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Preparation method of in-capillary immobilized enzyme microreactor

A microreactor, immobilized enzyme technology, applied in the direction of enzyme production/bioreactor, biochemical equipment and method, bioreactor/fermenter for specific purposes, etc., can solve the problem of poor reactor stability and easy washing of enzymes Take off and other problems to achieve the effect of convenient operation, shortening the effective length, enhancing stability and service life

Inactive Publication Date: 2014-03-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The physical adsorption method has mild fixation conditions, can retain most of the activity of the enzyme, and the reactor can also be repeatedly prepared. The disadvantage is that the stability of the reactor is poor, and the enzyme is easily eluted.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0032] A quartz capillary with an inner diameter of 50 μm and a length of 35.5 cm was selected, and was first washed with 1 M NaOH for 2 h, and then washed with H 2 Wash with O for 30 min and pure methanol for 30 min; then fix the capillary on a capillary electrophoresis apparatus, inject a 30% by volume methanol solution of 3-aminopropyltetraethoxysilane from the outlet of the capillary with a pressure of 50 mbar 6 s, after standing at room temperature for 1 h, then rinse with pure methanol from the inlet end of the capillary for 5 min, H 2 Rinse with O for 3 min, pH7.0 Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was washed for 5 min; then 2.5% glutaraldehyde solution was injected from the outlet end of the capillary with a pressure of 50 mbar for 6 s, and left at room temperature for 1 h, then Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was washed for 5 min; finally, 0.1 U / mL neuraminidase solution was injected from the outlet end of the capillary with a pressure of 50 ...

Embodiment 2

[0035] A quartz capillary with an inner diameter of 50 μm and a tube length of 38.5 cm was selected, firstly rinsed with 1 M NaOH for 2 h, and then washed with H 2 Wash with O for 30 min and pure methanol for 30 min; then fix the capillary on a capillary electrophoresis apparatus, inject a 30% by volume methanol solution of 3-aminopropyltetraethoxysilane from the outlet of the capillary with a pressure of 50 mbar 12s, after standing at room temperature for 1 h, then rinse with pure methanol from the capillary inlet for 5 min, H 2 O rinse for 3 min, pH 7.0 Na 2 HPO 4 -NaH 2 PO 4The buffer solution was washed for 5 min; then, 3% glutaraldehyde solution was injected from the outlet end of the capillary with a pressure of 50 mbar for 12 seconds. 2 HPO 4 -NaH 2 PO 4 The buffer solution was washed for 5 min; finally, 0.1 U / mL neuraminidase solution was injected from the outlet end of the capillary with a pressure of 50 mbar for 12 s, left at room temperature for 30 min, and s...

Embodiment 3

[0038] A quartz capillary with an inner diameter of 50 μm and a length of 40.5 cm was selected, and was first washed with 1 M NaOH for 2 h, and then washed with H 2 Wash with O for 30 min and pure methanol for 30 min; then fix the capillary on a capillary electrophoresis apparatus, inject a 30% by volume methanol solution of 3-aminopropyltetraethoxysilane from the outlet of the capillary with a pressure of 50 mbar 18 s, after standing at room temperature for 2 h, then rinse with pure methanol from the inlet end of the capillary for 5 min, H 2 Rinse with O for 3 min, pH 7.0 Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was washed for 5 min; then, 5% glutaraldehyde solution was injected from the outlet of the capillary with a pressure of 50 mbar for 18 s, left at room temperature for 3 h, and then injected with pH 7.0 Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was washed for 5 min; finally, 0.1 U / mL neuraminidase solution was injected from the outlet end of the capillary wit...

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Abstract

The invention provides a preparation method of an in-capillary immobilized enzyme microreactor. A glutaraldehyde bonding method is used for fixing neuraminidase at the outlet end of a capillary. According to the method, the glutaraldehyde bonding method is used for fixing the neuraminidase on the inner wall of the capillary for the first time, and the immobilized enzyme microreactor can be continuously used for more than 1 month; remarkable reduction of the activity of the neuraminidase is avoided, and the stability of the neuraminidase is greatly enhanced, so that the using amount of the neuraminidase is reduced to a great extent; the enzyme microreactor is fixed at the outlet end of the capillary during separation, so that the effective length of sample separation is decreased, and the separation efficiency is greatly improved; the preparation method comprises simple steps, is convenient to operate and high in screening speed, and can be widely applied to the screening of neuraminidase inhibitor.

Description

technical field [0001] The invention relates to the field of influenza virus drug screening, in particular to a preparation method of an enzyme microreactor immobilized in a capillary. Background technique [0002] In the 21st century, influenza is still a major killer that endangers human health. Neuraminidase, a glycoprotein found on the surface of influenza viruses, catalyzes the hydrolysis of specific linkages to sialic acid receptors and releases viral particles to infect new cells. At the same time, it can also promote the rapid entry of viral particles into lung glandular epithelial cells, resulting in severe lung disease, resulting in the death of patients. Because it plays an extremely critical role in the replication, transmission, infection and pathogenicity of influenza virus, the application of neuraminidase inhibitors is considered to be the main way to prevent and treat influenza. [0003] Currently, the most widely used neuraminidase inhibitors are oseltam...

Claims

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Application Information

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IPC IPC(8): C12M1/40C12M1/34C12Q1/34
CPCC12M21/18C12M23/16
Inventor 陈子林赵海燕
Owner WUHAN UNIV
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