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Trivalent europium-beta-diketone fluorescent label and uses thereof

A fluorescent marker, trivalent europium technology, used in biological testing, material testing products, measuring devices, etc., can solve the problems of expensive reagents and low sensitivity, and achieve low cost, high sensitivity, and simple reaction and separation operations. Effect

Inactive Publication Date: 2004-03-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its advantage is that it does not need to use solid phase materials, no binding / free separation (B / F separation) operation and washing steps, simple operation and easy automation, but the disadvantage is low sensitivity and expensive reagents

Method used

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  • Trivalent europium-beta-diketone fluorescent label and uses thereof
  • Trivalent europium-beta-diketone fluorescent label and uses thereof
  • Trivalent europium-beta-diketone fluorescent label and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Synthesis of 4-dentate β-diketone ligands

[0039] (1) 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedione-6"-yl)chloride Synthesis of sulfonyl-o-diphenylbenzene (abbreviated as BHHCT). BHHCT press figure 2 The synthetic route shown is synthesized, and the specific operation process is as follows.

[0040] (i) Synthesis of 4,4'-Diacetyl-o-diphenylbenzene (Compound I)

[0041] Under external ice-water bath cooling, dissolve 14 g of anhydrous aluminum trichloride and 8.1 g of acetyl chloride in 100 ml of dry dichloromethane, and add 11.5 g of o-diphenylbenzene in 50 ml of dichloromethane with stirring. The solution. The reaction solution was stirred for 30 minutes under cooling in an ice-water bath, then stirred at room temperature for 24 hours, and then back distilled for another 2 hours. The reaction solution was poured into a mixture of ice and hydrochloric acid, and after dichloromethane was distilled off, the precipitate was collected by filtration. After the pr...

Embodiment 2

[0051] Use BHHCT-Eu 3+ Marker protein

[0052] (1) Use BHHCT-Eu 3+ Mark BSA

[0053] Dissolve 5 mg of BSA in 1 ml of 0.1 mol / L sodium bicarbonate buffer solution with a pH of 9.3, and add 3.5 mg of BHHCT in 200 microliters of DMF dropwise with stirring. After stirring for 1 hour at room temperature, the labeled BSA and unreacted BHHCT were separated using Sephadex G-50 gel column, and developed with 0.05 mol / L ammonium bicarbonate solution. Measure the absorbance of the labeled BSA solution at 330nm, and use the molar absorption coefficient of BHHCT at 330nm (3.41×10 4 cm -1 mol -1 L) Calculate the concentration of BHHCT in the labeled BSA solution, and then calculate the labeling rate (the ratio of the concentration of BHHCT to the concentration of BSA). The labeling rate of labeled BSA prepared by this method is 35. Add EuCl equal to BHHCT in the solution 3 After that, the fluorescent labeled BSA solution BSA (BHHCT-Eu 3+ ) 35 . Add NaN to the solution 3 (0.1%) After adjusting t...

Embodiment 3

[0061] BHHCT-Eu 3+ Fluorescence properties of labeled BSA solution

[0062] Such as image 3 As shown, BHHCT-Eu 3+ The maximum fluorescence excitation wavelength is 330nm, the maximum emission wavelength is 614nm, and its emission peak shape is a sharp emission peak characteristic of europium complexes. Use BHHCT-Eu 3+ BHHCT-Eu measured with BSA solution 3+ In the 0.05mol / L Tris-HCl buffer solution with pH 7.8, the fluorescence lifetime is 380 microseconds, the fluorescence quantum yield is 0.27, and it contains 1.0×10 -5 The fluorescence lifetime in a 0.1 mol / L sodium carbonate solution of mol / L trioctyl phosphorus oxide and 0.05% sodium dodecyl sulfonate is 641 microseconds, and the fluorescence quantum yield is 0.76.

[0063] BHHCT-Eu 3+ The time-resolved fluorescence measurement results of the labeled BSA serial dilution solution are as follows Figure 4 As shown, using 2 times the standard deviation of the background signal to calculate the lowest detection limit, BSA (BHHCT-...

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Abstract

The invention discloses a trivalent europium - betta -tdiketone fluorescent label object and use thereof, wherein the four-tooth betta - diketone group ligand includes double betta - diketone group substituted orthobiphenyl benzene skeleton construction, and functional substituent group capable of bonding directly with the biomolecules, whose betta - diketone position can coordinate with the trivalent europium ion and form stable europium complex having strong fluorescence, the complex can bond in covalence with protein, amino acid, polypeptide, nucleic acid, nucleotide and organic compounds to mark these substances by the functional substituent groups contained therein, thus then invention can be used for the time measurement of these articles.

Description

Technical field [0001] The invention relates to a preparation method of trivalent europium and tetradentate β-diketone ligand fluorescent label and its application in the relevant technical field of time-resolved fluorescence biological detection. Background technique [0002] As a method for measuring trace physiologically active substances in biological samples (cell tissue, blood, urine, etc.), immunoassays, DNA hybridization assays, etc. have been widely used in various clinical assays. In these assays, certain markers are needed to label substances such as antibodies, antigens, nucleotides, and nucleic acids. Substances used as labels include radioactive elements, enzymes, fluorescent compounds, chemiluminescent compounds, and the like. [0003] Although the method of using radioactive markers is highly sensitive, there are many inconveniences and troubles in the storage, transportation, use, and waste disposal of radioactive markers and their labeled products, and they can ...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/533G01N33/68
Inventor 袁景利王桂兰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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