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Fluorescently labeled polysaccharide as well as preparation method and application thereof

A fluorescent labeling and polysaccharide technology, applied in the field of medical biological detection, can solve the problems of unstable fluorescent signal, inability to effectively distinguish different fluorescent signals, small Stokes shift, etc., to increase the variety of probes, low toxicity, and selectivity. high effect

Active Publication Date: 2018-10-16
SUZHOU BAIYUAN GENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of small Stokes shift, unstable fluorescent signal and inability to effectively distinguish different fluorescent signals in the prior art of fluorescently labeled polysaccharides.

Method used

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  • Fluorescently labeled polysaccharide as well as preparation method and application thereof
  • Fluorescently labeled polysaccharide as well as preparation method and application thereof
  • Fluorescently labeled polysaccharide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] This embodiment provides a fluorescent dye having a structure represented by the following formula B:

[0054]

[0055] The fluorescence synthesis route shown in formula B is as follows:

[0056]

[0057] (1) Preparation of Intermediate I’-1

[0058] Add phenylhydrazine to glacial acetic acid, stir, slowly add 3-methyl-2-butanone dropwise, heat to 62.5°C after dropwise addition, react for 3-4 hours, extract, concentrate, and refine to obtain intermediate I'- 1;

[0059] Among them, the molar ratio of phenylhydrazine and 3-methyl-2-butanone is 1:1.1;

[0060] (2) Preparation of Intermediate I’-2

[0061] Add Intermediate I'-1 and 1,2-Dibromoethylene into toluene, protected by nitrogen, heated and refluxed for 17 hours, cooled, and precipitated a solid, namely Intermediate I'-2;

[0062] Among them, the molar ratio of intermediate Ⅰ-1 and 1,2-dibromoethylene is 1:1.75;

[0063] (3) Preparation of Intermediate I’-4

[0064] Add dry N,N-dimethylformamide to dry dichloromethane, add phos...

Embodiment 2

[0075] This embodiment provides a fluorescently labeled polysaccharide, wherein the polysaccharide is dextran (purchased from sigma, catalog number 00268), and the molecular structure of the fluorescent dye is as follows:

[0076]

[0077] The preparation method of fluorescently labeled polysaccharide includes the following steps:

[0078] 1. Prepare saturated cyanogen bromide solution and 0.2M NaOH solution;

[0079] 2. Weigh 20mg of dextran, dissolve the dextran in 1ml of distilled water, fully stir to dissolve; add 4ml of cyanogen bromide solution, add 25μI of NaOH solution each time, continue to add (ensure pH> 10) Continue for 7 minutes and observe the change of the solution. If precipitation occurs, the experiment will fail and repeat;

[0080] 3. Dialysis the reaction solution obtained in the previous step against distilled water, dialysis for 1-2 days, replace the dialysis external fluid every 2-3h; concentrate the solution in the bag to dryness, and add 0.2M Na with pH=8 2 B 4...

Embodiment 3

[0083] This embodiment provides a fluorescently labeled polysaccharide, wherein the polysaccharide is chitosan (purchased from sigma, catalog number 448869), and the molecular structure of the fluorescent dye is as follows:

[0084]

[0085] The preparation method of fluorescently labeled polysaccharide includes the following steps:

[0086] 1. Prepare saturated cyanogen bromide solution and 0.2M NaOH solution;

[0087] 2. Weigh 10mg of chitosan, dissolve the chitosan in 1ml of distilled water, fully stir to dissolve; add 4ml of cyanogen bromide solution, add 25μI of NaOH solution each time, add continuously (ensure pH> 10) Continue for 9 minutes and observe the change of the solution. If precipitation occurs, the experiment will fail and repeat;

[0088] 3. Dialysis the reaction solution obtained in the previous step against distilled water, dialysis for 1-2 days, replace the dialysis external fluid every 2-3h; concentrate the solution in the bag to dryness, and add 0.2M Na with pH=8 ...

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Abstract

The invention discloses fluorescently labeled polysaccharide. The fluorescently labeled polysaccharide is formed by covalently coupling polysaccharide to a fluorescent dye, and the fluorescent dye adopts the structure shown as a formula I in the specification. Polysaccharide molecules and fluorescent dye molecules form stable covalent bonds, so that the fluorescently labeled polysaccharide has high stability in serum and other detection environments, has high biocompatibility, and is suitable for detection of saccharide molecules inside and outside cells. Due to large Stokes shift of the fluorescent dye molecules, the fluorescently labeled polysaccharide also has the advantages of good fluorescent stability, high fluorescence quantum yield, and high signal-to-noise ratio of an imaging result. The invention discloses a preparation method of the fluorescently labeled polysaccharide. With the preparation method, a reaction condition is mild, the operation is simple, the reaction selectivity is high, and the fluorescently labeled polysaccharide with high yield can be prepared.

Description

Technical field [0001] The invention belongs to the technical field of medical biological detection, and specifically relates to a fluorescently labeled polysaccharide and a preparation method and application thereof. Background technique [0002] Fluorescent labeling technology refers to labeling a substance that emits fluorescence on a certain group of the researched molecule through covalent bonding or physical adsorption, and its fluorescence characteristics are used to reflect the information of the research object. The fluorescence characteristics of the research object (polysaccharides, nucleic acids, proteins, peptides, etc.) are adsorbed or covalently combined with fluorescent labeling reagents, which reflects the information about the performance of the research object. With the continuous development of modern medicine and biological technology, the discovery of new fluorescent labeling dyes and the application of various advanced fluorescent detection technologies and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/02C08B37/08C09K11/06G01N21/64G01N30/02G01N21/33
CPCG01N21/33G01N21/6486G01N30/02C09K11/06C08B37/0006C08B37/003C09K2211/1088C09K2211/1092C09K2211/1029C09K2211/1007C09K2211/1014G01N2030/027C08L5/00C08L5/08C09B23/086G01N33/582G01N33/66G01N2400/10C09B23/0066C09B23/0041C09K2211/1018
Inventor 车团结
Owner SUZHOU BAIYUAN GENT CO LTD
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