CN104535754A - Preparation method for N-methylisatoic anhydride rapidly-labeled polysaccharide - Google Patents
Preparation method for N-methylisatoic anhydride rapidly-labeled polysaccharide Download PDFInfo
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- CN104535754A CN104535754A CN201410748956.XA CN201410748956A CN104535754A CN 104535754 A CN104535754 A CN 104535754A CN 201410748956 A CN201410748956 A CN 201410748956A CN 104535754 A CN104535754 A CN 104535754A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
A preparation method for N-methylisatoic anhydride rapidly-labeled polysaccharide belongs to the technical field of fluorescent labeling. The preparation method employs a fluorescence tracer N-methylisatoic anhydride for labeling polysaccharide, and the method comprises: performing incubating on a N-methylisatoic anhydride stock solution and a polysaccharide solution under the condition of 35 DEG C for 15-20 min, and performing separation purification on the fluorescence-labeled polysaccharide through an organic solvent precipitation process. The reaction is mild in reaction conditions and short in reaction time, and the fluorescence-labeled polysaccharide separation purification process is simple. Polysaccharide labeled by fluorescence can be detected by using a routine fluorescence detector, and research on polysaccharide functions and mechanism is facilitated.
Description
Technical field
The invention belongs to fluorescent labelling techniques field, be specifically related to a kind of preparation method of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide.
Technical background
In life science, polysaccharide is indispensable biomacromolecule, but lacks luminophore because of it, is therefore not easy to detect, therefore hampers its research.Its fluorescence radiation group can be attached on sugar chain molecule by fluorescent marker to chemically, thus makes polysaccharide with fluorophor, and conventional sense means easy to use can detect polysaccharide, is conducive to studying its mechanism of action further.
According to bibliographical information, radioactive isotope is usually used in marking polysaccharide, carrys out the biologically active of spike polysaccharide, and then study its mechanism of action with this.But, because radioactive isotope can cause test organism to produce bad reaction, be not easy to the activity research being applied in polysaccharide.But its fluorescence radiation group can be attached on sugar chain by fluorescent marker to chemically, makes polysaccharide have certain fluorescence.The fluorophor be attached on polysaccharide has the advantages that to excite with emission wavelength, therefore uses conventional fluorescence detector just can detect and spike polysaccharide.And having bibliographical information, its biologically active of the polysaccharide after fluorescence labeling and former polysaccharide do not have notable difference, and can also directly detect it.The domestic fluorescent marker for polysaccharide is mainly fluorescein isothiocynate (FITC) at present, FITC main mark glycosaminoglycan and lipopolysaccharides, and the polysaccharide of not all can mark.
Based on above-mentioned present situation, the present invention, marks the hydroxyl specificity of polysaccharide for fluorescent marker with N-methyl-isatin acid anhydrides, and reaction conditions is gentle, the reaction time is short, label separation and purification is simple.Polysaccharide after fluorescence labeling, can use conventional fluorescence detector to detect, and is convenient to study the function of polysaccharide and mechanism.
Summary of the invention
The invention provides a kind of preparation method of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide.Use fluorescent label polysaccharide, polysaccharide can be detected with fluorescence detector easily, thus be convenient to study the function of polysaccharide and mechanism.
