Protein in situ expression chip, and constructing method and application thereof

A protein and chip technology, applied in biological testing, material inspection products, etc., can solve the problems of template molecules and capture molecules that cannot be fixed in an orderly and gradual manner, spatial conformation changes, and loss of activity.

Inactive Publication Date: 2012-07-11
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Above these methods have advantages respectively, but all there is same shortcoming, one, the protein immobilization amount that adopts these methods to obtain is little (less than 50pmoles / cm 2 ); secondly, the spatial conformation of the protein changes due to direct adsorption on the substrate surface, resulting in the loss of activity; thirdly, the template molecules and capture molecules cannot be fixed on the substrate surface in an orderly and step-by-step manner.

Method used

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  • Protein in situ expression chip, and constructing method and application thereof
  • Protein in situ expression chip, and constructing method and application thereof
  • Protein in situ expression chip, and constructing method and application thereof

Examples

Experimental program
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Embodiment 1

[0093] Embodiment 1: the three-dimensional protein chip of detecting the content of hepatitis B virus core antibody (HBc Ab), hepatitis B virus e antibody (HBe Ab) and hepatitis B virus surface antibody (HBs Ab) in the body of hepatitis B patient

[0094] Evaporate 2nm of chromium and 40nm of gold on the surface of a glass slide with a high refractive index (the glass slide is 75mm*25mm), and modify the surface with long and short chain PEG molecules with mercapto groups, in which the first chain molecule PEG 45 ends at a carboxyl group , the second chain molecule PEG 6 terminal is a hydroxyl group, mixed at a ratio of 1:10, self-assembled on the gold surface, the total concentration is 1mM, the dosage is 100ul, and evenly spread on the surface of the gold sheet. After that, add Ni-NTA-modified α-cyclodextrin molecular solution (concentration: 100ug / ml, 40ul), and let it stand for 1 day, so that the cyclodextrin molecules self-assemble on the first chain molecule PEG to form ...

Embodiment 2

[0095] Example 2: Anti-protein diffusion chip containing four characteristic proteins of p21, p31, gp41 and gp120

[0096] On the gold-coated glass substrate, the surface is modified with long and short chain molecule dithiol molecules with sulfhydryl groups, in which the end of the first chain molecule dithiol (Mw: 8000) is a carboxyl group, and the second chain molecule Molecular dithiol Mw: 1500 terminal is a hydroxyl group, the structural formula is as follows. Among them, the first chain molecule R is a carboxyl group, and n is 45; the second chain molecule R is a hydroxyl group, and n is 6:

[0097]

[0098] Mixed at a ratio of 1:50, self-assembled onto the gold surface at a total concentration of 1 mM. After that, add 50ug / ml, 50ul Ni-NTA-modified α-cyclodextrin molecular solution, and let it stand for 1 day, so that the cyclodextrin molecules self-assemble on the first chain molecule dithiol to form a polyrotaxane structure . After using NHS / EDC solution to act...

Embodiment 3

[0099] Example 3: Discovery of Biomarkers for Autoimmune Diseases

[0100] 2nm of chromium and 40nm of gold are vapor-deposited on the surface of the glass slide with a high refractive index, and the surface is modified with long and short chain PEG molecules with mercapto groups, in which the end of the first chain molecule PEG45 is a carboxyl group, and the second chain molecule PEG6 The terminal is a hydroxyl group, mixed at a ratio of 1:10, self-assembled on the gold surface, the total concentration is 1mM, and the dosage is 100μl, and evenly spread on the surface of the gold sheet. Afterwards, a saturated concentration of α-carboxymethylcyclodextrin molecular solution (125mg / ml, 50ul) was added and allowed to stand for 1 day to allow the cyclodextrin molecules to self-assemble on the first chain molecule PEG to form a polyrotaxane structure. After using NHS / EDC solution to activate the carboxyl group on the first chain molecule PEG 45 and carboxymethyl cyclodextrin, add...

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Abstract

The invention provides a three-dimensional protein in situ expression chip with the effects of high fixed quantity, high protein activity and ordered fixation. The chip comprises a base (1), a surface modification layer and protein (5) fixed on the surface modification layer, wherein the surface finish layer comprises second chain molecules (4) and polyrotaxane; the polyrotaxane comprises first chain molecules (3) and cyclodextrin (2); and the second chain molecules and the polyrotaxane are fixed on the base through fixed groups at one ends of the second and the first chain molecules. The invention further provides a method and a device for building the chip as well as the application of the chip, the method and the device.

Description

technical field [0001] The invention relates to the field of biochips, in particular to the field of protein in situ expression chips constructed by a cell-free expression system and a three-dimensional self-assembled supramolecular structure. Background technique [0002] Protein arrays provide high-throughput methods and means for studying the interactions between protein molecules, protein molecules, nucleic acids, and other small molecules. However, due to the lack of protein amplification methods similar to the PCR amplification technology in DNA arrays, the construction of highly active functional proteins on the array surface has always been a difficulty in studying the interaction of functional proteins. The cell-free expression system provides a new method for constructing protein arrays - protein in situ expression array (In Situ Protein Array). The principle of this method is as follows: First, it constructs an expressible gene array on the array surface, includin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 朱劲松程志强王艳梅阿密达・兰德哈维尔・巴蒂斯塔・佩雷斯宋炉胜周大苏
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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