CN106645038A - Quantitative detection method of O-GlcNAc - Google Patents
Quantitative detection method of O-GlcNAc Download PDFInfo
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- CN106645038A CN106645038A CN201611226996.3A CN201611226996A CN106645038A CN 106645038 A CN106645038 A CN 106645038A CN 201611226996 A CN201611226996 A CN 201611226996A CN 106645038 A CN106645038 A CN 106645038A
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- China
- Prior art keywords
- glcnac
- wheat germ
- signal
- spr
- germ agglutinin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Abstract
The invention discloses a quantitative detection method of O-GlcNAc, comprising the following steps: detecting O-GlcNAc according to SPR signals recorded according to change of the refractive index caused by the inter combination effect of wheat germ agglutinin WGA and O-GlcNAc on a gold film; cutting O-GlcNAcase (OGA) by using the specificity of O-GlcNAc, wherein signal decrease after cutting is related to the concentration of O-GlcNAc; and according to change of signal delta R before and after enzyme digestion, realizing accurate and quantitative detection of O-GlcNAc. The quantitative detection method of O-GlcNAc has low requirements on samples which can be detected after simple treatment, is simple in operation, has a wide linear range relative to the previously reported work, and can be applied to detection of O-GlcNAc in cancer cells.
Description
Technical field
The invention belongs to medicine technology field, more particularly to a kind of O-GlcNAc quantitative detecting methods.
Background technology
The N-acetyl-glucosamine of oxygen connection（Abbreviation O-GlcNAc）It is by single N-acetyl-glucosamine（GlcNAc, monose）With
The serine/threonine of O-glycosides key and protein portion（Ser/Thr）The hydroxyl oxygen atom covalent bond of residue is formed.O-GlcNAc
Modification is a kind of protein post-translational modification of generally existing, and numerous studies prove the level and neurological of O-GlcNAc modifications
The diseases such as property disease, diabetes, cancer are closely related.But the modification glycosidic bond of O-GlcNAc is easy to fracture, in sub- chemistry
Stoichiometric level, with dynamic, protein mostly is low abundance proteinses, and this makes troubles for the foundation of detection method, particularly
Accurate quantitative determination.At present the method for detection O-GlcNAc is mostly used for quantitative and semi-quantitative analysis, exist end other
The interference of GlcNAc sugar, low sensitivity, sample early-stage preparations complexity, difficult expensive, data processing, needs mark and enrichment etc. are asked
Topic.Therefore, in the urgent need to setting up the method that sensitive, label-free and enrichment accurate quantitative analysis detect O-GlcNAc, glycosuria is contributed to
Disease, cancer etc. seriously threaten the early diagnosis of the disease of human health.
Surface plasma body resonant vibration instrument（SPR）Due to determinand it is label-free, sensitivity is high, real-time monitoring reaction, the back of the body
The advantages of scape interference is little is widely used in the fields such as physics, chemistry, biology and life science.But SPR instrument be difficult to it is less
The detection of variations in refractive index, which greatly limits its practical application.In order to solve this problem, many amplifying techniques
It is introduced in the research of surface plasmon resonance biosensor to improve the sensitivity of its detection.
The content of the invention
It is an object of the invention to provide a kind of O-GlcNAc quantitative detecting methods, it is intended to solve the side of detection O-GlcNAc
Method is mostly used for quantitative and semi-quantitative analysis, and interference, the sensitivity that there is other GlcNAc of end sugar is low, sample early-stage preparations are answered
Miscellaneous, expensive, data processing is difficult, need the problems such as mark and enrichment.