Technical scheme of the present invention:
(1) sample preparation: dissolve with the dimethyl sulphoxide solution that the borate buffer solution of 0.1mol/L, PH 8.0 and 5 ~ 10mL concentration w/v are 50% and be mixed with 40 ~ 80mg/mL polysaccharide solution; Described polysaccharide solution is gumwater or maltodextrin solution;
(2) preparation of fluorescence labeling storing solution: N-methyl-isatin acid anhydrides dmso solution is mixed with 20mg/mL solution;
(3) joined by the fluorescence labeling storing solution that step (2) obtains in the polysaccharide solution that step (1) obtains, hatch 15 ~ 20min at 35 DEG C, the mass ratio of fluorescent marker N-methyl-isatin acid anhydrides and polysaccharide is 1:50-1:60;
(4) separation of fluorescence labeling polysaccharide: adopt organic solvent precipitation method, absolute ethyl alcohol is added by step (3) reacted solution, utilize the character of polysaccharide alcohol precipitation by separation of polysaccharides, the volume ratio of fluorescence labeling polysaccharide solution and absolute ethyl alcohol is 1:4 ~ 1:8, centrifugal 10 ~ the 20min of polysaccharide 6000 × g of alcohol precipitation, the centrifugal supernatant that obtains and absolute ethyl alcohol volume ratio are 1:2 ~ 1:4, and alcohol precipitation is centrifugal again;
(5) purifying of fluorescence labeling polysaccharide: the precipitation mixing obtained twice, redissolves with distilled water, and use core filtration devices to remove the water-insoluble N-methyl-isatin acid anhydrides and impurity that do not react with polysaccharide, filtrate freeze-drying, can obtain fluorescence labeling polysaccharide.
Beneficial effect of the present invention:
1, the fluorescent marker N-methyl-isatin acid anhydrides (as FITC, Nile red etc.) compared with other fluorescent markers selected by the present invention is cheap.
2, the reaction conditions of fluorescent marker N-methyl-isatin acid anhydrides of the present invention and polysaccharide is gentle, the reaction time is short, fluorescence labeling separation of polysaccharides purification process is simple.
3, the polysaccharide after fluorescence labeling, can use conventional fluorescence detector to detect, and is convenient to study the function of polysaccharide and mechanism.
Accompanying drawing explanation
Fig. 1 is the fluorescence spectrum that fluorescence labeling Arabic gum and Arabic gum excite at 360nm place;
Fig. 2 is the fluorescence spectrum that fluorescence labeling maltodextrin and maltodextrin excite at 360nm place;
Fig. 3 is the distribution of fluorescence labeling maltodextrin in microcapsules of confocal laser scanning microscope;
Fig. 4 is the fluorescence intensity of fluorescence labeling maltodextrin in microcapsules.
Embodiment
embodiment 1
(1) sample preparation: dissolve with the borate buffer solution 5 ~ 10mL of 0.1mol/L, PH 8.0 and the dimethyl sulphoxide solution of concentration w/v 50% and be mixed with 40 ~ 80mg/mL gumwater.
(2) preparation of fluorescence labeling storing solution: N-methyl-isatin acid anhydrides dmso solution is mixed with 20mg/mL solution.
(3) the fluorescence labeling storing solution prepared is joined in gumwater according to the ratio of the mass ratio 1:50 of fluorescent marker N-methyl-isatin acid anhydrides and Arabic gum at 35 DEG C, hatch 15min.
(4) separation of fluorescence labeling polysaccharide: the Arabic gum organic solvent precipitation method of N-methyl-isatin acid anhydrides mark is separated, absolute ethyl alcohol is added in the polysaccharide solution after mark, utilize the character of polysaccharide alcohol precipitation by separation of polysaccharides, fluorescence labeling polysaccharide solution: absolute ethyl alcohol volume ratio 1:4, centrifugal 10 ~ the 20min of polysaccharide 6000 × g of alcohol precipitation, the centrifugal supernatant obtained: absolute ethyl alcohol volume ratio 1:2 ~ 1:4, alcohol precipitation is centrifugal again.
(5) purifying of fluorescence labeling polysaccharide: the precipitation mixing that twice is obtained, redissolve with distilled water, use core filtration devices to remove the water-insoluble N-methyl-isatin acid anhydrides and impurity that do not react with polysaccharide, filtrate freeze-drying, can obtain fluorescence labeling Arabic gum.
embodiment 2
(1) sample preparation: dissolve with the borate buffer solution 5 ~ 10mL of 0.1mol/L, PH 8.0 and the dimethyl sulphoxide solution of w/v 50% and be mixed with 40 ~ 80mg/mL maltodextrin solution.
