Pollitt, Michael John; (2007) Protein coating of nanoparticles. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

This thesis investigates protein antibody adsorption to gold colloids, the specific application being a diagnostic test kit for malaria. For the antibody to be active, it needs to adsorb in the end-on orientation with the antibody binding fragment able to interact with the antigen. Thus, orientation is important, which can be implied from the coating thickness measured by particle size changes. Another aim was to thermodynamically characterise the adsorption process. Gold particles were prepared and characterised initially by transmission electron microscopy and visible spectroscopy. The difficulties of measuring by photon correlation spectroscopy are discussed. Attempts were made to measure the size using multi-angle light scattering. However, simulations using Mie theory showed that the angular dependence of scattered light intensity for gold is not the same as for ordinary materials. It was not possible to accurately measure the size using this technique and a current instrument. A protein typically undergoes structural change when it adsorbs onto a particle, which has an associated enthalpy change. It was shown that the gold colloid is too dilute to measure this interaction by titration calorimetry. Thus a model system of BSA and polystyrene was used. However, the dilution enthalpy of the BSA was larger than the adsorption enthalpy so made the adsorption enthalpy measurement inaccurate. To explain the dilution enthalpy results, the states of BSA in solution were explored using field-flow fractionation. Field-flow fractionation is by itself a separation technique for colloids and macromolecules and was utilised to study the gold nanoparticles and conjugates. It was successfully used to separate gold particle - antibody conjugates from unbound antibody. However, the technique could not be used for quantification because much of the sample adsorbed to the FIFFF membrane. Unexpectedly, unbound antibody was detected, which would reduce the sensitivity of the malaria diagnostic kits. Gold nanoparticles are red or purple in colour due to surface plasmon resonance. Theory predicts that the colour depends on the size of the particles, which was verified experimentally. The surface plasmon resonance depends on the local environment of the gold, so is influenced by coating. Antibody adsorption was shown to have a slight influence on the colour of the gold. The difficulties of linking spectral changes to adsorption thickness are discussed but the measurements suggested a coating thickness of approximately 10 nm, which would correspond to end-on orientation of the antibody. By this method, the plateau coverage was approximately 3 mg m-2, which is broadly the same as measurements reported in the literature.

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