CN110954517A - One-step fluorescence derivatization method of reducing sugar and its application - Google Patents
One-step fluorescence derivatization method of reducing sugar and its application Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
本发明公开了一种还原性糖的一步荧光衍生方法及其应用。该方法以还原性糖为原料，与荧光标记试剂荧光素‑5‑氨基‑硫脲，在室温下搅拌反应，生成糖‑FTSC衍生物，实现了乳糖、糖醛酸、麦芽糊精以及中药来源还原性多糖等多种类型糖的标记；对所得到的产物进行质谱结构解析、荧光光谱和显微活细胞成像研究。结果显示，标记得到的糖腙衍生物稳定性良好，并且有很高的荧光产率，可用于还原性中性、酸性寡糖的荧光衍生标记及显微荧光成像检测。本发明方法反应条件温和，反应产率高、操作简单，可用于功能性寡糖探针的规模化合成制备；同时适用于研究还原性寡糖、多糖分子的质谱、色谱、荧光、显微荧光成像和流式细胞分析等，在生命科学领域有广阔的应用前景。
The invention discloses a one-step fluorescence derivatization method of reducing sugar and its application. The method uses reducing sugar as raw material, reacts with fluorescent labeling reagent fluorescein-5-amino-thiourea under stirring at room temperature to generate sugar-FTSC derivatives, and realizes the source of lactose, uronic acid, maltodextrin and traditional Chinese medicine Labeling of various types of sugars such as reducing polysaccharides; mass spectrometry structure analysis, fluorescence spectroscopy and microscopic live cell imaging studies of the obtained products. The results show that the labeled glycohydrazone derivatives have good stability and high fluorescence yield, and can be used for fluorescent derivatization labeling and microscopic fluorescence imaging detection of reducing neutral and acidic oligosaccharides. The method of the invention has mild reaction conditions, high reaction yield and simple operation, and can be used for the large-scale synthesis and preparation of functional oligosaccharide probes; at the same time, it is suitable for studying the mass spectrometry, chromatography, fluorescence and microfluorescence of reducing oligosaccharide and polysaccharide molecules. Imaging and flow cytometry have broad application prospects in the field of life sciences.
Description
Technical Field
The invention belongs to the field of biotechnology and sugar biology analysis and detection, and relates to a fluorescent labeling method of reducing sugar and application thereof in fluorescence, mass spectrum and microscopic imaging research.
Background
The reducing sugar not only serves as an energy source and a structural component of an organism, but also plays an extremely important role in cell biological functions, such as participating in the growth and differentiation, development, cell recognition and signal transduction, immune response and other life processes of cells. Many studies show 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 action mechanism and pathogenic mechanism of reducing saccharides is still in the research stage, so the development of fluorescent labeling technology for the analysis and detection of saccharides and the research on structure-activity relationship is very important.
The fluorescence labeling technology has the advantages of high sensitivity, quick response, visualization and the like, is an important technology for researching the metabolism and positioning of target molecules in a body and the interaction between the target molecules and cells, proteins and the like, and is expected to provide an effective way for researching the action mechanism of saccharides. The technology is widely applied to the analysis and detection research of nucleic acid and protein, and becomes an indispensable tool for high-sensitivity analysis and detection of biomacromolecules.
However, because the functional sugar substances in the nature have complex structures, low expression level and no template guidance for synthesis, no complete fluorescence labeling technology for the fluorescence labeling of the sugar substances exists at present.
At present, the fluorescent labeling of saccharides is mainly performed based on chemical derivatization, and the existing fluorescent labeling technologies for saccharides have the following two types:
labeling directly at the reducing end: commonly used derivatization labeling reagents are 2-aminopyridine, 2-aminobenzoic acid, carbazole-9-ethoxycarbohydrazide, 2-aminoacridone, 1-aminopyrene-1, 3, 6-trisulfonic acid, 3-amino-9-ethylcarbazole, etc., mainly based on reductive amination. However, the above-mentioned derivatized labeling reagents are commonly used for chromatographic separation analysis of saccharide molecules, and microscopic fluorescence imaging studies of saccharide substances have not been achieved.