The present invention is achieved in that a kind of O-GlcNAc quantitative detecting methods, the O-GlcNAc quantitative detecting methods
Including：The O-GlcNAc of variable concentrations is fixed in golden film, the wheat germ agglutinin of flow injection nano gold mark is solidifying according to wheat germ
Collection element WGA and O-GlcNAc be combined with each other to act in golden film and cause refractive index to change to detect the spr signal of record
O-GlcNAc, the molecular mass that the change of refractive index is combined with golden film surface is directly proportional, and variations in refractive index is bigger, the signal of SPR
Change is bigger；
The O-GlcNAc of variable concentrations is fixed in golden film, using the selectivity shearing enzyme N-Acetyl-D-glucosamine sugar of O-GlcNAc
Glycosides enzyme（OGA）The digestion O-GlcNAc 16h under the conditions of 37 DEG C, then the wheat germ agglutinin WGA of flow injection nano gold mark, cut
The decline for cutting rear spr signal is related to the concentration of O-GlcNAc；
The signal △ R of the SPR obtained after ferment treatment are deducted according to the signal of the SPR obtained before ferment treatment（Namely believe after digestion
Number change）, realize that the accurate quantitative analysis to O-GlcNAc are detected.
Further, the change that the interaction in golden film between biomolecule causes is biological by the SPR that nm of gold is amplified
Sensor is detected.
Further, it is described that folding is caused according to wheat germ agglutinin WGA and O-GlcNAc effects of be combineding with each other in golden film
Penetrate rate change record spr signal to detect O-GlcNAc in, wheat germ agglutinin WGA need to be carried out nano gold mark realize spirit
Quick detection.
Another object of the present invention is to provide it is a kind of using above-mentioned O-GlcNAc quantitative detecting methods based on wheat germ
The surface plasma body resonant vibration instrument of the O-GlcNAc of agglutinin recognition reaction.
Present invention utilizes wheat germ agglutinin（WGA）O-GlcNAc specific recognitions are acted on, a kind of nm of gold is devised
Amplify surface plasmon resonance biosensor, in order to avoid end others GlcNAc disturbs the Accurate Determining of O-GlcNAc, present invention employs
The selectivity shearing enzyme N-Acetyl-D-glucosamine glycosidase of O-GlcNAc（OGA）, the decline of signal is with O-GlcNAc's after shearing
Concentration is related.According to the change △ R of signal before and after digestion, the quantitative detecting method of O-GlcNAc is established, realized to several
The detection of the content of O-GlcNAc in complex sample cancer cell.
The present invention is based on the quantitative detecting method of the O-GlcNAc of wheat germ agglutinin recognition reaction to the less demanding of sample,
Jing simple process just can be detected, simple to operate.
Nm of gold amplifying technique is present invention employs, the sensitivity and detection range for making detection method improves ten times of left sides
The right side, relatively before the work of report has the wider range of linearity.
Present invention employs enzyme incision technology, it is to avoid end others GlcNAc interference, being capable of accurate quantitative determination cancer
The content of intracellular O-GlcNAc.
Description of the drawings
Fig. 1 is O-GlcNAc quantitative detecting methods flow chart provided in an embodiment of the present invention.
Fig. 2 is the glycosylated reference polypeptides of O-GlcNAc of fixed variable concentrations provided in an embodiment of the present invention, flowing note
Penetrate the signal graph of the SPR of WGA/AuNPs.
Fig. 3 is the glycosylated reference polypeptides of O-GlcNAc of fixed variable concentrations provided in an embodiment of the present invention, after digestion,
The spr signal figure of flow injection WGA/AuNP again.
Fig. 4 is working curve diagrams of the O-GlcNAc provided in an embodiment of the present invention in the range of 0.0465nM-465nM.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to only to explain the present invention
Limit the present invention.
The two big advantages of the present invention:Nm of gold is amplified（It is sensitive）And digestion（Accurately）.The present invention is special using O-GlcNAc's
One property shears enzyme N-Acetyl-D-glucosamine glycosidase（OGA）, according to the change △ R of signal before and after digestion, realize to O-GlcNAc
Accurate quantitative analysis detection.
The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.
