Neves_Moco_Alves_whole_thesis_ex_pub_mat.pdf (5.57 MB)
Nanoparticle and cell modifications using capillary electrophoresis
thesisposted on 2023-05-28, 11:48 authored by Alves, MN
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.
Rights statementCopyright 2020 the author The author's name on the thesis is M‚àö‚â•nica Filipa Neves Mo‚àövüo Alves, but the author publishes under the name M‚àö‚â•nica N. Alves Chapter 1 is the following fublished article: Alves, M. N., Mir‚àö‚â•, M., Breadmore, M. C., Macka, M. 2019. Trends in analytical separations of magnetic (nano)particles, TrAC trends in analytical chemistry, 114, 89-97 Chapter 2 is the following published article and has been removed for copyright reasons: Alves, M. N., Nesterenko, P. N., Paull, B., Haddad, P. R., Macka, M. 218. Separation of superparamagnetic magnetite nanoparticles by capillary zone electrophoresis using non-complexing and complexing electrolyte anions and tetramethylammonium as dispersing additive, Electrophoresis, 39(12), 1429-1436 Chapter 6, Isotachophoresis for rapid transformation of Escherichia coli, has been removed for proprietary reasons