Labeling with a fluorescent labeling reagent with high fluorescence intensity: in order to improve the quantum yield and the fluorescence intensity of a fluorescence labeling product, a fluorescence labeling reagent with high fluorescence intensity commonly used in proteomics, such as fluorescamine (fluorescamine), Fluorescein Isothiocyanate (FITC) and other fluorescence reagents, is selected to label saccharide substances.
When the polysaccharide is marked by the fluorescamine, hydroxyl (-OH) of the polysaccharide needs to be activated by cyanogen bromide (CNBr), and then the fluorescamine is added, wherein most of the marked polysaccharide-OH groups are damaged, and the natural structure of the polysaccharide is damaged to a certain extent, so that the biological activity of the polysaccharide is greatly influenced.
When the carbohydrate is labeled by using FITC, a free amino group is connected to the tail end of a sugar chain through a reductive amination reaction and then reacts with a protein fluorescent reagent FITC.
Therefore, a derivatization method which is mild in conditions and can simultaneously detect neutral sugar chains and acidic sugar chains is found, and the method has important significance for analysis and detection of reducing sugar chains and research of functional mechanisms. Based on the method, the invention develops a novel fluorescent derivation method of reducing sugar and application of the novel fluorescent derivation method to microscopic imaging detection of sugar molecules in cells.
Disclosure of Invention
In order to solve the problems of low labeling efficiency, complex steps, long time consumption and the like of a method for labeling saccharides by fluorescence in the background art and overcome the influence of a labeling process on the biological activity of the saccharides, the invention provides a method for efficiently labeling saccharides by fluorescence in one step, which directly performs fluorescence labeling, analysis, detection, research and application on reducing saccharides by using fluorescein with hydrazine groups and fluorescein-5-thiosemicarbazide (FTSC).
The technical scheme of the invention is to provide a one-step fluorescence labeling method for reducing sugar, which adopts fluorescein-5-thiosemicarbazide (FTSC) as a fluorescence reagent, and utilizes hydrazine group of the FTSC to perform one-step chemical derivatization reaction on C1-hemiacetal of the reducing sugar, so as to realize high-efficiency labeling and form the sugar-FTSC sugar hydrazone fluorescence labeling derivative. Wherein the structural formula (I) of fluorescein-5-thiosemicarbazide (FTSC) is as follows:
further, the reaction molar ratio of the reducing sugar to the FTSC is 1:3-1:6, and the reaction solvent is methanol, water and acetic acid; wherein the volume ratio of the methanol to the water to the acetic acid is 6:3: 1.
Furthermore, the chemical derivatization reaction temperature is 30-50 ℃, and the reaction time is 1-2 h.
Further, the reaction temperature was 40 ℃, the reaction time was 1h, and the pH was 4.5.
Further, the specific method for the further fluorescent labeling of the reducing sugar comprises the following steps:
s1, dissolving reducing sugar and FTSC in a molar ratio of 1:3 in a mixed solvent, wherein the mixed solvent is methanol: water: acetic acid is 6:3:1, volume ratio;
s2, reacting the mixed solution obtained in the step S1 at 40 ℃ for 1 hour to obtain a green reaction solution; then decompression and pumping;
s3, purifying the product obtained in the step S2 through a filter paper chromatography method to obtain the sugar-FTSC sugar hydrazone fluorescent labeling derivative.
Further, the chromatography of the filter paper sheet is specifically as follows:
adding a small amount of water into the reaction solution after evaporation to dryness for dissolving, transferring the dissolved solution onto a filter paper sheet rinsed with acetonitrile in advance, sequentially rinsing the filter paper sheet with 3 times of pure acetonitrile in the volume of the filter paper sheet and 5 times of acetonitrile 96% in the volume of the filter paper sheet, and discarding the filtrate; then, 1mL of deionized water is used for rinsing the filter paper sheet, the rinsing liquid is collected, rotary evaporation and pumping are carried out, and the solid is collected, so that the pure sugar-FTSC sugar hydrazone fluorescent labeling derivative is obtained.
The invention also provides a sugar-FTSC sugar hydrazone fluorescent labeling derivative, which has the structural formula as follows:
wherein R is1OH or sugar; r2OH or sugar; r3＝CH2OH，COOH。
The invention also provides application of the sugar-FTSC sugar hydrazone fluorescent labeling derivative in fluorescence imaging analysis.
