One-step fluorescence derivation method of reducing sugar and application thereof

A fluorescence and derivative technology, applied in fluorescence/phosphorescence, material analysis through optical means, material excitation analysis, etc., can solve the problems of complex steps, low labeling efficiency, and long time consumption, achieve good compatibility and simplify fluorescent labeling The effect of the process

Inactive Publication Date: 2020-04-03
NORTHWEST UNIV(CN)
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In order to solve the problems of low labeling efficiency, complicated steps, and long time-consuming in the method of fluorescently labeling carbohydrates in the background technology, and to overcome the influence of the labeling process on the biological activity of glycosides, the present invention provides a one-step reaction and high-efficiency fluorescently labeled carbohydrates The material method, using a fluorescein with a hydrazine group, fluorescein-5-thiosemicarbazide (FTSC), directly fluorescently labels reducing sugars and analyzes and detects research applications

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  • One-step fluorescence derivation method of reducing sugar and application thereof
  • One-step fluorescence derivation method of reducing sugar and application thereof
  • One-step fluorescence derivation method of reducing sugar and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] In this embodiment, lactose is selected as a reducing sugar, wherein lactose is a neutral sugar, and the steps for fluorescently labeling lactose with a fluorescent reagent fluorescein-5-thiosemicarbazide (FTSC) are as follows:

[0045] 1. Weigh 3.42mg of lactose and 12.63mg of fluorescein-5-thiosemicarbazide FTSC (the molar ratio is 1:3), put them in a 1.5mL centrifuge tube, add 0.3mL of methanol and 0.15mL of water to centrifuge Add 0.05mL of glacial acetic acid to the tube, shake it fully, dissolve it, put the reaction solution in a water bath at about 40°C and heat it for 1h, after the reaction is completed, dry it by rotary evaporation, add a small amount of water (50μL) to dissolve, and then go through filter paper chromatography purification.

[0046] 2. Chromatographic purification method of filter paper: 5 pieces of circular filter paper (diameter 20mm) are laid flat in a glass tube, add 10mL deionized water to rinse, and then add 5mL acetonitrile to rinse, add t...

Embodiment 2

[0058] This embodiment selects oligomeric uronic acid trisaccharide (GlcA 3 ) is a reducing sugar, in which oligomeric uronic acid trisaccharide (GlcA 3 ) is an acidic sugar, and the oligomeric uronic acid trisaccharide (GlcA 3 ) to carry out the steps of fluorescent labeling as follows:

[0059] 1. Weigh 5.4mg of oligouronic acid trisaccharide and 12.63mg of fluorescein-5-thiosemicarbazide (molar ratio is 1:3), put them in a 1.5mL centrifuge tube, add 0.3mL of methanol and 0.15 Put 0.05 mL of water in the centrifuge tube, then add 0.05 mL of glacial acetic acid, shake fully to dissolve, heat the reaction solution in a 40°C water bath for 1 h, after the reaction is completed, dry it by rotary evaporation, add a small amount of water (50 μL) to dissolve, and then Purified by filter paper disc chromatography.

[0060] 2. Chromatographic purification method of filter paper: 5 pieces of circular filter paper (diameter 20mm) are laid flat in a glass tube, add 10mL deionized wate...

Embodiment 3

[0068] In this example, the neutral oligosaccharide maltodextrin Maltodextran was selected as the reducing sugar, and the oligosaccharide maltodextrin Maltodextran was fluorescently labeled with the fluorescent reagent fluorescein-5-thiosemicarbazide (FTSC). Proceed as follows:

[0069] 1. Weigh 5.0mg of neutral oligosaccharide maltodextrin Maltodextran and 6mg of fluorescent reagent FTSC (molar ratio is 1:3-1:6), put them in a 1.5mL centrifuge tube, add 0.3mL methanol and 0.15 Put 0.05 mL of water in the centrifuge tube, then add 0.05 mL of glacial acetic acid, shake fully to dissolve, heat the reaction solution in a 40°C water bath for 1 h, after the reaction is completed, dry it by rotary evaporation, add a small amount of water (50 μL) to dissolve, and then Purified by filter paper disc chromatography.

[0070] 2. Chromatographic purification method of filter paper: 5 pieces of circular filter paper (diameter 20mm) are laid flat in a glass tube, add 10mL deionized water t...

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Abstract

The invention discloses a one-step fluorescence derivation method of reducing sugar and application thereof. By adopting reducing sugar as a raw material, the reducing sugar and a fluorescence labeling reagent fluorescein-5-amino-thiourea are subjected to a stirring reaction at room temperature to generate a sugar-FTSC derivative so that and various types of sugar such as lactose, uronic acid, maltodextrin and traditional Chinese medicine source reducing polysaccharide are labelled. Mass spectrum structure analysis, fluorescence spectrum and microscopic living cell imaging research are performed on an obtained product. Results show that the carbohydrazone derivative obtained by labeling has good stability and high fluorescence yield, and can be used for fluorescence derivative labeling andmicroscopic fluorescence imaging detection of reducible neutral and acidic oligosaccharides. The method has the advantages of mild reaction conditions, high reaction yield and simple operation, and can be used for large-scale synthesis and preparation of functional oligosaccharide probes; meanwhile, the method is suitable for researching mass spectrometry, chromatography, fluorescence, microscopic fluorescence imaging, flow cytometry and the like of reductive oligosaccharides and polysaccharide molecules, and has a wide application prospect in the field of life science.

Description

technical field [0001] The invention belongs to the field of biotechnology and glycobiology analysis and detection, and relates to a fluorescent labeling method of reducing sugar and its application in fluorescence, mass spectrometry and microscopic imaging research. Background technique [0002] Reducing sugars not only serve as energy sources and structural components of organisms, but also undertake extremely important cell biological functions, such as participating in cell growth, differentiation, development, cell recognition and signal transduction, immune response and other life processes. Many studies have shown that the abnormal expression of sugar chains is closely related to the occurrence and development of several malignant diseases such as tumors, but the research on the mechanism of action and pathogenesis of reducing sugars is still in the exploratory stage, so the development of fluorescent labeling technology is applied to The analysis and detection of car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N30/06G01N27/62
CPCG01N21/6428G01N21/6458G01N30/06G01N27/62G01N2021/6439
Inventor 张英薛向东王仲孚李娇唐梦君李澄
Owner NORTHWEST UNIV(CN)
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