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Method for synthesizing integral bed for plasmid analysis

A synthesis method and integrated bed technology, applied in the field of biomolecular analysis and chromatographic analysis, can solve the problems of inability to apply plasmid analysis, poor mass transfer performance, blockage of chromatographic columns, etc., and achieve good pollution resistance, low cost, and mass transfer speed. quick effect

Inactive Publication Date: 2012-05-30
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since this type of chromatographic medium is developed for protein separation analysis, the pore size in the monolithic bed is 1-2 μm. Although it can have good mass transfer performance in protein separation, because the length of the plasmid can reach hundreds of nanometers, the narrow part can be Therefore, the mass transfer performance of this type of monolithic bed is poor in plasmid analysis, and the chromatographic column is prone to clogging, so it cannot be applied to the analysis of plasmids.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Weigh monomer glycidyl methacrylate, crosslinking agent divinylbenzene and trimerized isocyanurate triallyl urate (1: 0.04: 0.01mol / mol / mol); porogen toluene and n-heptane ( Pore ​​agent / reaction mixture 80vol%, toluene / n-heptane 3: 2vol / vol); Initiator azobisisobutyronitrile (initiator / monomer 0.005: 1mol / mol), mix well, pack into a 50 ×4.6mm stainless steel tube, the other end was sealed, and reacted at 50°C for 72 hours. Connect the monolithic bed to the pump and flush with ethanol. Then diethylamine is used as a modifier, ethanol is used as a solvent, the volume content of diethylamine is 25%, the modification reaction is controlled at 80° C., and the reaction time is 12 hours. After the reaction, the monolithic bed was rinsed with ethanol. More than 75% of pores in the monolithic bed synthesized by the present invention have a diameter of more than ten microns, and the modification density of the monolithic bed is about 2.0 mmol / g monolithic bed.

Embodiment 2

[0038] Weigh monomer epoxy ethylene methacrylate, crosslinking agent ethylene glycol dimethacrylate and trivinylbenzene (1: 0.03: 0.06mol / mol / mol); porogen ethylbenzene and n-octane ( Porogen / reaction mixture 75vol%, ethylbenzene / n-octane 1:1vol / vol); Initiator azobisisobutyronitrile (initiator / monomer 0.01:1mol / mol), mix well, put into one end and seal In a 50×4.6mm stainless steel tube, the other end was sealed and reacted at 55° C. for 36 hours. Connect the monolithic bed to the pump and flush with THF. Then, dimethylamine was used as a modifier, tetrahydrofuran was used as a solvent, the volume content of dimethylamine was 75%, the modification reaction was controlled at 70° C., and the reaction time was 24 hours. After the reaction, the monolithic bed was flushed with tetrahydrofuran. More than 75% of pores in the monolithic bed synthesized by the present invention have a diameter of more than ten microns, and the modification density of the monolithic bed is about 2.3m...

Embodiment 3

[0040] Weigh monomer glycidyl acrylate, cross-linking agent divinylbenzene and trimerized isocyanurate triallyl urate (1: 0.06: 0.03mol / mol / mol); porogen xylene (porogen / reaction Mixture 60vol%); Initiator azobisisoheptanonitrile (initiator / monomer 0.02: 1mol / mol), mix homogeneously, pack in a 50×4.6mm stainless steel tube with one end sealed, and seal the other end, at 60°C React for 12 hours. Connect the monolithic bed to the pump and flush with methanol. Then, ethylamine was used as a modifying agent, methanol was used as a solvent, the volume content of ethylamine was 30%, the modification reaction was controlled at 75° C., and the reaction time was 18 hours. After the reaction, the monolithic bed was flushed with methanol. More than 75% of pores in the monolithic bed synthesized by the present invention have a pore diameter of more than ten microns, and the modification density of the monolithic bed is about 2.1 mmol / g monolithic bed.

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Abstract

The invention relates to a method for synthesizing an integral bed for plasmid analysis, which belongs to the technical fields of biomelecule analysis and chromatographic analysis. The method comprises the following steps: mixing metacrylic acid ester containing an epoxy terminal group or an acrylic ester monomer containing the epoxy terminal group with cross-linking agent, pore-forming agent andinitiating agent to obtain a reaction mixture; uniformly mixing the mixture in an ultraphonic way, adding the mixture into a chromatographic column pipe and carrying out in-situ polymerization to form an integral bed; after polymerization reaction is finished, washing the integral bed by organic solvent; modifying the integral bed by amine modifying agent dissolved in the organic solvent; and after modification reaction is finished, washing the integral bed by the organic solvent. The integral bed synthesized by the method has favorable pollution resistance, high plasmid transfer speed, low backpressure, convenient and rapid analyzing process and low cost in plasmid analysis.

Description

technical field [0001] The invention relates to a method for synthesizing an integral bed for plasmid analysis, and belongs to the technical fields of biomolecular analysis and chromatographic analysis. Background technique [0002] The application research of gene therapy and DNA vaccine in the diagnosis and treatment of major diseases such as cancer, single-gene genetic disease, AIDS, cardiovascular disease and arthritis is attracting more and more attention. At present, plasmids have gradually become the most important gene delivery system for gene therapy, so the large-scale preparation of pharmaceutical plasmids used in gene therapy has become its basis. One of the keys to the large-scale preparation process of pharmaceutical plasmids is how to monitor the purity of the plasmid, monitor the performance of the plasmid preparation process, and evaluate the final product quality and product specifications, which are very important for process development, certification, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/50G01N30/56C08F220/32C08J9/12C08F8/32
Inventor 张敏莲刘孟儒孔宪
Owner TSINGHUA UNIV
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