The invention has the beneficial effects that:
1. the invention utilizes the characteristic that a hydrazine group of fluorescein-5-thiosemicarbazide (FTSC) can form a sugar hydrazone derivative with a hemiacetal group at C1 position of sugar, and the preparation method is characterized in that the reaction is carried out in the presence of methanol: water: in an acetic acid system, the reducing saccharides are directly marked to form a stable saccharide-FTSC saccharide hydrazone fluorescent marking derivative, the saccharide marking can be completed through one-step reaction, the marking yield is higher than 90%, and the saccharide fluorescent marking process is simplified.
2. The sugar-FTSC sugar hydrazone fluorescent labeled derivative has similar fluorescent characteristics with commonly used 5-Fluorescein Isothiocyanate (FITC), so that the derivative has good compatibility with fluorescent equipment commonly used in proteomics and is suitable for subsequent microscopic fluorescence imaging analysis.
3. Experiments prove that the sugar-FTSC sugar hydrazone fluorescent labeling derivative has no toxicity to cells basically.
4. The fluorescence labeling method provided by the invention is suitable for fluorescence labeling and microscopic imaging detection analysis of reducing sugar such as neutral reducing sugar, acidic reducing sugar and the like. The method provides an important means for preparing the saccharide fluorescent probe and further clarifying the action mechanism research of target saccharide molecules in a body.
Drawings
FIG. 1 is a schematic diagram of a one-step fluorescence labeling method and application of reducing sugar and FTSC;
FIG. 2 is an ESI-MS spectrum of a lactose-FTSC glycohydrazone fluorescent label derivative;
FIG. 3 is an HPLC chromatogram of a lactose-FTSC glycohydrazone fluorescent labeled derivative;
FIG. 4 is GalA acid oligosaccharide3-ESI-MS profile of FTSC glycohydrazone fluorescent labeled derivative;
FIG. 5 is an ESI-MS spectrum of a neutral oligosaccharide maltodextrin-FTSC glycohydrazone fluorescent label derivative;
FIGS. 6a and 6b are fluorescence spectra of lactose-FTSC sugar hydrazone fluorescence labeled derivatives, gynostemma pentaphyllum polysaccharide GPS-FTSC sugar hydrazone fluorescence labeled derivatives and FTSC;
FIG. 7 is the cytotoxicity analysis of gynostemma pentaphylla polysaccharide GPS-FTSC glycohydrazone fluorescence labeling derivative on mouse splenocytes;
FIG. 8 is an image of viable cells of Gynostemma pentaphyllum polysaccharide GPS-FTSC glycohydrazone fluorescent labeled derivatives.
Detailed Description
The examples of the present invention are a one-step fluorescence labeling method for reducing sugars and mass spectrometry, chromatography and microscopic fluorescence analysis for sugar-FTSC sugar hydrazone fluorescence labeled derivatives, the study strategy is shown in fig. 1. FTSC is used as a derivatization reagent for the first time, and the structure of the FTSC has hydrazine groups, so that the FTSC can be labeled with hemiacetal of reducing sugars such as neutrality, acidity and the like, and after derivatization, the FTSC is favorable for subsequent mass spectrum, chromatography and fluorescence analysis, and the method has strong universality and is simpler and more efficient compared with the existing method.
FTSC is selected in the chemical derivatization reaction, the whole labeling reaction condition is mild, and the derivatization condition has important significance for maintaining the structural integrity of the derivative with unstable functional groups such as carboxyl; all sugar chain raw materials are labeled, so that the fluorescence labeling efficiency of the sugar chains can be obviously improved, and the fluorescence spectrum and the fluorescence quantum yield are similar to those of a fluorescence reagent FITC commonly used in proteomics.
The invention is further described with reference to the following figures and specific embodiments.
