Lectin simulant preparation method and application based on molecular imprinting technique

A technology of molecular imprinting and lectin, which is applied in the field of molecularly imprinted functional materials, and achieves wide application prospects, low cost, and good recognition ability

Active Publication Date: 2015-08-05
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many literatures have reported molecularly imprinted materials using monosaccharides as templates [G.Wulff, R.Grobe-Einsler, W.Vesper, A.Sarhan, Makromol.Chem. "Macromolecular Chemistry" 1977,178,2817- 2825; Z.L.Cheng, E.K.Wang, X.R.Yang, Biosens.Bioelectron. "Biosensing and Bioelectronics" 2001, 16, 179-185 W.J.Wizeman, P. Kofinas, Biomaterials "Biomaterials" 2001, 22, 1485-1491] , however, molecularly imprinted materials with lectin molecular recognition properties, that is, the ability to specifically recognize both the complete glycoprotein and the characteristic fragments of the glycoprotein have never been reported.
In addition, molecularly imprinted polymers using sugar chains as templates have not yet been reported

Method used

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  • Lectin simulant preparation method and application based on molecular imprinting technique
  • Lectin simulant preparation method and application based on molecular imprinting technique
  • Lectin simulant preparation method and application based on molecular imprinting technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Obtaining of sugar chains (imprinted template molecules)

[0036] Firstly, the intact glycoprotein was dissolved in 10mM ammonium bicarbonate buffer solution (pH 7.5) to prepare a 1mg / mL mixed solution. Take 100 μL of the above mixed solution and immerse it in a boiling water bath for 10 minutes, take it out and cool it down to 25°C naturally, then add 10 units of peptide-N-glycosidase F and react in a water bath at 37°C for 24 hours. After the reaction, sugar chains were separated from the reaction system by centrifugation at a speed of 14,000 rpm for 30 minutes with an ultrafiltration tube with a molecular weight cutoff of 3,000 Daltons. The obtained sugar chains were stored at -20°C until use.

[0037] The above-mentioned intact glycoprotein is ribonuclease B, or transferrin; or other glycoproteins.

[0038] The above-mentioned peptide-N-glycosidase F is an enzyme used to enzymatically cut off the sugar chain on the complete glycoprotein.

Embodiment 2

[0039] Example 2: Preparation of 2,4-difluoro-3-formyl-phenylboronic acid modified magnetic nanoparticles

[0040] First prepare an amino-modified magnetic nanoparticle [for the preparation method, see but not limited to the following documents L.Wang, J.Bao, L.Wang, F.Zhang, Y.Li, Chem.Eur.J. "European Chemistry" 2006 , 12, 6341-6347]. 200 mg of amino-modified magnetic nanoparticles were added to 40 mL of anhydrous methanol, followed by 400 mg of 2,4-difluoro-3-formyl-phenylboronic acid and 400 mg of sodium cyanoborohydride. The mixture was stirred and reacted at 25°C for 24 hours. The obtained product was washed three times with water and ethanol respectively, and dried under vacuum at 50° C. for 12 hours. That is, magnetic nanoparticles modified with 2,4-difluoro-3-formyl-phenylboronic acid are obtained.

Embodiment 3

[0041] Embodiment 3: the investigation of imprinted coating thickness change relation with time

[0042] Silver nanoparticles with a particle size similar to that of the magnetic nanoparticles were added to a mixed solution of 40 mL of absolute ethanol and 0.7 mL of concentrated ammonia water (37%). Finally, 10 mL of 10 mM tetraethyl orthosilicate in ethanol was added. Stir at 25°C, from 10 minutes to 60 minutes after stirring, samples are taken every 5 minutes, and the thickness of the imprinted coating on the surface is characterized by a transmission electron microscope. The result is as figure 2 . The linear correlation coefficient r in the figure 2 =0.99, the slope s=0.04nm / min, which shows that the thickness of the polymer has a good linear correlation with the time growth, which provides a guarantee for the controllability of the technology.

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Abstract

The invention discloses a lectin simulant preparation method based on a molecular imprinting technique. The preparation method includes the steps: firstly, acquiring a carbohydrate chain on complete glycoprotein as a template molecule by enzyme digestion reaction; secondly, fixing the template molecule on the surface of a boric acid functional substrate material by the aid of a boric affinity function; thirdly, performing condensation reaction by the aid of silanization reagents to form an imprinting layer; finally, removing a template under the acid conditions to form an imprinting cavity to obtain a lectin simulant based on molecular imprinting. The lectin simulant is simple to prepare, low in cost, stable in nature, excellent in specificity and affinity and high in anti-jamming capacity, a recognized target object is more easily released, integrated glycoprotein can be recognized, characteristic fragments such as glycopeptides and glycan of the glycoprotein can be recognized, and the lectin simulant still can keep single-minded recognized capacity in used for complex biological samples and has an excellent application prospect in aspects such as glycoproteomics, metabonomics, glycobiology, disease diagnosis and analeptic inspection.

Description

technical field [0001] The invention belongs to the field of molecularly imprinted functionalized materials, and specifically relates to the preparation of a lectin mimic based on molecularly imprinted technology and its specific recognition and separation of glycoproteins and their characteristic fragments in complex samples, as well as their use in Applications in proteomics, metabolomics, glycobiology, disease diagnosis, and doping detection. Background technique [0002] Antibodies are a class of protein molecules that can specifically recognize specific molecules (antigens), and have important applications in the fields of biochemistry, biomedicine, and clinical diagnosis. Antibodies usually only recognize the complete antigen molecule, and when the antigen molecule is enzymatically hydrolyzed or degraded, the antibody usually cannot recognize the fragments produced after the fragmentation of the antigen. Therefore, the specific recognition of itself and its characteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07K1/14
CPCY02P20/582G01N33/6848C07K1/14G01N2333/4724
Inventor 刘震别子俊陈阳
Owner NANJING UNIV
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