CN110215738B - Preparation method of graphene polymer hydrophilic pretreatment material - Google Patents
Preparation method of graphene polymer hydrophilic pretreatment material Download PDFInfo
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- CN110215738B CN110215738B CN201910637334.2A CN201910637334A CN110215738B CN 110215738 B CN110215738 B CN 110215738B CN 201910637334 A CN201910637334 A CN 201910637334A CN 110215738 B CN110215738 B CN 110215738B
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a preparation method of a graphene polymer hydrophilic pretreatment material. The method comprises the following specific steps: (1) suspending a functional monomer 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT) and a graphene material (GO/CS) for modifying chitosan in a solvent, and oscillating for 10-70h at room temperature. (2) And after the reaction is finished, centrifuging the obtained material for 5min, washing the material by using 5% hydrochloric acid, and drying the washed material in vacuum to obtain the hydrophilic pretreatment material for the graphene polymer.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a preparation method of a graphene polymer hydrophilic pretreatment material.
Background
The post-translational modifications of proteins are complex and diverse, and are an important part of proteomics, and the commonly used classes are Phosphorylation (Phosphorylation), Methylation (Methylation), Acetylation (Acetylation), Ubiquitination (Ubiquitination), Glycosylation (Glycosylation), etc. [1 ]. The glycosylation of protein is one of the most important and most common post-translational modifications of protein, more than half of the protein in human body has glycosylation modification [2], the modified glycoprotein is widely distributed in various tissues and cells and participates in a plurality of physiological processes such as regulation of cell growth, recognition, adhesion, migration, protein folding, transportation and the like [3-4], and the occurrence and development of a plurality of major diseases of human are closely related to the glycosylation abnormity of protein. Therefore, glycoproteomics research is also important for the discovery of diagnostic markers and therapeutic targets of diseases while providing important information for the study of biological mechanisms [5 ]. However, glycoproteins have microheterogeneity [6], and most glycoproteins are low-abundance proteins that are easily masked by other non-glycoproteins during mass spectrometric detection. Therefore, the efficient and selective separation and enrichment of glycoproteins with different glycoform structures is the key point for realizing high-sensitivity glycoprotein identification. Various methods such as lectin affinity method, hydrazine chemistry method, hydrophilic interaction method, boric acid affinity method, size exclusion method and the like have been developed for separation and enrichment of glycoproteins.
Hydrophilic interaction chromatography (HILIC) is a chromatography mode using a polar stationary phase (e.g. silica gel, derivatized silica gel) and an organic solvent (e.g. acetonitrile) -water as the mobile phase, and has a mobile phase similar to that of reverse phase liquid chromatography, and the elution order of the compounds is similar to that of normal phase chromatography. HILIC can effectively retain incompletely or unretained strong polar compounds in reversed phase chromatography, has good separation effect, and is a reliable means for solving the problem of separating various strong polar compounds from hydrophilic compounds. The method uses a relatively simple mobile phase system, has mild separation conditions, wide application range and less sample loss, and can enrich glycopeptides with various structures, so that the method is rapidly developed in the fields of pharmaceutical analysis, food science and the like [7 ].
Graphene (Graphene) material is a novel carbon nano material with a single atomic layer thickness and sp2 hybridization, and the unique physicochemical properties of the Graphene (Graphene) material attract the attention of scientists in various fields and show huge application potential [8-10 ]. The derivative Graphene Oxide (GO) is prepared by adding a strong oxidant and strong acid graphite oxide [11], a large number of oxygen-containing groups improve the chemical stability and the hydrophilicity of GO, reaction sites [12-14] are provided for preparing a Graphene oxide matrix composite material, and the Graphene oxide matrix composite material is a carbon material which has good biocompatibility, large specific surface area, wide pH tolerance range and easily modified surface, and is widely applied to the aspects of analysis and detection, drug delivery, bioimaging, biosensors, diagnosis and treatment of cancers and the like.
Chitosan (Chitosan, Cs), also known as Chitosan, is a cheap and easily available basic polysaccharide, and is ubiquitous in nature. Chitosan has good biocompatibility, low toxicity, enzymatic degradability, etc., which are attributed to the presence of a large number of amino groups in its molecule [15 ]. Meanwhile, the macromolecular chain of the chitosan contains a large amount of amino groups, so that the chitosan has higher polarity and can be used for preparing hydrophilic separation materials.
The 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT) has a unique triazole ring structure, has certain alkalinity, is easily protonated under acidic conditions to carry positive charges, and shows electrostatic attraction or repulsion. AHMT is a zwitterion, with both positive and negative charge centers, and has good hydrophilicity [16 ].
The technology adopts a rapid, simple and cheap method, takes the graphene oxide as a substrate to modify, and provides a novel graphene polymer hydrophilic pretreatment material. The material has novel structure, definite acting force and stronger application prospect.
Reference to the literature
[1] Zhangfeng, Wangxinghua, proteomics research and its revelation on the development of traditional Chinese medicine [ J ]. Proc. Guangzhou university of traditional Chinese medicine, 2004,21(6): 486-.