Example one
In this embodiment, lactose is selected as reducing sugar, wherein lactose is neutral sugar, and the step of fluorescently labeling lactose with a fluorescent reagent fluorescein-5-thiosemicarbazide (FTSC) is as follows:
1. weighing 3.42mg of lactose and 12.63mg of fluorescein-5-thiosemicarbazide FTSC (molar ratio is 1:3), placing the mixture into a 1.5mL centrifuge tube, respectively adding 0.3mL of methanol and 0.15mL of water into the centrifuge tube, then adding 0.05mL of glacial acetic acid, fully shaking and dissolving, placing the reaction solution into a water bath at about 40 ℃ for heating for 1h, performing rotary evaporation and draining after the reaction is finished, adding a trace amount of water (50 mu L) for dissolving, and purifying by using a filter paper chromatography method.
2. A filter paper sheet chromatographic purification method: 5 round filter paper sheets (diameter 20mm) are laid in a glass tube, 10mL deionized water is added for rinsing, 5mL acetonitrile is added for rinsing, a sample to be purified is added on the filter paper sheets, standing is carried out for 5min, 10mL acetonitrile is added for rinsing the filter paper sheets, 10mL 96% acetonitrile is used for rinsing the filter paper sheets, 5mL deionized water is used for rinsing the filter paper sheets, an aqueous solution is collected, rotary evaporation drying is carried out, and the lactose-FTSC glycosyl hydrazone fluorescent labeled derivative is obtained, wherein the yield is 91.23 mg.
3. And (3) carrying out mass spectrum structure analysis on the sample obtained in the step (2):
3.1, dissolving: 0.05mg of the purified lactose-FTSC glycohydrazone fluorescent labeled derivative is weighed, and 0.2mL of ultrapure water is added for dissolution for later analysis.
3.2, mass spectrum data acquisition: mass spectral data were collected on a Thermo Scientific LTQ-XL ion-trap electrospray ionization mass spectrometer (ESIMS).
And (3) directly carrying out mass spectrometry on the sample liquid obtained in the step (3.1).
The mass spectrum data acquisition conditions were as follows, spray voltage: 5.0 kV; sheath gas (N)2) Flow rate: 40.0 arb; auxiliary gas (N)2) Flow rate: 5.0 arb; purge gas (N)2) Flow rate: 0.5 arb; capillary temperature: 340 ℃; tube lens voltage: 48V. Mass spectrometry scan molecular weight range is medium molecular weight end: 200-1500; mobile phase: 30% methanol; flow rate of mobile phase: 200 mu L/min; and (3) sample introduction mode: 2 μ L of quantitative loop sample. The detection mode is a positive ion mode, electrospray ion source.
The analysis results are shown in fig. 2, lactose was subjected to derivatization treatment and purification according to the derivatization method of the present invention, and only the lactose-FTSC glycohydrazone fluorescent-labeled derivative was detected, and no by-product was found. This method is described as being suitable for the fluorescent labeling of neutral reducing sugars.
4. Purity analysis
The sample purity analysis data was collected on a Thermo Scientific LTQ-XL ion-trap LC-MS.
And (4) carrying out liquid chromatography-mass spectrometry analysis on the sample liquid obtained in the step 3.1. Liquid chromatography conditions: liquid chromatography (surfyor); PDA detector (detection wavelength 520 nm); a chromatographic column: RP-C18(5 μm, 250 mm. times.4.6 mm); sample loading temperature: 25 ℃; mobile phase A: 0.01mol/L ammonium acetate (pH7.5, filtered through a 0.45 μm filter); mobile phase B: acetonitrile (HPLC grade); sample introduction amount: 10 mu L of the solution; gradient elution mode: the mobile phase A is from 90% to 70% for 0-90 min; flow rate: 1 mL/min. When liquid chromatography is used in conjunction with mass spectrometry, the mobile phase is split 1:9 using a T-type valve after passing through the PDA detector, only 1/10 flow is guaranteed to pass through the ESIMS for mass spectrometry data acquisition.
The chromatographic analysis result is shown in fig. 3, lactose is subjected to derivatization treatment and purification according to the derivatization method of the invention, a chromatographic peak is detected by HPLC, and the lactose-FTSC glycohydrazone fluorescent labeled derivative is verified by subsequent mass spectrometry analysis, and no by-product is found. The method provided by the invention has high reaction efficiency, can be used for efficiently preparing the sugar hydrazone derivative, and is suitable for the fluorescent labeling and chromatographic analysis of neutral reducing sugar.
