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Rare earth fluorescent marker and its application

A rare earth fluorescence and marker technology, applied in the field of determination of trace physiologically active substances, can solve the problems of high measurement sensitivity, low sensitivity and high sensitivity, and achieve the effects of large fluorescence quantum yield, wide application range and good water solubility.

Inactive Publication Date: 2004-12-22
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

Their disadvantages are: 1) the method of using radioactive markers, radioactive markers and their marked products have many inconveniences and troubles in storage, transportation, use, waste liquid treatment, etc., and can cause environmental pollution, and radioactive markers The self-attenuation problem of the marker leads to a short storage time
2) In the method of using enzyme markers, the molecular weight of enzyme markers is too large, and its activity is easily affected by temperature, storage conditions, acidity, heteroions, preservatives, etc., resulting in poor reproducibility of the method
3) When the determination method using organic fluorescent markers is used for the determination of biological tissues, serum and other samples, due to the influence of the scattered light of the excitation light and the strong background fluorescence produced by the sample, the sensitivity of this method is low, and it is only suitable for Determination of High Concentration Substances
The sensitivity of the DELFIA assay is high, but its disadvantage is that the solution of the fluorescence assay of this method contains a large excess of β-diketone ligands and trioctylphosphine oxide, which is easily affected by the assay environment, assay samples and The pollution of metal ions in the solution used for the measurement has extremely strict requirements on the measurement operating environment and the reagents used; moreover, this system can only be used for liquid phase fluorescence measurement, and its application range is greatly limited
This method has high sensitivity and does not need to use fluorescence enhancement solution. Its disadvantage is that the markers have poor water solubility and are not suitable for labeling small molecular substances.

Method used

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  • Rare earth fluorescent marker and its application
  • Rare earth fluorescent marker and its application
  • Rare earth fluorescent marker and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Marker 4'-(2-thienyl)-2,2':6',2"-biteropyridine-6,6"-dimethylaminetetraacetic acid mono-N-hydroxysuccinamide ester (referred to as NHS-TTTA )Synthesis. The marker NHS-TTTA was synthesized by the synthetic route shown below,

[0038]

[0039]

[0040] The specific operation process is as follows:

[0041] (1) Synthesis of 4'-(2-thienyl)-2,2':6',2-biterpyridine (Compound 1)

[0042] Add 23.1 grams of ammonium acetate, 16.3 grams of N-[2-(pyrid-2'-yl)-2-oxoethyl]pyridinium iodide (N-[2-(2'-pyridyl)-2-oxo Ethyl]pyridine iodide, 50mmol), and 10.76 grams of (E)-3-(2"-thenyl)-1-(pyrid-2'-yl)prop-2-enone((E)-3-( 2"-thienyl)-1-(2'-pyridyl)-2-propenone, 50 mmol), and the solution was stirred and back-distilled for 24 hours. After the reaction solution was cooled to room temperature, it was placed at -15°C for 1 hour, and the precipitate was collected by filtration, washed thoroughly with cold methanol (about -15°C), and the product was recrystallized with acetonitril...

Embodiment 2

[0061] Marker N, N, N 1 , N 1 -[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-(2"-thienyl)pyridine]tetraacetic acid mono-N-hydroxysuccinamide ester (referred to as NHS-BTTA) synthesis:

[0062] The marker NHS-BTTA was synthesized by the synthetic route shown below,

[0063]

[0064] The specific operation process is as follows:

[0065] (1) Synthesis of 2,6-dibromo-4-(2'-thienyl)pyridine (compound I)

[0066] Add 3.25 grams of 4-amino-2,6-dibromopyridine (12.9 mmol) and 12.9 grams of thiophene to 250 ml of acetic acid. After stirring and dissolving, add 1.93 grams of nitrosyl isopentyl ester dissolved in 13 ml of acetic acid dropwise under stirring. solution. After the reaction solution was stirred at room temperature for 24 hours, the stirring reaction was continued at 50° C. for 3 hours. The solvent was distilled off under reduced pressure, the product was neutralized with 40 ml of 10% potassium carbonate, and the product was extracted with 4×60 ml of chloroform. After th...

Embodiment 3

[0080] TTTA and BTTA and rare earth ions (Eu 3+ and Tb 3+ ) Determination of fluorescence properties of complexes

[0081] 1. Fluorescence spectrum, fluorescence intensity and fluorescence lifetime

[0082] In aqueous solution, TTTA and BTTA can quickly form stable complexes after mixing with rare earth ions. TTTA-Eu was determined by using 0.05mol / L boric acid buffer solution with pH value of 9.1 as solvent 3+ , TTTA-Tb 3+ and BTTA-Eu 3+ , BTTA-Tb 3+ UV-Vis spectrum, fluorescence spectrum, molar absorptivity (ε), fluorescence quantum yield (φ) and fluorescence lifetime (τ) in the solvent. The instrument used for the determination of ultraviolet-visible spectrum is Tianmei UV 7500 spectrophotometer. The instrument used for fluorescence measurement is a Perkin Elmer LS 50B fluorescence spectrophotometer. Quantum yield measurement using 4’-phenyl-2,2’:6’,2”-biteropyridine-6,6”-dimethylaminetetraacetic acid and Eu 3+ and Tb 3+ The complex was measured as a standard (its...

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Abstract

A novel rare-earth fluorescent marker is a fluorescent match of the RE ions (Eu, Sm, Gd, Tb and Dy) and the ligand containing 2,2':6,2''-bitripyridine skeleton structure or 2,6-dipyrazole pyridine skeleton structure. It can bind with protein, amino acid, polypeptide, nucleic acid, nucleotide, and organic compound via its function group to mark them for fluorometry.

Description

technical field [0001] The invention relates to a measurement technology of trace physiologically active substances, in particular to a rare earth fluorescent marker and its application. Background technique [0002] Immunoassays, DNA hybridization assays, and the like have been widely used in various clinical assays as methods for assaying trace amounts of physiologically active substances in biological samples (cell tissue, blood, urine, etc.). In these assays, certain markers are required to label substances such as antibodies, antigens, nucleotides, and nucleic acids. Substances currently used as markers include radioactive elements, enzymes, fluorescent compounds, chemiluminescent compounds, and the like. Their disadvantages are: 1) the method of using radioactive markers, radioactive markers and their marked products have many inconveniences and troubles in storage, transportation, use, waste liquid treatment, etc., and can cause environmental pollution, and radioacti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06G01N33/533
Inventor 袁景利谭明乾王桂兰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI