Part I: Surface Modification of Magnetic Nanoparticles for the Recovery of Plasmid DNA and Exclusion of Endotoxins in Recombinant DNA Assays; Part II: Biosafety and Environmental Health Program Implementation Related to Manufacturing and Research of Medical Diagnostic Systems Focusing on Biopsy-Based Cancer Tests and Reagents

摘要

Part I: Surface Modification of Magnetic Nanoparticles for the Recoveryudof Plasmid DNA and Exclusion of Endotoxins in Recombinant DNAudAssays / udAbstract: udPlasmids are small circular double- stranded DNA molecules found naturally in bacterialudcells. The genes that bacterial plasmids carry give the bacteria certain genetic advantages, suchudas being antibiotic resistant. As a bacterium divides the plasmids that it contains are copied andudpassed on to each daughter cell. Due to this unique nature of bacterial plasmids, scientists andudresearchers have employed the use of plasmids to clone, transfer and alter genes. Although quiteuduseful for various molecular techniques, plasmid DNA which is destined for use in in vivoudresearch pharmaceutical applications requires additional considerations due to other bacterialudcompounds bacterial cells possess. Endotoxin is a unique bacterial compound present in the outer membrane of Gramnegativeudbacteria. Endotoxin serves as a protective outer membrane permeability barrier, whichudgives the bacteria a defense mechanism in its environment. The endotoxin is released during theudlysis of bacterial cells, as endotoxins can be hazardous and even deadly in certain concentrations.udIt is important that specialized techniques are utilized to purify plasmids. One particularudtechnique developed by Ferradigm, a small Tucson, AZ based startup company, for purifyingudplasmids from bacterial cells, known as the Plasmid FlexPrep Kit, utilizes magneticudnanoparticles as a mode of extraction for plasmids. The Plasmid FlexPrep Kit used by Ferradigmudinvolves basic DNA isolation chemistry. PEG (polyethylene glycol) is used to precipitate theudplasmids out of solution, followed by a solid phase binding system of paramagneticudnanoparticles. This binding system allows the user to separate the bonded material out of solution using a magnet. Since plasmids (DNA) are negatively charged along with endotoxinsudthere are issues of specificity.udThus for my internship project, I explored surface modification of the magneticudnanoparticles to increase specificity, which would in turn increase purity. Four different avenuesudwere addressed including: affinity-ligand binding utilizing adsorption chemistry, nanoparticleudmetal typing, manufacture of magnetic nanoparticles utilizing a microwave in the presence of audproposed ligand, and surface coating of magnetic nanoparticles with humic acid to increase theudability of the nanoparticles to remain colloidal in solution.udResults from these four different areas of investigation set the stage for furtherudinvestigation aimed at the surface of magnetic nanoparticles and improving the specificity of theudplasmid preparation assays. The concept of magnetic nanoparticle surface modification couldudenable scientist and researchers to specialize plasmid assays, and prepare plasmids for specificuddownstream in vivo experiments or potential pharmaceutical research applications, such as geneudtherapy.