Example two
This example selects oligosaccharidouronic acid trisaccharide (GlcA)3) Is reducing saccharide, wherein oligouronic acid trisaccharide (GlcA)3) For acidic sugars, fluorescein-5-thiosemicarbazide FTSC was used as a fluorescent reagent for oligosaccharide uronic acid trisaccharide (GlcA)3) The steps for carrying out the fluorescent labeling are as follows:
1. weighing 5.4mg of oligosaccharin aldehydic acid trisaccharide and 12.63mg of fluorescein-5-thiosemicarbazide (molar ratio is 1:3), placing the mixture into a 1.5mL centrifuge tube, respectively adding 0.3mL of methanol and 0.15mL of water into the centrifuge tube, then adding 0.05mL of glacial acetic acid, fully shaking and dissolving, placing the reaction solution into a 40 ℃ water bath for heating for 1h, after the reaction is finished, performing rotary evaporation and suction drying, adding a trace amount of water (50 mu L) for dissolving, and purifying by a filter paper sheet chromatography.
2. A filter paper sheet chromatographic purification method: 5 round filter paper sheets (diameter 20mm) are laid in a glass tube, 10mL of deionized water is added for rinsing, 5mL of acetonitrile is added for rinsing, a sample to be purified is added on the filter paper sheets, the filter paper sheets are kept stand for 5min, 10mL of acetonitrile is added for rinsing the filter paper sheets, 10mL of 96% acetonitrile is used for rinsing the filter paper sheets, 5mL of deionized water is used for rinsing the filter paper sheets, an aqueous solution is collected, and the mixture is rotated, evaporated and dried to obtain 8.61mg of the oligouronic acid trisaccharide-FTSC glycohydrazone fluorescent labeled derivative (yield is 90.5%).
3. And (3) carrying out mass spectrum structural analysis on the product obtained in the step (2):
(1) dissolving: after the filter paper sheet is purified, 0.05mg of the sample is weighed, and 0.2mL of ultrapure water is added for dissolution for later analysis.
(2) Collecting mass spectrum data: mass spectral data were collected on a Thermo Scientific LTQ-XL ion-trap electrospray ionization mass spectrometer (ESIMS).
And (2) directly carrying out mass spectrometry on the sample liquid obtained in the step (1).
The mass spectrum data acquisition conditions were as follows, spray voltage: 5.0 kV; sheath gas (N)2) Flow rate: 40.0 arb; auxiliary gas (N)2) Flow rate: 5.0 arb; purge gas (N)2) Flow rate: 0.5 arb; capillary temperature: 340 ℃; tube lens voltage: 48V. Mass spectrometry scan molecular weight range is medium molecular weight end: 200-1500; mobile phase: 30% methanol; flow rate of mobile phase: 200 mu L/min; and (3) sample introduction mode: 2 μ L of quantitative loop sample. The detection mode is positive ion mode, and electrospray ion source analysis is carried out.
The results of mass spectrometry are shown in FIG. 4, and the acid saccharide oligouronotriose GlcA3Derivatization treatment and purification are carried out according to the derivatization method of the invention, the oligouronic acid trisaccharide-FTSC glycohydrazone fluorescent marker is detected, no by-product is found, and the carboxyl structure in the sugar chain structure is kept intact. This method is illustrated for the fluorescent labeling of acidic sugars.
EXAMPLE III
In this example, neutral oligosaccharide maltodextrin maltodextrine is selected as reducing sugar, and the step of fluorescently labeling oligosaccharide maltodextrin maltodextrine with fluorescent reagent fluorescein-5-thiosemicarbazide (FTSC) is as follows:
1. weighing 5.0mg of neutral oligosaccharide maltodextrin maltodextrine and 6mg of fluorescent reagent FTSC (molar ratio is 1:3-1:6), placing the mixture into a 1.5mL centrifuge tube, respectively adding 0.3mL of methanol and 0.15mL of water into the centrifuge tube, then adding 0.05mL of glacial acetic acid, fully shaking and dissolving, placing the reaction solution into a 40 ℃ water bath for heating for 1h, after the reaction is finished, performing rotary evaporation and suction drying, adding a trace amount of water (50 mu L) for dissolving, and purifying by a filter paper chromatography.
