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Study on the Separation and Purification of Plasimd DNA Based on Functionalized Silica Nanoparticles

Author: HuoHaiLing
Tutor: WangKeMin；HeXiaoXiao
School: Hunan University
Course: Analytical Chemistry
Keywords: Amino-silica coated nanoparticles (ASNPs) Amino-silica coated magnetic nanoparticles (ASMNPs) Electrostatic adsorption Purification Plasmid DNA
CLC: TB383.1
Type: Master's thesis
Year: 2008
Downloads: 107
Quote: 0
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Abstract


The demand for efficient production methods of plasmid DNA has increased vastly for widely applications in gene therapy, gene vaccine, DNA sequence and so on. As a kind of new functionalized nanomaterials, silica coated nanoparticles have received more and more attentions in the field of bio-separation for their biochemical stability, large surface area and good biocompatibility. In this study, we provided a new method for the purification of plasmid DNA based on the eletrostatic adsorption principles of amino-silica coated (magnetic) nanoparticles with plasmid DNA. This method avoids the disadvantages of phenol/chloroform technique such as toxicity and complexes operation. The results reported here open up new perspectives for amino-silica coated (magnetic) nanoparticles in the field of biomedicine and will have great potential in purificaton of genome. And a novel method for monitoring cancer was developed based on the the silica coated fluorescent nanoparticles.1. Research on plasmid DNA purification using amino-silica coated nanoparticles.Based on the electrostatic adsorption between the amino-silica coated nanoparticles (ASNPs) with positive surface charge and plasmid DNA with negative charge at neutral pH, an approach for the purification of pEGFP-N3 plasmid has been developed. The adsorption performance of ASNPs and the plasmid DNA isolated from the ASNPs-plasmid DNA complexes have been investigated. Meanwhile, the bioactivity of purified plasmid DNA using ASNPs was also studied. The results indicated that the plasmid DNA can be absorbed to the ASNPs very well to form ASNPs-plasmid DNA complexes. And the plasmid DNA was isolated from the ASNPs-plasmid DNA complexes successfully. Furthermore, the bioactivetity of purified plasmid DNA using ASNPs was very well.2. Research on plasmid DNA purification using amino-silica coated magnetic nanoparticles.Based on the electrostatic adsorption between the amino-silica coated magnetic nanoparticles (ASMNPs) with positive surface charge and plasmid DNA with negative charge at neutral pH, an approach for the purification of pEGFP-N3 plasmid without centrifiguation has been developed. The adsorption performance of ASMNPs and the factors that would affect the result of isolation were studied. Meanwhile, the purified efficiency of this method was compared with the traditional method. At last, the bioactivity of purified plasmid DNA using ASMNPs was studied. The results indicated that the ASMNPs-plasmid DNA complex can be separated quickly under the magnet. And the plasmid DNA was isolated successfully from the ASMNPs-plasmid DNA complexes. We also found that the result of isolation did not obviously relied on the temperature, condition, time and times of the isolation. In addition, the efficiency of the plasmid DNA purification using ASMNPs was better than traditional method. And the bioactivetity of purified plasmid DNA using ASMNPs was very well.3. Research on the interation between cancer cells with silica coated fluorescent nanoparticles-glucosamine complexes (FSNPs-DG).A novel method for monitoring cancer was developed based on the rapid metabolization of glucose in cancer cells and the silica coated fluorescent nanoparticles label method. The silica coated fluorescent nanoparticles-glucosamine complexes (FSNPs-DG) was prepared by physical adsorption. Three kinds of cancer cells and one kind of normal cell were chose for the analysis, respectively. The recognition of FSNPs-DG to immobilized cancer cells was investigated based on the principle that glucose transportor high express on the membrance of cancer cells. And then uptake of FSNPs-DG by the cancer cells was further studied. The results indicated that the FSNPs-DG can recognize the immobilized cancer cells very well, and the uptake of FSNPs-DG by cancer cells was more clearly than the normal cells.

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