mol/L sodium borate buffer solution with a pH value of 9.1 as a solvent, the UV-visible spectrum, fluorescence spectrum, molar extinction coefficient (ε), fluorescence quantum yield (φ) and fluorescence of ATTA-Eu ³⁺ in this solvent were measured. Lifetime (τ). EP-ATTA-Eu ³⁺ is prepared by ATTA-Eu ³⁺ and Na ₂ MoO ₄ /H ₂ O ₂ in 0.1mol/L NaHCO ₃ -NaOH buffer solution with pH value 10.5, and then with 0.05mol/L pH value 9.1 After the sodium borate buffer solution was diluted 10 times, its fluorescence properties in the solvent were measured. The instrument used for the determination of ultraviolet-visible spectrum is Tianmei UV7500 spectrophotometer. The fluorescence measurement instrument is a Perkin Elmer LS 50B fluorescence spectrophotometer; the fluorescence quantum yield measurement uses 4'-phenyl-2,2':6',2"-bitripyr-6,6"-dimethylaminetetraacetic acid The europium complex was measured as a standard (document 13: M.Latva, H.Takalo, VMMukkala, C.Matachescu, JCRodriguez-Ubis, J.Kankare, J.Lumin., 1997, 75, 149-169), calculated The formula is φ ₁ ＝I ₁ ε ₂ C ₂ φ ₂ /I ₂ ε ₁ C ₁ , where I ₂ , ε ₂ , C ₂ , and φ ₂ are the fluorescence intensity, molar extinction coefficient, concentration and fluorescence quantum yield of the standard. I ₁ , ε ₁ , C ₁ , and φ ₁ are the fluorescence intensity, molar extinction coefficient, concentration and fluorescence quantum yield of the analyte.

The measurement results are shown in Table 1.

Table 1. Absorption and fluorescence properties of ATTA-Eu ³⁺ and EP-ATTA-Eu ³⁺ in sodium borate buffer solution

compound Maximum absorption wavelength (nm) Molar extinction coefficient, 335nm(mol ^-1 Lcm ^-1 ) Maximum fluorescence emission wavelength (nm) Fluorescence quantum yield (%) Fluorescence lifetime (ms) ATTA-Eu ³⁺ 294,335 17200 615 0.58 0.989 EP-ATTA-Eu ³⁺ 294,335 14500 615 10.0 1.209

It can be seen from Table 1 that the fluorescence intensity of the probe ATTA-Eu ³⁺ has changed greatly before and after the reaction with ¹ O ₂ , the fluorescence of ATTA-Eu ³⁺ is very weak, while the fluorescence of EP-ATTA-Eu ³⁺ is very strong . The fluorescence spectra of the two compounds are shown in Fig. 4.

(2) Effect of solution pH on the fluorescence properties of EP-ATTA-Eu ³⁺

Dissolve EP-ATTA-Eu ³⁺ with 0.05mol/L Tris-HCl buffer solution with different pH values, and measure its fluorescence intensity and fluorescence lifetime at different pH values. The results are shown in Figure 5; it can be seen from the figure that at pH In the range where the value is greater than 3, the fluorescence intensity and fluorescence lifetime of EP-ATTA-Eu ³⁺ are not greatly affected by the pH value, indicating that the probe can be used in weakly acidic, neutral and weakly alkaline environments.

(3) Photostability of EP-ATTA-Eu ³⁺

The aqueous solutions of fluorescein and EP-ATTA-Eu ³⁺ were irradiated under a 30W deuterium lamp for 65 minutes, and the fluorescence intensity was recorded every 5 minutes. The results are shown in Figure 6; it can be seen from Figure 6 that EP-ATTA- Eu ³⁺ has better photostability than fluorescein probes, and is more suitable for fluorescence microscopy imaging of ¹ O ₂ in biological systems.

Example 5. Quantitative determination of ¹ O ₂ produced by Na ₂ MoO ₄ /H ₂ O ₂ system using ATTA-Eu ³⁺

Add ATTA-Eu ³⁺ (100nmol/L), Na ₂ MoO ₄ (10mmol/L) and a series of concentrations of H ₂ O ₂ to a 0.1mol/L NaHCO ₃ -NaOH buffer solution with a pH value of 10.5 (the system 1 molecule of ¹ O ₂ is generated for every 2 molecules of H ₂ O ₂ ); after standing at 37° C. for 18 hours, the reaction solution was subjected to time-resolved fluorescence measurement. The measuring instrument is a Wallac Victor 1420 multi-label counter (produced by Perkin Elmer Life Sciences), and the measuring conditions are: excitation wavelength, 340nm; detection wavelength, 615nm; delay time, 0.2ms; window time, 0.4ms; cycle time, 1.0 ms.

As shown in Figure 7, the amount of ¹ O ₂ has a good linear relationship with the fluorescence intensity, indicating that ¹ O ₂ can be quantitatively detected by the probe ATTA-Eu ³⁺ . Using 3 times the standard deviation of the background signal to calculate the minimum detection limit, the minimum detection limit of ATTA-Eu ³⁺ for ¹ O ₂ is 2.8nmol/L, which is 28 times lower than the reported chemiluminescence method.

Example 6. Selective detection of ATTA-Eu ³⁺ to ¹ O ₂

Add 10 μmol/L H ₂ O ₂ , 10 μmol/L H ₂ O ₂ and 10 μmol/L ferrous ammonium sulfate (OH) to 0.1 mol/L NaHCO ₃ buffer solution at pH 8.4 containing 100 nmol/LATTA-Eu ³⁺ and 10μmol/L KO ₂ (O ₂ ^- ), and then observe its fluorescence change; after the probe ATTA-Eu ³⁺ reacted with the above active oxygen components, the fluorescence intensity increased by 12%, 76% and 6%, which was far less than ¹ O ₂ (1246% increase), indicating that ATTA-Eu ³⁺ has a very high selectivity for ¹ O ₂ .

Claims (5)

1. a singlet oxygen europium coordination compound fluorescent probe is characterized in that: be with trivalent europium ion Eu ³⁺With contain functional groups 2,2 ': 6 ' 2 "-complex that ter cycloheptapyridine skeleton structure class ligand forms, wherein said ligand structure formula is:

R＝H，C _nH _2n+1，C ₆H ₅

2. according to the described singlet oxygen europium coordination compound fluorescent probe of claim 1, it is characterized in that: n=1～6 wherein.

3. the application of the described singlet oxygen europium coordination compound fluorescent probe 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 change greatly, then by fluorometry measure probe with ¹O ₂The variable quantity of fluorescence intensity is measured before and after the effect ¹O ₂Concentration, determine ¹O ₂Generation and growing amount.

Europium coordination compound fluorescent probe of the present invention can

4. according to the application of the described singlet oxygen europium coordination compound 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 singlet oxygen europium coordination compound fluorescent probe of claim 1, it is characterized in that: be used to be prepared into and contain claim 1 described singlet oxygen europium coordination compound fluorescent probe ¹O ₂Detectable and kit.

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