2. A filter paper sheet chromatographic purification method: 5 circular filter paper sheets (the diameter is 20mm) are laid in a glass tube, 10mL of deionized water is added for rinsing, 5mL of acetonitrile is added for rinsing, a sample to be purified is added on the filter paper sheets, standing is carried out for 5min, 10mL of acetonitrile is added for rinsing the filter paper sheets, 10mL of 96% acetonitrile is used for rinsing the filter paper sheets, 5mL of deionized water is used for rinsing the filter paper sheets, an aqueous solution is collected, and rotary evaporation and drying are carried out to obtain the maltodextrin Maltodextran-FTSC glycosyl hydrazone fluorescent labeling derivative.
3. Carrying out mass spectrum structure analysis on the maltodextrin Maltodextran-FTSC sugar hydrazone fluorescence labeling derivative:
(1) dissolving: after the filter paper sheet is purified, 0.05mg of the sample is weighed, and 0.2mL of ultrapure water is added for dissolution for later analysis.
(2) Collecting mass spectrum data: mass spectral data were collected on a Thermo Scientific LTQ-XL ion-trap electrospray ionization mass spectrometer (ESIMS).
And (2) directly carrying out mass spectrometry on the sample liquid obtained in the step (1).
The mass spectrum data acquisition conditions were as follows, spray voltage: 5.0 kV; sheath gas (N)2) Flow rate: 40.0 arb; auxiliary gas (N)2) Flow rate: 5.0 arb; purge gas (N)2) Flow rate: 0.5 arb; the capillary temperature was 340 ℃; tube lens voltage: 48V. Mass spectrometry scan molecular weight range is medium molecular weight end: 200-1500; mobile phase: 30% methanol; flow rate of mobile phase: 200 mu L/min; and (3) sample introduction mode: 2 mu L of pyridineAnd (5) measuring and injecting samples. The detection mode is positive ion mode, and electrospray ion source analysis is carried out.
The mass spectrometry results are shown in fig. 5, and maltodextrin Maltodextran was subjected to derivatization treatment and purification according to the derivatization method of the present invention, and a series of molecular ion peaks ([ M + Na ] 584.55, 746.64, 908.55, 1070.64, 1232.64, 1394.64, 1556.73, 1718.73, 1880.82, 2042.73, 2204.75, 2366.87, 2528.91, 2691.00, 2853.09, etc. were detected]+) The structure of the derivative is analyzed to be maltodextrin Maltodextran-FTSC sugar hydrazone fluorescent labeled derivative (G1-15-FTSC; g1 is dextran monosaccharide, G2 is dextran disaccharide, G3 is dextran trisaccharide, G15 is dextran pentadecaose), each sugar chain is labeled with one molecule of FTSC molecule, no by-product or unlabeled oligosaccharide chain is found, indicating that the method is suitable for the fluorescent labeling of neutral oligosaccharides.
Example four
This example analyzes the fluorescence properties of the sugar-FTSC sugar hydrazone fluorescently labeled derivatives.
1. Fluorescent labeling of sugars
Respectively weighing 3.42mg and 5.0mg of lactose and neutral oligosaccharide maltodextrin, respectively adding 12.43mg of fluorescent reagent FTSC, respectively placing two groups of reactants into a 1.5mL centrifuge tube, respectively adding 0.3mL of methanol and 0.15mL of water into the centrifuge tube, then adding 0.05mL of glacial acetic acid, fully shaking and dissolving, respectively placing two groups of reaction solutions into a 40 ℃ water bath and heating for 1h, respectively performing rotary evaporation and draining after the reaction is finished, adding trace water (50 mu L) for dissolving, and respectively performing filter paper chromatography purification. The chromatographic purification of the filter paper sheets is carried out as described in example one. Obtaining the lactose-FTSC and maltodextrin Maltodextran-FTSC sugar hydrazone fluorescent labeling derivative.