As shown in figure 1, O-GlcNAc quantitative detecting methods provided in an embodiment of the present invention, including：
S101:The change of refractive index is caused according to the wheat germ agglutinin WGA and O-GlcNAc effect of be combineding with each other in golden film
The spr signal of record is detecting O-GlcNAc；
S102：Enzyme N-Acetyl-D-glucosamine glycosidase is sheared using the selectivity of O-GlcNAc（OGA）, the decline of signal after shearing
It is related to the concentration of O-GlcNAc；
S103：According to the change △ R of signal before and after digestion, realize that the accurate quantitative analysis to O-GlcNAc are detected.
Further, golden film detection signal amplifies surface plasmon resonance biosensor and is detected by nm of gold.
Further, it is described that folding is caused according to wheat germ agglutinin WGA and O-GlcNAc effects of be combineding with each other in golden film
Penetrate rate change record spr signal to detect O-GlcNAc in, nano gold mark need to be carried out to wheat germ agglutinin WGA.
Below in conjunction with the accompanying drawings and specific embodiment to the present invention application principle be further described.
Embodiment 1：
First mercaptan acid is modified in golden film by gold and the interaction of sulfydryl, then by amino carboxyl cross-linking reaction by difference
The glycosylated reference polypeptides of O-GlcNAc of concentration are modified in golden film, in the WGA of flow injection nano gold mark, are utilized
Navi-200SPR instrument, the effect of be combineding with each other between on-line real time monitoring WGA and O-GlcNAc.
Embodiment 2：
Because WGA can not only recognize O-GlcNAc, additionally it is possible to other glycan that end is GlcNAc are recognized, in order to avoid end
The interference of GlcNAc, present invention employs enzyme incision technology.Modify mercaptan acid in golden film first, then it is anti-by the crosslinking of amino carboxyl
The glycosylated reference polypeptides of O-GlcNAc of variable concentrations, then the digestion 16h under the conditions of 37 DEG C should be modified, then flow injection is received
The WGA of rice gold mark, using Navi-200SPR instrument tracer signals, according to the change △ R and O-GlcNAc of signal before and after digestion
Concentration establishes the working curve of detection O-GlcNAc.Corresponding spr signal is with working curve as shown in 2 ~ accompanying drawing of accompanying drawing 4.
Fig. 2 is the glycosylated reference polypeptides of O-GlcNAc of fixed variable concentrations provided in an embodiment of the present invention, flowing note
Penetrate the signal graph of the SPR of WGA/AuNPs.
Fig. 3 is the glycosylated reference polypeptides of O-GlcNAc of fixed variable concentrations provided in an embodiment of the present invention, after digestion,
The spr signal figure of flow injection WGA/AuNP again.
Fig. 4 is working curve diagrams of the O-GlcNAc provided in an embodiment of the present invention in the range of 0.0465nM-465nM.
The present invention is based on the quantitative detecting method of the O-GlcNAc of wheat germ agglutinin recognition reaction to the less demanding of sample,
Jing simple process just can be detected, simple to operate.
Nm of gold amplifying technique is present invention employs, the sensitivity and detection range for making detection method improves ten times of left sides
The right side, relatively before the work of report has the wider range of linearity.
Present invention employs enzyme incision technology, it is to avoid end others GlcNAc interference, being capable of accurate quantitative determination cancer
The content of intracellular O-GlcNAc.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (4)
1. a kind of O-GlcNAc quantitative detecting methods, it is characterised in that the O-GlcNAc quantitative detecting methods include：In golden film
The O-GlcNAc of upper fixed variable concentrations, the wheat germ agglutinin of flow injection nano gold mark, according to wheat germ agglutinin WGA and O-
The GlcNAc effects of be combineding with each other in golden film cause the change of refractive index to detect the spr signal O-GlcNAc of record, folding
Penetrate the molecular mass that the change of rate combined with golden film surface to be directly proportional, variations in refractive index is bigger, and the signal intensity of SPR is bigger；
The O-GlcNAc of variable concentrations is fixed in golden film, using the selectivity shearing enzyme N-Acetyl-D-glucosamine sugar of O-GlcNAc
Glycosides enzyme（OGA）The digestion O-GlcNAc 16h under the conditions of 37 DEG C, then the wheat germ agglutinin WGA of flow injection nano gold mark, cut
The decline for cutting rear spr signal is related to the concentration of O-GlcNAc；
The signal △ R of the SPR obtained after ferment treatment are deducted according to the signal of the SPR obtained before ferment treatment, is realized to O-GlcNAc
Accurate quantitative analysis detection.
