Fluorescent probe for detecting generation of nitric oxide and use thereof

A fluorescent probe, nitric oxide technology, applied in the field of biological detection and clinical medical detection, can solve the problems of inability to realize real-time three-dimensional detection of NO, lack of specificity of NO detection, slow progress of NO detection, etc., and achieve enhanced fluorescence intensity. , good selectivity, good stability

Inactive Publication Date: 2008-11-12
TIANJIN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since 1995, laboratories around the world have devoted themselves to the development of bioluminescence detection reagents for NO, such as diaminonaphthalene and its derivatives (Diaminonaphthalene, DAN), dichlorofluorescin (DCFH), ferrous iron and cobalt ions. Complexes, diaminofluoresceins (DAFs) and diaminorhodamines (DARs), etc., but there are various defects in the current fluorescent detection reagents that hinder its further application, such as lack of specificity for NO detection (DAN and DCFH), low detection sensitivity (complexes

Method used

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  • Fluorescent probe for detecting generation of nitric oxide and use thereof
  • Fluorescent probe for detecting generation of nitric oxide and use thereof
  • Fluorescent probe for detecting generation of nitric oxide and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the common synthetic route of compound 3a-Cu, 3b-Cu, 3c-Cu, 3d-Cu, 3h-Cu

[0051] Dissolve 300mmol of substituted salicylaldehyde (1a~1d) in 125ml of methanol under stirring, then add 150mmol of substituted o-phenylenediamine (2a or 2b), stir at room temperature for 1 hour, and generate the corresponding precipitate, which is collected by filtration The precipitate was washed with methanol and dried in vacuo to obtain ligands Aa~Ah.

[0052] Dissolve 13mmol of ligands Aa~Ah in 100ml of chloroform, and then add 2.5g of Mn(II)Cl 2 4H 2 O was dissolved in the prepared solution of 40ml ethanol to generate a brown solution, and then this solution was stirred for 12 hours under the situation of feeding air to obtain a brown solution, stirred at room temperature for 24 hours and concentrated to generate a brown precipitate, which was filtered out. Wash with DMF, then with a small amount of ethanol, and dry in vacuum to obtain the corresponding trivalent mangane...

Embodiment 2

[0067] Embodiment 2: the common synthetic route of compound 4a, 4b, 4c, 4d, 4e, 4f and its copper complex:

[0068] Dissolve 300 mmol of substituted salicylaldehyde (1a-1f) in 125 ml of methanol under stirring, then add 150 mmol of o-naphthalene diamine (2), and stir at room temperature for 1 hour to generate a corresponding precipitate, which is collected by filtration. Washed with methanol and dried in vacuum to obtain the ligands Da~Df.

[0069] Dissolve 13mmol of ligands Da~Df in 100ml of chloroform, and then add 2.5g of Mn(II)Cl 2 4H 2 O was dissolved in the prepared solution of 40ml ethanol to generate a brown solution, and then this solution was stirred for 12 hours under the situation of feeding air to obtain a brown solution, stirred at room temperature for 24 hours and concentrated to generate a brown precipitate, which was filtered out. Wash with DMF, wash with a small amount of ethanol, and dry in vacuum to obtain trivalent manganese complexes Ea-Ef.

[0070] Di...

Embodiment 3

[0075] Embodiment 3: the selectivity of complex to NO

[0076] Take 3a-Cu, 3d-Cu, 3h-Cu, 4b-Cu and dissolve them in DMSO to a final concentration of 1 mmol / L. Add various copper complexes to the 96-well plate, respectively add nitrite, ammonium, nitrate, hydrogen peroxide, ONOO - , NO, and then add deionized water to all the samples, so that the probe concentration in each well is 100 μmol / L. Nitrite, Ammonium, Nitrate, Hydrogen Peroxide, ONOO - , the molar amount of NO is 100 times that of the corresponding probe, respectively. After reacting for 1 hour, the fluorescence intensity was measured on a microplate reader, and the fluorescence intensity after the reaction was divided by the fluorescence intensity of the original copper ion complex to normalize the fluorescence value. The results are shown in Figure 1, where A is 3a-Cu, B is 3d-Cu, C is 3h-Cu, and D is 4b-Cu.

[0077] It can be seen from the figure that the probes 3a-Cu, 3d-Cu, 3h-Cu, 4b-Cu have high selectivity...

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Abstract

The invention relates to a fluorescent probe for measuring generation of nitric monoxide and application of the fluorescent probe. The fluorescent probe is copper complex formed by Cu<2+> and organic ligand 2-(2-hydroxy phenyl) benzimadazoles derivatives, and has the structure of a formula (I) or a formula (II). The complex can simply and quickly enter into viable cells for specific acquisition of cells or active NO in tissues, thereby the fluorescence intensity of the complex is obviously increased, consequently the complex can be applied to the detection of NO in a chemical system, analytic detection and fluorescence imaging detection of NO in viable cells or living tissues of organisms, the detection of NO in lesion tissues clinically, and real-time detection of NO of living biological systems. The fluorescent probe has high NO measurement sensitivity; the minimum lower detectable limit of the fluorescent probe is 17 nmol/L; the fluorescent probe has good selectivity on the NO; fluorescence change is quick before and after the reaction with the NO; and the fluorescence is stable. Simultaneously, the probe has good stability, can be stored for use for a long time, and is suitable for various environments of neutrality, subacidity and alkalinity.

Description

【Technical field】: [0001] The invention belongs to the technical field of biological detection and clinical medical detection, and relates to the measurement of NO in living cells or tissues, in particular to a copper complex fluorescent probe which can be used for imaging measurement of NO in living cells and its application. 【Background technique】: [0002] NO is a gas with small molecules and simple structure. It is insoluble in water and easily diffuses through cell membranes. Because there is an unpaired electron in its molecule, it has a free radical structure and is extremely unstable, and it is easy to react with oxygen, superoxide free radicals, and transition metal ions. The half-life in the tissue is extremely short, generally less than 1 min, but the half-life can be extended to about 4 min outside the tissue or under the condition of oxygen. NO can be quickly oxidized to nitrate or nitrite, combined with superoxide ions, hemoglobin and heme-containing proteins,...

Claims

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Application Information

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IPC IPC(8): C09K11/06G01N21/64G01N33/80
Inventor 欧阳杰张峻峰洪浩沈红芹张有来赵勇李明亮
Owner TIANJIN UNIVERSITY OF TECHNOLOGY
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