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Bilayer lipid membrane surface modified protein chip and its manufacturing method and use

A protein chip and surface modification technology, applied in the field of protein chips, can solve the problems of non-specific adsorption, reduced biological activity, poor stability, etc., and achieve the effect of maintaining biological activity, high protein selectivity, and good stability

Active Publication Date: 2007-09-19
INST OF MECHANICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of non-specific adsorption, poor stability, and reduced biological activity in the method of immobilizing ligand molecules in the existing protein chip technology, thereby providing a method that can maintain the biological activity of ligand molecules. A protein chip that is stably immobilized on the surface of the substrate and can effectively inhibit non-specific adsorption of the double lipid membrane surface

Method used

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  • Bilayer lipid membrane surface modified protein chip and its manufacturing method and use
  • Bilayer lipid membrane surface modified protein chip and its manufacturing method and use
  • Bilayer lipid membrane surface modified protein chip and its manufacturing method and use

Examples

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Effect test

Embodiment 1

[0027] Example 1. Preparation of PEGylated phospholipids—p-nitrophenyl ester group-polyethylene glycol-(1,2-bisoleoyl-3-glycerol phosphatidylethanolamine)

[0028] According to the reference: p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specifc ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups, synthesize pNP-PEG-DOPE

[0029] 1) Add 80 μl triethylamine and 5ml polyethylene glycol-bis-p-nitrophenyl ester solution to 480 μl 1,2-bisoleoyl-3-glycerophosphatidylethanolamine solution, and protect the mixed solution with argon Stand overnight at room temperature;

[0030] The 1,2-bisoleoyl-3-glycerophosphatidylethanolamine solution is configured by adding 50 mg of 1,2-bisoleoyl-3-glycerophosphatidylethanolamine per 1 ml of chloroform;

[0031] The polyethylene glycol-bis-p-nitrophenyl ester solution is prepared by adding 1g of polyethylene glycol-bis-p-nitrophenyl ester to every 5ml of chloroform;

[00...

Embodiment 2

[0033] Embodiment 2, preparation double lipid film surface modification protein chip of the present invention

[0034] 1) Cut the silicon substrate into 10×20mm2 Small pieces, put in 30wt% H 2 o 2 Soak in a mixed solution of concentrated sulfuric acid (1:3 by volume) for 30 minutes to hydroxylate the substrate, and wash it with water and ethanol in turn; after drying the hydroxylated substrate, put 5wt% 3-aminopropyl-3 ethyl In a mixed solution of oxysilane and 95wt% absolute ethanol, react at room temperature for 2 hours to form a monomolecular self-assembled film with amino groups at the end, wash with ethanol and water three times in turn; finally, use 2.5wt% pentadiene Soak in PBS solution of aldehyde for 1 hour, wash with PBS buffer solution, obtain the substrate with active aldehyde group formed on the surface;

[0035] 2) In the microfluidic system for preparing biochips disclosed in Chinese patent 03102659.1, step 1) the substrate with active aldehyde groups formed o...

Embodiment 3

[0040] Embodiment 3, the application of the double lipid membrane surface modified protein chip of the present invention as an ellipsometric imaging biosensor,

[0041] In the microfluidic system for preparing biochips disclosed in Chinese Patent 03102659.1, human blood immunoglobulin G (IgG) was immobilized on the phospholipid double membrane layer prepared in Example 2 according to the following method and then mixed with human blood Immunoglobulin antibody (anti-IgG) response:

[0042] The IgG (0.1 mg / ml), anti-IgG (10 wt%) and bovine serum albumin (BSA, 10 mg / ml) solutions used were all prepared from PBS buffer.

[0043] 1) The IgG solution is transported to the surface of the protein chip successfully modified through the phospholipid double membrane layer in Example 2 by microfluidic microtubes. After 30 minutes, the amino group of the IgG molecule and the pNP- on the double lipid membrane The active groups at the ends of PEG-DOPE molecules are covalently and stably bon...

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Abstract

The present invention relates to a bilayer lipids membrane surface modified protein chip, a silicon substrate being a solid substrate on which being, in turn, a L-alpha-phosphatidylethanolamine monomolecular layer linked to the silicon substrate through covalent bonds and a p-nitrophenyl ester-polyethylene glycol-(1,2-dioleoyl-3-glycerophosphatide ethanolamine) layer associated with the phosphatidylethanolamine monomolecular layer by a hydrophobic effect. The protein chip is obtained by fixing the L-alpha-phosphatidylethanolamine monomolecular layer at the surface of the silicon substrate through covalent bonds in a micro- runner system and then adsorbing the p-nitrophenyl ester-polyethylene glycol-(1,2-dioleoyl-3-glycerophosphatide ethanolamine) layer. Said protein chip is capable of applied in an imaging ellipsometry biosensor to explore target molecules capable of associating specifically therewith by covalently fixing ligands molecules at terminal tetrafunctional radicals of the protein chip surface PEG, wherein the surface morphology changes of the protein chip are capable of being observed in said imaging ellipsometry system, after both are associated. The bilayer lipids membrane surface modified protein chip in accordance with the present invention is capable of avoiding effectively a nonspecific adsorption, with its ligands molecules having excellent stability and being kept high biological activities.

Description

technical field [0001] The invention relates to a protein chip modified on the surface of a double lipid film, a preparation method thereof, and the application of the protein chip in ellipsometric imaging biosensors. Background technique [0002] As protein chip sensing technology is widely applied to more and more fields, some problems that need to be solved are also exposed. A commonly used protein chip is to immobilize protein molecules on the surface of a solid substrate as a ligand to identify biomolecules that specifically bind to it. Protein molecules as ligands are immobilized on the surface of the chip with a certain density, and there are gaps between the molecules. When the surface of such a protein chip contacts the solution of the molecules to be tested, the molecules to be tested and the ligand molecules are specifically combined to produce At the same time as the specific signal, it can also be combined with the chip substrate in the molecular gap of the lig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/48
Inventor 靳刚王战会梁伟刘妍
Owner INST OF MECHANICS - CHINESE ACAD OF SCI
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