Europium complex-based nitric oxide fluorescent probe and application thereof

Abstract

The invention relates to a novel europium complex fluorescent probe capable of being used for fluorescent measurement of the specificity of nitric oxide and application thereof. The probe is a complex (called DATTA-Eu<3+> for short) which is formed from trivalent europium ions and an organic ligand 4'-[4-(3,4-diamidophenoxy)phenyl]-2,2':6',2''-terpyridine-6,6''-dimethylamine tetraacetic acid, wherein the organic ligand has a structural formula shown in the specification; and the probe can specifically react with the nitric oxide in oxygen-containing aqueous solution with a physiological pH value, so that the fluorescence intensity of the probe is increased by over 47 times; and thus the probe realizes selective fluorescent measurement of the nitric oxide in the system.

Description

technical field The invention relates to the technical field of fluorescence measurement of nitric oxide, in particular to a europium complex fluorescent probe which can be used for the fluorescence measurement of nitric oxide in aqueous solution and biological system and its application. Background technique In biological systems, nitric oxide (NO) can be automatically synthesized by organisms and quickly react with other free radical molecules or metalloproteins in animals, plants, fungi, and bacteria, thus playing important physiological and pathological roles. In organisms, low-concentration NO plays an important role in blood circulation, immunity and nervous system as a signal transduction molecule between cells and cells (document 1: R.M.J.Palmer, A.G.Ferrige, S.Moncada, Nature1987, 327, 524; Literature 2: S.H.Snyder, Science1992, 257, 494; Literature 3: V.Holan, M.Krulova, A.Zajicova, J.Pindjakova, Mol.Immunol.2002, 38, 989; Literature 4: S.Jasid, M.Simontacchi, C.G...

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Problems solved by technology

(1) Using NO as a free radical characteristic magnetic resonance spectroscopy (document 6: T.Yoshimura, H.Yokoyama, S.Fujii, F.Takayama, K.Oikawa, H.Kamada, Nat.Biotechnol.1996, 14,992; Literature 7: Y.Katayama, N.Soh, M.Maeda, Chem.Phys.Chem.2001, 2, 655), however, because NO has a very short half-life in the liquid phase, it is easily oxidized into other Nitrogen oxides, making the method slightly insufficient in sensitivity, specificity, stability and application range

(2) Spectrophotometry, using NO to be easily oxidized into NO 2 - , and then react with Griess reagent under acidic conditions to generate the method of diazo compound (document 8: L.C.Green, D.A.Wagner, J.Glogowski, P.L.Skipper, J.S.Wishnok, S.R.Tannenbaum, Anal.Biochem.1982, 126, 131), This method is simple to operate, but the accuracy and sensitivity are low

(3) Electrochemical method, utilize NO to be easily oxidized and electrochemical reaction takes place, thus the method (document 9: T.Malinski, Z.Taha, Nature1992,358,676; document 10: F.Bedioui that NO concentration is measured , N.Villeneuve, Electroanal.2003, 15, 15), this method has high sensitivity, good electrode stability, and long service life, but it is difficult to make an electrode that can be used for intracellular NO measurement, and inserting this electrode into the cell will cause damage to the cell. cause greater damage

(4) Chemiluminescence method, which uses oxidant to oxidize NO into nitrogen dioxide and then utilizes the method of releasing energy and emitting light when the excited state nitrogen dioxide returns to the ground state (document 11: J.F.Brien, B.E.McLaughlin, K.Nakatsu, G.S.Marks, Methods Enzymol.1996,268,83), this method has high sensitivity, but the operation is complicated, and the oxidants used in the operation process such as ozone, hydrogen peroxide, etc. have great damage to the cells, so it is difficult to be used for the determination of NO in the cell system

However, the fluorophores used in the existing NO fluorescent probes are all organic fluorescent molecules. These organic fluorophores have poor photostability, are easy to be photobleached, and have small Stokes shifts. Disadvantages such as background fluorescence interference of coexisting impurities (Literature 15: Z. Zhang, S. Achilefu, Org. Lett. 2004, 6, 2067)

In addition, although these fluorescent probe molecules can pass through the cell membrane and react with NO in the cell to generate fluorescent signals, the reaction products of these probe molecules and NO can easily overflow from the cell (Document 16: L.E.McQuade, S.J.Lippard, Curr.Opin.Chem.Biol.2009, 14, 1), resulting in errors in fluorescence measurement

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