[2]Zhang Y,Jiao J,Yang P,et al.Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery[J].Clinical Proteomics,11,1(2014-05-05),2014,11(1):18.
[3]Helenius A,Aebi M.Intracellular functions of N-linked glycans.[J].Science,2001,291(5512):2364-9.
[4]Prabhjit K Grewal,Satoshi Uchiyama,David Ditto,et al.The Ashwell receptor mitigates the lethal coagulopathy of sepsis[J].Naturemedicine,2008,14(6):648-655.
[5] Chimonanthus praecox, Van super, Huangjunjie, et al, preparation of graphene oxide immobilized lectin and application in glycoprotein/glycopeptide enrichment [ J ]. chromatography, 2015,33(2): 116-.
[6] Royal and Li, glycopeptide enrichment and identification new technology based on functionalized nano materials and biological mass spectrometry, new method [ D ]. Compound denier university, 2014.
[7] The development of Jinxiaoqing, Yaohixin, Xue Kunpeng, hydrophilic interaction chromatography outlines [ J ] chemical engineering management, 2014(27):70-71.
[8]Georgakilas V,Otyepka M,Bourlinos A B,et al.Functionalization of graphene:covalent and non-covalent approaches,derivatives and applications.[J].Chemical Reviews,2012,112(11):6156-214.
[9]Hong Y M,Laurent S,Wei C,et al.Graphene:Promises,Facts,Opportunities,and Challenges in Nanomedicine[J].Chemical Reviews,2013,113(5):3407.
[10]Allen M J,Tung V C,Kaner R B.Honeycomb carbon:a review of graphene[J].Chemical Reviews,2010,110(1):132.
[11]Dikin D A,Stankovich S,Zimney E J,et al.Preparation and characterization of graphene oxidepaper[J].Nature,2007,448(7152):457.
[12]Zhang LN,Deng H H,Lin F L,et al.In situ growth of porous platinum nanoparticles on graphene oxide for colorimetric detection of cancer cells[J].Analytical Chemistry,2014,86(5):2711-8.
[13]Qin Y,Li J,KongY,et al.In situ growth of Au nanocrystals on graphene oxide sheets.[J].Nanoscale,2014,6(3):1281-5.
[14]Kuo T R,Wang D Y,ChiuY C,et al.Layer-by-layer thin film of reduced graphene oxide and gold nanoparticles as an effective sample plate in laser-induced desorption/ionization mass spectrometry.[J].Analytica Chimica Acta,2014,809:97-103.
[15] Preparation and application research of water-soluble chitosan derivatives from Wangzhong [ D ]. Qingdao university of science and technology, 2010.
[16] Guo Shi, Zhang Xiuli, Xuqing, Lixin, 2815640 hydrophilic interaction chromatography stationary phase and its application in traditional Chinese medicine separation [ J ]. chromatography, 2009,27(05): 675) 681.
Disclosure of Invention
The invention aims to provide a preparation method of a graphene polymer hydrophilic pretreatment material, which comprises the following specific steps:
(1) suspending a functional monomer 4-amino-3-hydrazino-5-sulfydryl-1, 2, 4-triazole and a graphene material for modifying chitosan in a solvent, and oscillating for 10-70h at room temperature;
(2) and after the reaction is finished, centrifuging the obtained material for 5min, washing with 5% hydrochloric acid, and drying the product after washing to obtain the hydrophilic pretreatment material for the graphene polymer.
In one embodiment of the invention, the material in step 1 is a graphene material modified with chitosan, which is self-made in a laboratory, and the preparation and characterization methods are all referred to in the literature (analytical Chimica Acta 850 (2014)) 49-56, and some modifications are made on the basis; the solvent is 5% hydrochloric acid solution.
In another embodiment of the present invention, the weight ratio of 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole to the graphene material and solvent of the modified chitosan is 1: 0.01-0.5: 20 to 200 parts. Preferably 1:0.05: 100.
in yet another embodiment of the present invention, the parameter for centrifugation in step (2) is 16000r/min, 5 min.
Detailed Description
The invention will be further illustrated below. It should be noted that the following description is only an illustration of the claimed technical solutions, and does not limit these technical solutions in any way. The scope of the present invention is defined by the appended claims.
Example 1
(1) 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT) (100mg) was weighed and dissolved in 5% hydrochloric acid (10mL), and then chitosan-modified graphene material (GO/Cs) (5mg) was added thereto and shaken at room temperature for 24 h.
(2) And after the reaction is finished, centrifuging at 16000rpm for 5min, washing with 5% hydrochloric acid to remove excessive reactants, and drying in vacuum for later use to obtain the material, namely the hydrophilic pretreatment material of the graphene polymer.
