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Chip enzymolysis micro-reactor based on functionalized magnetic microsphere and its preparation method and uses

A technology of magnetic microspheres and microreactors, applied in the field of biochemical analysis, can solve the problems of severe synthesis reaction conditions, easy loss of enzyme activity, scrapped enzyme reactors, etc., and achieve high sensitivity, no need for protein denaturants, and easy regeneration.

Inactive Publication Date: 2007-08-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the method of immobilizing enzymes is the covalent bond sum method. The advantage of this method is that the combination of the enzyme and the carrier is relatively firm. The disadvantage is that the preparation process of the enzyme reactor is quite cumbersome. At the same time, the synthesis reaction conditions are relatively severe, and the enzyme activity is easily lost.
Moreover, once the immobilized enzyme of this enzyme reactor is inactivated, it will not be able to regenerate and the entire enzyme reactor will be scrapped.

Method used

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  • Chip enzymolysis micro-reactor based on functionalized magnetic microsphere and its preparation method and uses
  • Chip enzymolysis micro-reactor based on functionalized magnetic microsphere and its preparation method and uses
  • Chip enzymolysis micro-reactor based on functionalized magnetic microsphere and its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Preparation of functionalized magnetic microspheres and immobilization of enzymes:

[0041] (1) 2.70 g FeCl 3 ·6H 2 O was dissolved in 100 mL of ethylene glycol under magnetic stirring, and after stirring for 0.5 h, 7.20 g of sodium acetate was added and stirred for another 0.5 h. The obtained solution was transferred to a 200 ml reaction kettle, reacted at 200° C. for 8 hours, and then cooled to room temperature. The ferroferric oxide magnetic microspheres produced by the reaction were collected with a magnet, washed with ethanol and water several times, and the product was vacuum-dried at 60° C. for 12 hours.

[0042] (2) 0.01 gram of ferroferric oxide magnetic microspheres are cleaned with 5 milliliters of 2mol / L hydrochloric acid solution for 5 minutes under ultrasonic, dispersed with 100 mL of ethanol / water (weight ratio 7: 3) solution containing 1% ammonia, and mechanically Under stirring, 0.05 g of ethyl orthosilicate was slowly added into the dispersion of...

Embodiment 2

[0052] 1. Preparation of functionalized magnetic microspheres and immobilization of enzymes:

[0053] (1) 3.8 g FeCl 3 ·6H 2 O was dissolved in 100 mL of ethylene glycol under magnetic stirring, and after stirring for 1 hour, 8.0 g of sodium acetate was added and stirred for another 1 hour. The reaction was carried out at 200° C. for 10 hours. All the other are with embodiment 1.

[0054] (2) 0.02 gram of ferroferric oxide magnetic microspheres were cleaned with 15 milliliters of 2mol / L hydrochloric acid solution for 10 min under ultrasonic, and under mechanical stirring, 0.10 gram of tetraethyl orthosilicate was slowly added in the magnetic microsphere dispersion liquid and heated at room temperature Stirring was continued for 12 hours. The particle size of the magnetic microspheres is 300nm. All the other are with embodiment 1.

[0055] (3) Using tricarboxymethylethylenediamine as a chelating agent, the dosage is 3.00 grams, γ-glycidyl ether propyl trimethoxysilane 1mL...

Embodiment 3

[0062] 1. Preparation of functionalized magnetic microspheres and immobilization of enzymes:

[0063] (1) 2.0 g FeCl 3 ·6H 2 0 was dissolved in 100 ml of ethylene glycol under magnetic stirring, and after stirring for 1 hour, 7.0 g of sodium acetate was added and reacted at 180°C for 6 hours. All the other are with embodiment 1.

[0064] (2) 0.03 g of ferroferric oxide magnetic microspheres were washed with 15 ml of 2 mol / L hydrochloric acid solution for 10 min under ultrasonic, and 0.15 g of ethyl orthosilicate was added under mechanical stirring. The particle size of the magnetic microspheres is 400nm. All the other are with embodiment 2.

[0065] (3) Use nitraminotriacetic acid as a chelating agent, the dosage is 5.00 grams, γ-glycidyl ether propyl trimethoxysilane 1.5mL, the stirring time in the ice-water bath is 12 hours, the heating and stirring time is 8 hours, and the rest are the same Example 1.

[0066] (4) The amount of magnetic microspheres is 0.04 g, the amo...

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Abstract

The invention belongs to biochemical analysis technology fiels, which in detail relates to a chip enzymolysis microreactor based on functional magnetic microsphere, the preparing method, regeneration method, and its application in protein biochemical analysis. The invention fixes protein enzyme on the surface of magentic microsphere with metal complexation method, and fixes in microchannel of microcurrent chip with its magnetism by using superparamagnetism of magetic microsphere, the protein liquid enters into microchannel under pump pressure for fast and effective enzymolysis, then enzymolysis product flows out of microchannel, and enters into mass analyzer for analysis. The invention aslo regenerates the magnetic microshpere lost enzymatic activity. The invention is characterized by realization of fast protein chip enzymolysis and mass spectrum determination, simple operation, high sensitivity, no protein denaturant, low energy consumption, easy regeneration, suitability for application in fast protein separation and determination in complicate biological sample, which possesses good practical and application value in protein histone research.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, and in particular relates to a chip enzymolysis microreactor based on functionalized magnetic microspheres, a preparation method and a regeneration method thereof, and an application in protein analysis. technical background [0002] With the successful completion of the Human Genome Project, life science research has entered the post-genome era, in which functional genomics has become the research focus, and proteomics is an important pillar of it. The development of modern biological mass spectrometry technology provides strong technical support for protein identification. Using biological mass spectrometry as a detector can not only provide accurate molecular weight information of protein molecules, but also provide accurate relative molecular mass information of peptides after proteolysis, so as to deduce the partial sequence of peptides. Identification is important. [0003] I...

Claims

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Application Information

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IPC IPC(8): C12N11/00C12Q1/25
Inventor 邓春晖李嫣张祥民徐秀青
Owner FUDAN UNIV
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