(2) preparation of fluorescence labeling storing solution: N-methyl-isatin acid anhydrides dmso solution is mixed with 20mg/mL solution.
(3) the fluorescence labeling storing solution prepared to be joined in maltodextrin solution 35 DEG C according to the ratio of the mass ratio 1:55 of fluorescent marker N-methyl-isatin acid anhydrides and maltodextrin and hatch 18min.
(4) separation of fluorescence labeling polysaccharide: the Arabic gum organic solvent precipitation method of N-methyl-isatin acid anhydrides mark is separated, absolute ethyl alcohol is added in the polysaccharide solution after mark, utilize the character of polysaccharide alcohol precipitation by separation of polysaccharides, fluorescence labeling polysaccharide solution: absolute ethyl alcohol volume ratio 1:6, centrifugal 10 ~ the 20min of polysaccharide 6000 × g of alcohol precipitation, the centrifugal supernatant obtained: absolute ethyl alcohol volume ratio 1:2 ~ 1:4, alcohol precipitation is centrifugal again.
(5) purifying of fluorescence labeling polysaccharide: the precipitation mixing that twice is obtained, redissolve with distilled water, use core filtration devices to remove the water-insoluble N-methyl-isatin acid anhydrides and impurity that do not react with polysaccharide, filtrate freeze-drying, can obtain fluorescence labeling maltodextrin.
embodiment 3
(1) sample preparation: dissolve with the borate buffer solution 5 ~ 10mL of 0.1mol/L, PH 8.0 and the dimethyl sulphoxide solution of w/v 50% and be mixed with 40 ~ 80mg/mL maltodextrin solution.
(2) preparation of fluorescence labeling storing solution: N-methyl-isatin acid anhydrides dmso solution is mixed with 20mg/mL solution.
(3) the fluorescence labeling storing solution prepared to be joined in maltodextrin solution 35 DEG C according to the ratio of the mass ratio 1:60 of fluorescent marker N-methyl-isatin acid anhydrides and maltodextrin and hatch 20min.
(4) separation of fluorescence labeling polysaccharide: the Arabic gum organic solvent precipitation method of N-methyl-isatin acid anhydrides mark is separated, absolute ethyl alcohol is added in the polysaccharide solution after mark, utilize the character of polysaccharide alcohol precipitation by separation of polysaccharides, fluorescence labeling polysaccharide solution: absolute ethyl alcohol volume ratio 1:8, centrifugal 10 ~ the 20min of polysaccharide 6000 × g of alcohol precipitation, the centrifugal supernatant obtained: absolute ethyl alcohol volume ratio 1:2 ~ 1:4, alcohol precipitation is centrifugal again.
(5) purifying of fluorescence labeling polysaccharide: the precipitation mixing that twice is obtained, redissolve with distilled water, use core filtration devices to remove the water-insoluble N-methyl-isatin acid anhydrides and impurity that do not react with polysaccharide, filtrate freeze-drying, can obtain fluorescence labeling maltodextrin.
(6) preparation of fluorescence labeling microcapsules: maltodextrin (maltodextrin containing part N-methyl-isatin acid anhydrides mark) is dissolved in 60 DEG C of distilled water with converted starch, add again and be dissolved with emulsifying agent grease, through high-pressure homogeneous, spraying dry, obtain fluorescence labeling microcapsules.
(7) observe: the fluorescence labeling microcapsules obtained are laid on microslide, ensure Granular composite.Use laser confocal microscope under 405nm excitation wavelength, observe the distribution situation of maltodextrin in microcapsules.
