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Nanoparticle and cell modifications using capillary electrophoresis

Neves Moco Alves, MF 2020 , 'Nanoparticle and cell modifications using capillary electrophoresis', PhD thesis, University of Tasmania.

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Abstract

Particle separation and manipulation is a strength of electrophoresis. In this dissertation, firstly, the power of electrophoresis as a microanalytical technique is demonstrated by separations of bare magnetic nanoparticles provided for the first time electropherograms exhibiting symmetrical and highly reproducible peaks, free of spurious spikes characteristic of nanoparticle clusters. This was achieved using non-complexing (nitrate) and complexing (chloride, citrate and phosphate) electrolyte ions with additions of tetramethylammonium hydroxide. This enabled the separation of bare and functionalised magnetic nanoparticles. Secondly, CE was coupled to a magnetic field and trapping of magnetic nanoparticles was demonstrated using purposely selected electrolyte compositions.
Just like nanoparticles, cells exhibit an electric surface charge due to exposed charged or chargeable functional groups. Therefore, they migrate under the influence of an electric field. Through isotachophoretic focusing, the electrostatic adsorption of functionalised magnetic nanoparticles to the cell wall of Escherichia coli TOP10 and their cellular uptake was studied. The same strategy was applied for the ITP transformation of E. coli TOP10 with plasmid DNA. Counter pressure-assisted isotachophoresis brought a large excess of plasmid DNA in contact with the cell surface allowing for a transformation rate 1,000-fold higher compared to electroporation and chemical transfection at survival rates greater than 60%. Based on the findings, the ITP method was adjusted for effective transfection of mammalian cells (Jurkat T) showing similar robustness to electroporation. This opens possibilities of using the developed method for the delivery of many other membrane impermeable solutes for screening of genes and drugs.

Item Type: Thesis - PhD
Authors/Creators:Neves Moco Alves, MF
Keywords: Capillary electrophoresis, Escherichia coli, green fluorescent protein, isotachophoresis, Jurkat cells, magnetic (nano)particles, transfection, transformation
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Copyright 2020 the author

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