2. O-GlcNAc quantitative detecting methods as claimed in claim 1, it is characterised in that the phase in golden film between biomolecule
The change that interaction causes is detected by the surface plasmon resonance biosensor that nm of gold is amplified.
3. O-GlcNAc quantitative detecting methods as claimed in claim 1, it is characterised in that described according to wheat germ agglutinin WGA
O-GlcNAc is detected with the spr signal of the change record that the O-GlcNAc effects of be combineding with each other in golden film cause refractive index
In, nano gold mark need to be carried out to wheat germ agglutinin WGA and realize Sensitive Detection.
4. the O- based on wheat germ agglutinin recognition reaction of the O-GlcNAc quantitative detecting methods described in a kind of utilization claim 1
The surface plasma body resonant vibration instrument of GlcNAc.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611226996.3A CN106645038A (en) | 2016-12-27 | 2016-12-27 | Quantitative detection method of O-GlcNAc |
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| CN201611226996.3A CN106645038A (en) | 2016-12-27 | 2016-12-27 | Quantitative detection method of O-GlcNAc |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108956540A (en) * | 2018-05-01 | 2018-12-07 | 中国科学院大学 | A kind of SPR method of quick screening charge reversal type cationic gene carriers |
| CN111443007A (en) * | 2020-04-13 | 2020-07-24 | 厦门大学附属厦门眼科中心有限公司 | Detection method for measuring concentration of hyaluronidase based on flow velocity of hydrogel composite membrane |
| CN116794287A (en) * | 2023-06-19 | 2023-09-22 | 中国科学院大学 | Fecal occult blood sensor with high scale resistance and high accuracy and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040005582A1 (en) * | 2000-08-10 | 2004-01-08 | Nanobiodynamics, Incorporated | Biospecific desorption microflow systems and methods for studying biospecific interactions and their modulators |
| CN101487794A (en) * | 2008-01-18 | 2009-07-22 | 周礼君 | Biosensing apparatus and system |
| CN105181658A (en) * | 2014-05-27 | 2015-12-23 | 中央研究院 | Sensing device, and sensing system and sensing method using the same |
-
2016
- 2016-12-27 CN CN201611226996.3A patent/CN106645038A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040005582A1 (en) * | 2000-08-10 | 2004-01-08 | Nanobiodynamics, Incorporated | Biospecific desorption microflow systems and methods for studying biospecific interactions and their modulators |
| CN101487794A (en) * | 2008-01-18 | 2009-07-22 | 周礼君 | Biosensing apparatus and system |
| CN105181658A (en) * | 2014-05-27 | 2015-12-23 | 中央研究院 | Sensing device, and sensing system and sensing method using the same |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108956540A (en) * | 2018-05-01 | 2018-12-07 | 中国科学院大学 | A kind of SPR method of quick screening charge reversal type cationic gene carriers |
| CN111443007A (en) * | 2020-04-13 | 2020-07-24 | 厦门大学附属厦门眼科中心有限公司 | Detection method for measuring concentration of hyaluronidase based on flow velocity of hydrogel composite membrane |
| CN116794287A (en) * | 2023-06-19 | 2023-09-22 | 中国科学院大学 | Fecal occult blood sensor with high scale resistance and high accuracy and preparation method and application thereof |
| CN116794287B (en) * | 2023-06-19 | 2024-01-23 | 中国科学院大学 | Fecal occult blood sensor with high scale resistance and high accuracy and preparation method and application thereof |
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