Claims (5)
1. a preparation method for N-methyl-isatin acid anhydrides Fast Labeling polysaccharide, is characterized in that:
(1) sample preparation: dissolve with the dimethyl sulphoxide solution that the borate buffer solution of 0.1mol/L, PH 8.0 and 5 ~ 10mL concentration w/v are 50% and be mixed with 40 ~ 80mg/mL polysaccharide solution;
(2) preparation of fluorescence labeling storing solution: N-methyl-isatin acid anhydrides dmso solution is mixed with 20mg/mL solution;
(3) the fluorescence labeling storing solution that step (2) obtains is joined in the polysaccharide solution that step (1) obtains and hatch;
(4) separation of fluorescence labeling polysaccharide: adopt organic solvent precipitation method, fluorescence labeling polysaccharide solution by step (3) adds absolute ethyl alcohol, utilize the character of polysaccharide alcohol precipitation by separation of polysaccharides, first time the centrifugal supernatant that obtains and absolute ethyl alcohol volume ratio be 1:2 ~ 1:4, alcohol precipitation is centrifugal again;
(5) purifying of fluorescence labeling polysaccharide: the precipitation mixing obtained twice, redissolves with distilled water, and use core filtration devices to remove the water-insoluble N-methyl-isatin acid anhydrides and impurity that do not react with polysaccharide, filtrate freeze-drying, can obtain fluorescence labeling polysaccharide.
2. the preparation method of a kind of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide according to claim 1, is characterized in that: described polysaccharide solution is gumwater or maltodextrin solution.
3. the preparation method of a kind of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide according to claim 1, is characterized in that: in step (3), the mass ratio of fluorescent marker N-methyl-isatin acid anhydrides and polysaccharide is 1:50 ~ 1:60.
4. the preparation method of a kind of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide according to claim 1, is characterized in that: the incubation condition described in step (3) is: hatch 15 ~ 20min at 35 DEG C.
5. a kind of method of N-methyl-isatin acid anhydrides Fast Labeling polysaccharide and separation purifying technique according to claim 1, it is characterized in that: in step (4), during first time centrifugal polysaccharide alcohol precipitation, fluorescence labeling polysaccharide solution and absolute ethyl alcohol volume ratio are 1:4 ~ 1:8, the centrifugal 10 ~ 20min of polysaccharide 6000 × g of alcohol precipitation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2683050A1 (en) * | 2017-03-22 | 2018-09-24 | Universidad De Murcia | New fluorescent compounds with affinity to biological molecules (Machine-translation by Google Translate, not legally binding) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101023973A (en) * | 2006-12-01 | 2007-08-29 | 广州中医药大学第二附属医院 | Extract of polysaccharide of lithospermum erthrorhizon and composition containing same and use thereof |
| CN104024849A (en) * | 2011-08-12 | 2014-09-03 | 生命科技公司 | Apparatuses, methods, computer program products, and kits for hi-throughput glycan analysis |
| WO2014184320A1 (en) * | 2013-05-15 | 2014-11-20 | Electrophoretics Limited | Mass labels |
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- 2014-12-10 CN CN201410748956.XA patent/CN104535754A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101023973A (en) * | 2006-12-01 | 2007-08-29 | 广州中医药大学第二附属医院 | Extract of polysaccharide of lithospermum erthrorhizon and composition containing same and use thereof |
| CN104024849A (en) * | 2011-08-12 | 2014-09-03 | 生命科技公司 | Apparatuses, methods, computer program products, and kits for hi-throughput glycan analysis |
| WO2014184320A1 (en) * | 2013-05-15 | 2014-11-20 | Electrophoretics Limited | Mass labels |
Non-Patent Citations (1)
| Title |
|---|
| PAUL L. DEANGELIS: "Polysaccharide Labeling with N-Methylisatoic Anhydride: Generation of Ultraviolet Chromophores and Blue Fluorophores", 《ANALYTICAL BIOCHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2683050A1 (en) * | 2017-03-22 | 2018-09-24 | Universidad De Murcia | New fluorescent compounds with affinity to biological molecules (Machine-translation by Google Translate, not legally binding) |
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Application publication date: 20150422 |