Example 2
A selective test for enriching glycoprotein was performed using a protein mixture solution containing horseradish peroxidase (HRP) and Bovine Serum Albumin (BSA) at a molar ratio of 1:10 (equivalent to HRP 5ug and BSA 75ug) as an object of examination. The graphene polymer hydrophilic pretreatment material (1.5mg) powder and 200 μ L of 80% ACN were added separately and vortexed for 30 s. 16000rpm for 5min, and discarding the supernatant. All three tubes were vortexed for 30s and incubated on a shaker for 1h, centrifuged at 16000rpm for 5min, and the supernatant was discarded. Wash again with 200. mu.L of 85% ACN/1% TFA. Vortex for 5min, incubate on shaker for 1h, centrifuge at 16000rpm for 5min, and discard the supernatant. And respectively adding 200 mu L of 70% ACN/1% FA into the three tubes, vortexing for 5min, incubating for 1h in a shaking table, ultrasonically oscillating for 5min, centrifuging for 5min at 16000rpm, reserving supernatant, respectively drying in a freeze dryer for later use, and characterizing by 12% SDS-PAGE gel electrophoresis.
In addition, the invention also sets the following comparative examples:
comparative example 1: the solvent was 20% hydrochloric acid, otherwise the same as in example 1;
comparative example 2: the weight ratio of the 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole to the graphene material of the modified chitosan to the solvent is 1:10:100, and the rest is the same as in example 1;
comparative example 3: after the reaction, the excess raw material was washed by centrifugation at 5000r/min for 5min, which was otherwise the same as in example 1.
The resulting analytical bands on the gel electrophoresis were subjected to a gray scale analysis.
Specific results are shown in table 1, and the results show that the HRP enrichment selectivity obtained by the material preparation conditions of example 1 is high.
TABLE 1 HRP Gray level analysis results
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (7)
1. A preparation method of a graphene polymer hydrophilic pretreatment material comprises the following specific steps:
(1) suspending a functional monomer 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT) and a graphene material (GO/CS) for modifying chitosan in a solvent, and oscillating for 10-70h at room temperature;
(2) and after the reaction is finished, centrifuging the obtained material for 5min, washing the material with 5% hydrochloric acid, and drying the washed material in vacuum to obtain the graphene polymer hydrophilic pretreatment material.
2. The preparation method of the hydrophilic pretreatment material for graphene polymers according to claim 1, wherein the solvent in the step (1) is hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 1% -20%.
3. The method for preparing the hydrophilic pretreatment material of the graphene polymer according to claim 2, wherein the solvent in the step (1) is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 5%.
4. The preparation method of the graphene polymer hydrophilic pretreatment material according to claim 1, wherein the weight ratio of the functional monomer 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole to the graphene material of the modified chitosan to the solvent in step (1) is 1: 0.01-0.5: 20-200.
5. The method for preparing the graphene polymer hydrophilic pretreatment material according to claim 4, wherein the weight ratio of the 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole to the graphene material of the modified chitosan to the solvent in step (1) is 1:0.05: 100.
6. The method for preparing the hydrophilic pretreatment material of the graphene polymer according to claim 1, wherein the reaction temperature is room temperature and the reaction time is 24 hours.
7. The method for preparing the hydrophilic pretreatment material of the graphene polymer according to claim 1, wherein the rotation speed of the centrifugation in the step (2) is 16000 r/min.
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| CN104148037A (en) * | 2014-08-08 | 2014-11-19 | 华东理工大学 | Arginine bonded type hydrophilic chromatography stationary phase and preparation method thereof |
| CN105310998A (en) * | 2015-11-03 | 2016-02-10 | 吉林大学 | Microcapsule containing functionalized graphene in capsule wall and preparation method of microcapsule |
| WO2016045032A1 (en) * | 2014-09-25 | 2016-03-31 | 深圳粤网节能技术服务有限公司 | Solid phase extraction column, preparation method therefor, and chemical sample pre-processing method based on solid phase extraction column |
| CN105669827A (en) * | 2014-11-20 | 2016-06-15 | 中国科学院高能物理研究所 | Application of oxidized graphene as protein adsorption medium material and protein separation method |
| CN108927116A (en) * | 2018-07-20 | 2018-12-04 | 大连医科大学 | A kind of preparation method for the stannic oxide/graphene nano material that phenyl boric acid functionalization is chitosan-modified |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104148037A (en) * | 2014-08-08 | 2014-11-19 | 华东理工大学 | Arginine bonded type hydrophilic chromatography stationary phase and preparation method thereof |
| WO2016045032A1 (en) * | 2014-09-25 | 2016-03-31 | 深圳粤网节能技术服务有限公司 | Solid phase extraction column, preparation method therefor, and chemical sample pre-processing method based on solid phase extraction column |
| CN105669827A (en) * | 2014-11-20 | 2016-06-15 | 中国科学院高能物理研究所 | Application of oxidized graphene as protein adsorption medium material and protein separation method |
| CN105310998A (en) * | 2015-11-03 | 2016-02-10 | 吉林大学 | Microcapsule containing functionalized graphene in capsule wall and preparation method of microcapsule |
| CN108927116A (en) * | 2018-07-20 | 2018-12-04 | 大连医科大学 | A kind of preparation method for the stannic oxide/graphene nano material that phenyl boric acid functionalization is chitosan-modified |
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