Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof

A technology of immobilized enzymes and magnetic nanoparticles, applied in the direction of immobilization on/in organic carriers, fermentation, etc., can solve problems such as sample loss, affecting the sensitivity and quantitative accuracy of protein identification, and limiting enzymatic hydrolysis efficiency. To achieve the effect of increasing the solid loading capacity

Active Publication Date: 2014-03-26
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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AI Technical Summary

Problems solved by technology

However, the currently widely used method of directly immobilizing enzymes in a single layer on the surface of the substrate makes the enzyme immobilization capacity per unit mass of the immobilized enzyme material limited by the surface area of ​​the substrate, thus limiting the further improvement of the enzymatic hydrolysis efficiency.
At the same time, the commonly used silicon or hydrophobic polymer matrix materials are difficult to avoid non-specific adsorption of proteins and their enzymatic hydrolysis products, resulting in sample loss, which affects the sensitivity of protein identification and the accuracy of quantification

Method used

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  • Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof
  • Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof
  • Hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and preparation method thereof

Examples

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Effect test

Embodiment 1

[0045] Example 1. Preparation of Hair-like Hydrophilic Polymer Hybrid Magnetic Nanoparticles Immobilized Protease

[0046] according to figure 1 The process flow shown was carried out.

[0047] 1.1 Synthesis of SI-ATRP initiator. Using 3-aminopropyltriethoxysilane and 2-bromoisobutyryl bromide to synthesize SI-ATRP with one end as a silane coupling agent capable of binding to the surface of silica-coated magnetic nanoparticles and the other end as an ATRP initiator Initiator. The details are as follows: 8mmol 3-aminopropyltriethoxysilane and 10mmol triethylamine were added to 12.5ml tetrahydrofuran, after mixing, nitrogen gas was introduced to remove oxygen while ice bathing was carried out for 30min, and then 10mmol 2-bromoisobutyryl bromide was slowly Add it dropwise into the mixture and stir vigorously for 4 hours (continuous nitrogen flow), and finally filter the solution and vacuum-dry it to 1 / 3 of the original volume to remove tetrahydrofuran and triethylamine, remove...

Embodiment 2

[0057] An appropriate amount of bovine serum albumin (BSA) standard protein (its sequence is shown in sequence 1) was weighed and dissolved in 50 mM ammonium bicarbonate solution (pH=8.0), with a final concentration of 2 μg / μl. Add 10 mM mercaptoethanol (DTT), according to the molar ratio of the final concentration of IAA and DTT is 5:1, add IAA solution, and place in dark place for 1 hour to denature the protein. Mix 50 μl of the denatured protein solution (concentration 2 μg / μl) with 10 mg of trypsin immobilized on hydrophilic polymer hybrid magnetic particles, place it in a water bath at 37°C for 1 minute, and then use a magnet to adsorb the enzyme immobilized on magnetic particles to the tube. wall, and the protein hydrolyzate in the supernatant was removed. Take the same amount of denatured protein solution, add trypsin at a mass ratio of 1:50 (trypsin: protein substrate) according to the conventional enzymatic hydrolysis conditions, place it in a water bath at 37°C for 1...

Embodiment 3

[0059] An appropriate amount of bovine serum albumin (BSA) standard protein was weighed and dissolved in 50 mM phosphate buffer (pH=7.8), with a final concentration of 2 μg / μl. Add 10 mM mercaptoethanol (DTT), according to the molar ratio of the final concentration of IAA and DTT is 5:1, add IAA solution, and place in dark place for 1 hour to denature the protein. Take 50 μl of the denatured protein solution (concentration 2 μg / μl) and mix it with 10 mg of hydrophilic polymer hybrid magnetic particle-immobilized endoproteinase Glu-C, place it in a water bath at 37°C for 1 minute and fix the magnetic particle with a magnet The enzyme is adsorbed on the tube wall, and the protein hydrolyzate in the supernatant is taken out. Take the immobilized enzyme hydrolyzate and spot it on the target of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer and let it air-dry naturally, then spot the CHCA matrix (5 mg / ml, dissolved in 50% acetonitrile, 0.1% TFA), a...

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Abstract

The invention discloses a hair-like hydrophilic polymer hybridization magnetic nanoparticle immobilized enzyme and a preparation method thereof. The preparation method of the hair-like hydrophilic polymer hybridization magnetic nanoparticle mmobilized enzyme comprises the following steps of carrying out in situ polymerization on the surface of a magnetic particle through an SI-ATRP (Surface-Initiated Atom Transfer Radical Polymerization) method to generate a polymethacrylamide glucose polymer chain, and generating an aldehyde group by utilizing an oxidative ring-opening polymer lateral chain glucose; then carrying out protease immobilization by carrying out covalent coupling on the aldehyde group and the N-end amido of protease. According to the invention, an enzymatic hydrolysate and the immobilized enzyme can be separated through an external magnetic field; the immobilized enzyme and a protein sample can complete protein enzymolysis by only hatching for 1-2 minutes under the condition of 37 DEG C after being mixed, so that the enzymolysis time greatly shortened; the problem of nonspecific adsorption of the traditional hydrophobic immobilized enzyme material on protein and the enzymatic hydrolysate thereof is solved, so that the sample recovery rate is enhanced. Finally, the immobilized enzyme disclosed by the invention is easy, convenient and fast to use and good in stability, can be repeatedly used, and has good application prospect in large-scale protein group sample analysis.

Description

technical field [0001] The invention relates to a hair-like hydrophilic polymer hybrid magnetic particle immobilized protease and a preparation method thereof. Background technique [0002] As a key research field in the post-genome era, proteomics research can not only provide a theoretical basis for the elucidation of the laws of life activities, but also provide an important basis for the early diagnosis of many diseases, the discovery of drug targets, the judgment of curative effect and prognosis. Since 2001, it was rated as the six hot research fields in the 21st century by "Science" magazine, and proteomics research has received extensive attention from scientists. At present, the proteome identification strategy based on the "shotgun method" is the most widely used and successful qualitative and quantitative research strategy for complex proteome samples. This strategy relies on the site-specific enzymatic hydrolysis reaction of proteases on proteins, so as to hydrol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12P21/06C12P19/14
Inventor 钱小红秦伟捷蔡耘徐龙范超
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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