2. Fluorescence spectrum analysis of sugar-FTSC sugar hydrazone fluorescence labeling derivative
Fluorescent labeled derivatives of Fluorescein Isothiocyanate (FITC), FTSC and neutral sugar-FTSC glycohydrazone are respectively prepared into 1x10-6M in water, the sample was shaken up. The excitation and emission spectra of FITC, FTSC and the fluorescent labeled derivative of the neutral sugar FTSC sugar hydrazone were measured and recorded on a FL-4500 fluorescence spectrophotometer, placed in a 1cm fluorescence cell, and sweptFluorescence spectra were drawn and fluorescence intensity values were measured. Excitation and emission slits are both 5nm, the temperature is controlled to be 20 ℃, and the fluorescence spectrum characteristics and fluorescence quantum yield phi of FITC, FTSC and neutral sugar-FTSC sugar hydrazone fluorescence labeling derivatives are respectively measured.
Fluorescence spectrum analysis shows that the fluorescence spectrum characteristics of the sugar-FTSC sugar hydrazone fluorescence labeling derivative are similar to those of a fluorescence reagent FITC commonly used in proteomics, the excitation wavelengths are all near 488nm, and the emission wavelength is 518 nm; and has high fluorescence quantum yield. According to the literature, the fluorescence quantum yield of fluorescein,. phi.0.95. + -. 0.03 (arthritis A. Kazarian, et al, James Suttil; Development of a novel fluorescent tag O-2- [ aminoethiol ] fluorescent for the electrophoretic separation of oligosaccharides, analytical Chimica Acta,662(2010) 206-. The fluorescence quantum yields of free FTSC, lactose-FTSC and maltodextrin-FTSC glycohydrazone fluorescent labeled derivatives are determined as shown in table 1, the quantum yield Φ of the free FTSC is 0.88, and the quantum yields Φ of the lactose-FTSC glycohydrazone fluorescent labeled derivatives and the maltodextrin-FTSC glycohydrazone fluorescent labeled derivatives are 0.81 and 0.77, respectively, which are 0.85 and 0.81 times of FITC. The above results show that the method is suitable for the fluorescent labeling of saccharides and the subsequent microscopic imaging analysis.
TABLE 1 fluorescence Spectroscopy of sugar-FTSC sugar Hydrazone fluorescent-labeled derivatives
EXAMPLE five
This example is fluorescence labeling and fluorescence imaging analysis of gynostemma pentaphylla polysaccharide.
1. Fluorescence labeling of gynostemma pentaphylla polysaccharide
Weighing 5mg of gynostemma pentaphylla polysaccharide GPS (prepared in a glycobiology laboratory of northwest university), adding 5mg of a fluorescent reagent FTSC, placing reactants in a 1.5mL centrifuge tube, respectively adding 0.3mL of methanol and 0.15mL of water in the centrifuge tube, then adding 0.05mL of glacial acetic acid, fully shaking and dissolving, placing reaction solutions in a water bath at 40 ℃ for heating for 1h, respectively performing rotary evaporation and suction drying after the reaction is finished, adding a trace amount of water (50 mu L) for dissolving, and performing filter paper chromatography purification. The chromatographic purification of the filter paper sheets is carried out as described in example one. Obtaining the gynostemma pentaphylla polysaccharide-FTSC glycohydrazone fluorescent labeling derivative.
2. Fluorescence spectrum analysis of FTSC (fluorine-doped stannic sulfide) glycohydrazone fluorescence labeling derivative of lactose Lac and gynostemma pentaphylla polysaccharide GPS (Global positioning System)
Respectively weighing 0.1mg lactose-FTSC sugar hydrazone fluorescence labeling derivative and 1mg gynostemma pentaphylla polysaccharide-FTSC sugar hydrazone fluorescence labeling derivative, dissolving in water to obtain a solution of 1x10-6The results of fluorescence spectrum analysis of the aqueous solution of M are shown in FIGS. 6a and 6 b. Fluorescence spectrum analysis shows that the fluorescence spectrum characteristics of the lactose-FTSC sugar hydrazone fluorescence labeling derivative and the gynostemma pentaphylla polysaccharide-FTSC sugar hydrazone fluorescence labeling derivative are very similar to those of common Fluorescein Isothiocyanate (FITC), the maximum excitation wavelength is 488nm, the maximum emission wavelength is 518nm, and the lactose-FTSC sugar hydrazone fluorescence labeling derivative and the gynostemma pentaphylla polysaccharide-FTSC sugar hydrazone fluorescence labeling derivative have good compatibility with existing microscopic analysis equipment and are suitable for subsequent microscopic imaging analysis.
3. Cytotoxicity assays
Murine thymocytes are a type of environmentally sensitive cells that are commonly used to examine the toxicity of drugs to living cells. In the embodiment, gynostemma pentaphylla polysaccharide GPS and gynostemma pentaphylla polysaccharide GPS-FTSC sugar hydrazone fluorescent labeled derivatives are respectively incubated with mouse thymocytes,3the H-TdR incorporation assay measures the rate of cell growth. The results are shown in FIG. 7. The result shows that the gynostemma pentaphylla polysaccharide GPS-FTSC sugar hydrazone fluorescence labeling derivative has the activity of promoting splenocyte proliferation compared with the free gynostemma pentaphylla polysaccharide GPS; but the activity intensity of the gynostemma pentaphylla polysaccharide GPS-FTSC glycohydrazone fluorescence labeling derivative is reduced to a certain extent and is probably related to the increase of the molecular weight after labeling. However, the gynostemma pentaphylla polysaccharide GPS marked by FTSC can stimulate splenocyte proliferation, which shows that the gynostemma pentaphylla polysaccharide GPS-FTSC glycosyl hydrazone derivatives have no cytotoxicity basically. Similarly, plant-derived polysaccharide oligogalacturonotriose-FTSC glycohydrazone derivatives and lactose, neutral oligosaccharide maltodextrin, Maltodextran, were also subjected to similar experimental data. The above experimental results show that the sugar-FTSC sugar hydrazone derivative obtained by reacting the reducing end of the sugar with the hydrazino fluorescent labeling reagent has no toxicity to cells.
4. Confocal laser imaging analysis
HepG2 cells were seeded in confocal laser culture plates (6X 10)5per well), in RPMI-1640 medium containing 10% fetal bovine serum FBS, 100U/mL penicillin (penicillin) and 0.10mg/mL streptomycin, at a culture temperature of 37 ℃ and 5% CO2The culture box of (1) was cultured overnight. One group was not treated, and the other group was incubated overnight for 12 hours with the addition of Gynostemma pentaphyllum polysaccharide GPS-FTSC glycohydrazone fluorescent-labeled derivative (10. mu.g/mL). The culture medium was removed, washed 2 times with PBS, DAPI nuclear staining was performed, and the cells were then infiltrated in 2mL of PBS. Fluorescence imaging under a confocal laser microscope (Fluoview FV1000 confocal microscope (Olympus, Tokyo, Japan), magnification x20, using a-krypton/argon laser source at 488and 568nm the results are shown in FIG. 8, left column for brightfield imaging; the middle group is fluorescence signals with the excitation wavelength of 488 nm; the right column is the superposition of the two groups of signals, the upper layer is fluorescence imaging of incubation of FTSC and HepG2 cells, the lower layer is fluorescence imaging analysis of incubation of gynostemma pentaphylla polysaccharide GPS-FTSC glycohydrazone derivatives and cells, after incubation of the cells and the gynostemma pentaphylla polysaccharide GPS-FTSC glycohydrazone derivatives, the experimental results show that the sugar-FTSC sugar hydrazone fluorescence labeling derivative can be used for live cell imaging microscopic fluorescence analysis.
In conclusion, the one-step fluorescence derivatization method for the reducing sugar chain takes FTSC as a derivatization reagent, and due to the structure of the FTSC has hydrazine groups, the FTSC can be derivatized with C1-position hemiacetal of reducing sugar to form sugar-FTSC sugar hydrazone fluorescence labeled derivatives, so that the method is beneficial to subsequent mass spectrum and chromatographic detection, and the fluorescence characteristics of the product are similar to that of a common fluorescence reagent FITC of proteomics, so that the method is suitable for microscopic imaging analysis. The method has strong universality, and is simpler, more convenient and more efficient.
In the derivatization process, the pH condition is weak acidity, the reaction is carried out at low temperature, and the derivatization condition of the invention can keep the stability of the structure of the derivative with unstable functional groups.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
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