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Method of labeling biomolecular by CdTe Nano crystal coupled to fluorescence of strepto affinant

A biomolecule and streptavidin-conjugated technology, applied in the field of fluorescent labeling, can solve the problems of narrow excitation spectrum, single emission wavelength, wide emission spectrum, etc., and achieve the effect of single component and no toxic and side effects

Inactive Publication Date: 2004-10-27
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of fluorescently labeling cells and biomolecules with organic fluorescent dyes has long been used here, but traditional fluorescent dyes have insurmountable defects: narrow excitation spectrum; broad emission spectrum; easy photobleaching and photolysis; nanocrystals have narrow symmetry Fluorescence peak, adjustable emission spectrum, its emission wavelength (color) can be single or multiple

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] 1: Protein biotinylation.

[0012] Dissolve 40 mg of hydroxysuccinimide biotin lipid in 1 ml of dimethylformamide; use 0.1M pH8.0 sodium carbonate solution to make the protein into a 10 mg / ml solution, add 50 μl of hydroxysuccinimide biotin to this solution Lipid solution, after mixing, react at room temperature for 1-3 hours; dialyze against 50mM phosphate buffer at 4°C for 12-24 hours, and freeze-dry.

[0013] 2: Covalent coupling of CdTe nanocrystals with streptavidin.

[0014] Get the CdTe nanocrystal colloid solution (10 -7 M) 1ml, add 0.5ml of 50mM N-hydroxysuccinimide solution, 1mg of streptavidin, and react for 1-4 hours under the condition of pH9-10. Dialyze against 50mM phosphate buffer at 4°C for 12-24 hours. Streptavidin labeled with fluorescent colors of various colors can be obtained.

[0015] 3: Mix the solutions prepared in 1 and 2 and incubate at 25°C-40°C for 1-2 hours to obtain proteins labeled with fluorescent colors of various colors.

Embodiment 2

[0017] 1: Biotinylation of monoclonal antibodies

[0018] Dissolve 40mg of hydroxysuccinimide biotin lipid in 1ml of dimethylformamide; use 0.1M pH8.0 sodium carbonate solution to make the monoclonal antibody into a 10mg / ml solution, add 50μl of hydroxysuccinimide to this solution Biotin lipid solution, react at room temperature for 1-3 hours after mixing; dialyze against 50mM phosphate buffer at 4°C for 12-24 hours, and freeze-dry.

[0019] 2: Covalent coupling of CdTe nanocrystals with streptavidin.

[0020] Get the CdTe nanocrystal colloid solution (10 -7 M) 1ml, add 0.5ml of 50mM N-hydroxysuccinimide solution, 1mg of streptavidin, and react for 2 hours under the condition of pH9.5. Dialyze against 50 mM PBS solution overnight at 4°C. Streptavidin labeled with fluorescent colors of various colors can be obtained.

[0021] 3: Mix the solutions prepared in 1 and 2, and incubate at 37°C for 1 hour to obtain monoclonal antibodies labeled with fluorescent colors of various c...

Embodiment 3

[0023] 1: Biotinylation of immunoglobulins

[0024] Dissolve 40mg of hydroxysuccinimide biotin lipid in 1ml of dimethylformamide; use 0.1M pH8.0 sodium carbonate solution to make immunoglobulin into a 10mg / ml solution, add 50μl of hydroxysuccinimide to this solution Biotin lipid solution, react at room temperature for 1-3 hours after mixing; dialyze against 50mM phosphate buffer at 4°C for 12-24 hours, and freeze-dry.

[0025] 2: Covalent coupling of CdTe nanocrystals with streptavidin.

[0026] Get the CdTe nanocrystal colloid solution (10 -6 M) 1ml, add 0.5ml of 50mM N-hydroxysuccinimide solution, 1mg of streptavidin, and react for 2 hours under the condition of pH9.5. Dialyze against 50 mM PBS solution overnight at 4°C. Streptavidin labeled with orange fluorescence can be obtained.

[0027] 3: Mix the solutions prepared in 1 and 2 and incubate at 37°C for 1 hour to obtain immunoglobulins labeled with fluorescent colors of various colors.

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Abstract

The method includes procedure of biotin forming of biomolecules-Nano crystal covalent coupled strepto affinant- fluorescence labeled biomolecules. Procedure of forming biotin includes stesps: adding sodium carbonate solution of biomolecule into fat solution of biotin of hydroxyl succinimide, mixed reaction for 1-3 hours. Procedure of Nano crystal covalent coupled strepto affinant includes steps: adding solution of N- hydroxyl succinimide and strepto affinant into colloid solution of luminous CdTe Nano crystal, reaction for 1-4 hours under pH 8-11. Finally, mixing biotin-formed product with product obtained from coupled strepto affinant obtains fluorescence labeled biomolecules. Luminous CdTe Nano crystal with no poison excels other Nano crystals in water-solubility, biocompatibility, fluorescent stability and luminous efficiency.

Description

technical field [0001] The invention belongs to a detection method of biomolecules, in particular to a fluorescent labeling method using CdTe nanocrystals with high luminous efficiency coupled with streptavidin to mark biomolecules. Background technique [0002] As a high-sensitivity non-isotopic fluorescence analysis method, nanocrystalline fluorescent labels with high luminous efficiency have attracted increasing attention for their advantages in real-time monitoring of protein-protein interactions in vivo, especially in cells, and in the study of cell differentiation and cell-cell interactions. . The method of fluorescently labeling cells and biomolecules with organic fluorescent dyes has long been used here, but traditional fluorescent dyes have insurmountable defects: narrow excitation spectrum; broad emission spectrum; easy photobleaching and photolysis; nanocrystals have narrow symmetry Fluorescence peak, adjustable emission spectrum, its emission wavelength (color...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/533
Inventor 王丽萍张皓房学迅李惟杨柏
Owner JILIN UNIV
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