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Biomolecule fluorescence marking method, fluorescence-marked biomolecule obtained thereby and application of fluorescence-marked biomolecule

A biomolecular and fluorescent labeling technology, applied in fluorescence/phosphorescence, chemical instruments and methods, analytical materials, etc., can solve the problems of poor water solubility, increase the difficulty of fluorescent dye synthesis and separation, and affect the activity of biomolecules, and achieve efficient labeling. , Avoid the influence of biomolecular activity, the effect of the method green environmental protection

Inactive Publication Date: 2016-07-06
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These fluorescent dyes need to be modified with specific reactive groups, which increases the difficulty of synthesis and separation of fluorescent dyes
Moreover, these fluorescent dyes have poor water solubility, and organic solvents need to be introduced when labeling antibodies, proteins, peptides and other biomolecules, which will affect the activity of biomolecules

Method used

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  • Biomolecule fluorescence marking method, fluorescence-marked biomolecule obtained thereby and application of fluorescence-marked biomolecule
  • Biomolecule fluorescence marking method, fluorescence-marked biomolecule obtained thereby and application of fluorescence-marked biomolecule
  • Biomolecule fluorescence marking method, fluorescence-marked biomolecule obtained thereby and application of fluorescence-marked biomolecule

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The structural formula of the cyanine dye compound used in this embodiment is

[0040]

[0041] where R 1 for -(CH 2 ) 5 COOH, R 2 is Cl, n=1.

[0042] Dissolve 1.0 μmol of glutathione (GSH) in a buffer solution of Tris-HCl (pH=7.4), and then add it into a buffer solution of 1.0 μmol of cyanine dye Tris-HCl (pH=7.4), avoid light , stirring at room temperature for 2 hours, after the reaction was completed, the reaction solution was added into a dialysis bag for dialysis separation, and then the solution in the dialysis bag was taken out and freeze-dried to obtain fluorescently labeled GSH. The molecular weight was identified by MALDI-TOF.

[0043] By H NMR spectroscopy (such as figure 2 Shown) the GSH that gained fluorescent label is good has been characterized, and the result is as follows:

[0044] 1 HNMR (400MHz, CD 3OD, 298K, TMS): δ = 8.79 (d, J = 12Hz, 2H, CH); δ = 7.53 (d, J = 8Hz, 2H, ArH); δ = 7.40 (t, J = 8Hz, 2H, ArH ); 7.31-7.23 (m, 4H, ArH); 6....

Embodiment 2

[0051] The structural formula of the cyanine dye compound used in this embodiment is where R 1 for -(CH 2 ) 5 COOH, R 2 is Cl, n=1.

[0052] Dissolve 1.0 μmol of the polypeptide ECGD (sequence Glu-Cys-Gly-Asp) in Tris-HCl (pH=7.4) buffer solution, and then add 2.0 μmol of cyanine dye Tris-HCl (pH=7.4) In the buffer solution, avoid light, and stir at 20°C for 6 hours. After the reaction is completed, the reaction solution is added to the dialysis bag for dialysis separation, and then the solution in the dialysis bag is taken out and freeze-dried to obtain the fluorescently labeled polypeptide ECGD. The molecular weight was identified by MALDI-TOF.

[0053] MALDI-TOF: Calculate C 56 h 73 N 6 o 13 S + The m / z is 1069.49, from Figure 6 The m / z obtained in the spectrum shown was 1069.6.

Embodiment 3

[0055] The structural formula of the cyanine dye compound used in this embodiment is where R 1 for -(CH 2 ) 5 COOH, R 2 is Cl, n=1.

[0056] Dissolve 1.0 μmol of the polypeptide DGRKLVFFCG (sequence Asp-Gly-Arg-Lys-Leu-Val-Phe-Phe-Cys-Gly) in a Tris-HCl (pH=7.4) buffer solution, and then add to a solution containing 4.0 In the buffer solution of μmol cyanine dye Tris-HCl (pH=7.4), avoid light and stir at 30°C for 0.5h. After the reaction is completed, the reaction solution is added to the dialysis bag for dialysis separation, and then the solution in the dialysis bag is taken out and freeze-dried to obtain Fluorescently labeled polypeptide DGRKLVFFCG. The molecular weight was identified by MALDI-TOF.

[0057] MALDI-TOF: Calculate C 94 h 131 N 16 o 17 S + The m / z is 1787.95, from Figure 7 The m / z obtained in the spectrum shown is 1787.2.

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Abstract

The invention provides a biomolecule fluorescence marking method, a fluorescence-marked biomolecule obtained thereby and application of the fluorescence-marked biomolecule.The method includes: dissolving fluorescence molecule and sulfhydryl-containing biomolecule in a buffering solution, stirring, and performing dialysis separation to obtain the fluorescence-marked biomolecule, wherein the fluorescent molecule is one or or a combination of at least two of cyanine dye compounds of the structure.By using the method, the cyanine dye compounds can be efficiently utilized to mark the biomolecule under mild conditions, organic solvents are not needed, so that the method is environment-friendly, and influence on biomolecule activity by certain organic solvents is avoided.The method is high in marking efficiency, simple to operate and wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of fluorescent labeling of biological materials, and relates to a method for fluorescent labeling of biomolecules, the obtained fluorescently labeled biomolecules and applications thereof. Background technique [0002] Fluorescent proteins (eg, phycoerythrin) or bioluminescent reporter systems are often used in some fluorescence-based systems used in life science research. However, these techniques are time-consuming, unable to detect multiple targets, and are less sensitive and photostable than synthetic fluorochromes. Fluorescent techniques using specific probes labeled with fluorochromes enable the detection of multiple targets in cell-based applications and are compatible with a variety of fluorescent devices. [0003] Fluorescent labeling is the process of covalently attaching fluorescent groups to molecules such as proteins and nucleic acids. Such processes are typically accomplished using derivative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/113C07K7/06C07K5/093C09K11/06G01N21/64
CPCC07K5/1021C07K5/0819C07K7/06C09K11/06C09K2211/1007C09K2211/1029G01N21/6428
Inventor 王浩安红维乔增莹乔圣林
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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