Nano-magnetic microsphere with amino having fixed protease at surface, preparing method and application thereof

A nano-magnetic and magnetic microsphere technology, applied in the field of biochemical analysis, can solve problems such as hindering applications

Inactive Publication Date: 2007-06-06
FUDAN UNIV
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nano-magnetic microspheres prepared by traditional methods have good monodispersity and crystallinity, however, before surface modification, these nano-magnetic microspheres prepared by thermal decomposition technology only have Very good dispersibility, which greatly hinders their application in the field of biotechnology

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Immobilization of protease on the surface of nano-magnetic microspheres with amino groups:

[0035] (1) Disperse 1 mg of dried nano-magnetic microspheres with amino groups with a particle size of 500 nm in 200 μL of 50 mM ammonium acetate buffer (CB, CaCl 2 1mM, MnCl 2 1mM, pH 8.3);

[0036] (2) Use a magnet to hold the nano-magnetic microspheres, remove the CB, redisperse them in 200 μL of glutaraldehyde (GA) CB solution (GA 5%-10%, pH 6.8), and activate at room temperature at 1.5 Hour;

[0037] (3) Disperse the nanomagnetic microspheres activated by GA in 200 μL of protease CB solution and soak at room temperature for 3 h to immobilize the enzyme, wherein the content of protease in CB solution is 5 mg / mL, NaCNBH 3 The content is 1%;

[0038] (4) Disperse the nano-magnetic microspheres immobilized with protease in the CB solution of 200 μL glycine and soak at room temperature for 1 hour to seal the aldehyde group of the unimmobilized enzyme on the magn...

Embodiment 2

[0040] Example 2. Application of nano-magnetic microspheres immobilized with protease to enzymatic hydrolysis of protein solution:

[0041] (1) Heat denaturation of 0.2mg / mL cytochrome C in CB solution at 95°C for 15 minutes;

[0042] (2) Disperse 1 mg of nano-magnetic microspheres immobilized with protease in 1 mL of CB, remove 10 μL of the magnetic microsphere dispersion and add 10 μL of denatured cytochrome C solution, and enzymatically hydrolyze at 37 ° C for 1 hour, wherein the magnetic microspheres are dissolved in the CB solution The content in it is 1-3mg / mL;

[0043] (3) After absorbing the magnetic microspheres with a magnet, spot the supernatant onto the target plate;

[0044] (4) Spot the acetonitrile solution containing α-cyano-4-hydroxycinnosilicate (CHCA) on the sample target, dry it and send it to a mass spectrometer for analysis.

[0045] The results of mass spectrometry analysis were similar to those obtained by traditional solution enzymatic hydrolysis. Th...

Embodiment 3

[0046] Example 3. Application of nano-magnetic microspheres immobilized with protease to protein chip enzymolysis:

[0047] (1) Place the magnet under the microchannel of the chip;

[0048] (2) The magnetic microsphere dispersion liquid immobilized by the pump flows through the microchannel of the chip under the push of the pump;

[0049] (3) The magnetic microspheres are fixed in the microchannel of the chip under the action of a magnetic field to form a packed bed of 1-3mm.

[0050] (4) Heat denaturation of 0.2 mg / mL cytochrome C in CB solution at 95°C for 15 minutes;

[0051](5) Push 0.5 μL of denatured cytochrome C solution into the chip enzyme reactor based on functionalized magnetic microspheres with a pump, seal both ends, and enzymolyze at 50°C for 5 minutes;

[0052] (6) Push out the enzymatic hydrolyzate in the chip enzyme reactor based on functionalized magnetic microspheres with a pump, collect and spot on the target plate;

[0053] (7) Spot the acetonitrile sol...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an nm magnetic micro ball with the amino which the surface is fixed with the protease and the preparation method, also it is applied in the protein enzymolysis. The micro ball is composed by the one-step hydro-thermal method and with the particle size of 15-200nm. The glutaraldehyde is as the cross linker and the protease is fixed on the surface of the nm magnetic micro ball by which the protein is solution, slug, target decomposed. The method of the invention is simple, so it is proper for separation and identification of the protein in complex biological sample and it has the good future in the protein study.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, and in particular relates to a nano-magnetic microsphere with amino groups immobilized on the surface of protease, a preparation method thereof and an application in proteolysis. technical background [0002] In the identification and analysis of proteins, enzymatic hydrolysis of proteins is a key step. The traditional solution enzymatic hydrolysis method is cumbersome and time-consuming, which is not conducive to automatic operation. With the current large-scale development of proteomics research in organisms around the world, it is urgent to develop more effective and simpler proteolysis technology. [0003] Immobilized enzymes instead of traditional solution enzymolysis have many advantages: (1) improve enzyme stability and can be reused; (2) increase enzyme concentration per unit volume, thereby increasing enzyme use efficiency and reducing enzymatic hydrolysis time; (3) It is ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N33/68C12Q1/00
Inventor 邓春晖李嫣张祥民徐秀青
Owner FUDAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap