<p>This thesis has exploited the use of gold nanoparticles (AuNPs)/DNA conjugates towards 1) the development of simple colorimetric assays to monitor DNA functions and relevant biological processes, and 2) the control the nanoassembly of AuNPs using biomolecules and biological processes.</p> <p>DNA has a number of attractive functions including specific biorecognition, catalysis and being manipulated by protein enzymes, etc. These characteristics were exploited to permit nanoassembly to be responsive to a specific stimulus and also ensure the specificity and precision in the construction of well-defined 3D nanostructures. Meanwhile, the assembly or disassembly of AuNPs, which results in distinct color changes due to the localized surface plasmon resonance, provides an excellent platform for the colorimetrically monitoring the DNA functions and the relevant biological processes.</p> <p>We have specifically investigated how the surface charges, the length and conformations of surface-tethered DNA polymers affect the assembly of AuNPs. We found that the colloidal stability of AuNPs can be well-tuned by nucleotides (small charged molecules) with various binding affinity to AuNP surface and/or different number of negatively-charged phosphate groups. This relies on the fact that nucleotides can bind to AuNP surface via nucleobase-Au interaction, and negatively charged phosphates stabilize AuNPs via electrostatic repulsion. This investigation allowed us to monitor protein enzymatic reactions where nucleotides are modified by alkaline phosphatase and to control the growth of AuNPs using nucleotides as capping ligands.</p> <p>We then investigated the effect of the length of DNA polymers on AuNP surface on AuNP colloidal stability. DNA-modified AuNPs are stabilized electrosterically at a relatively high salt concentration; the removal (or shortening) of the DNA molecules by enzymatic cleavage or the dissociation of DNA aptamers from AuNP surface upon binding to their target destabilizes AuNPs and results in AuNP aggregation. We attribute this to the loss of negatively-charged polymeric DNA molecules that initially served as colloidal stabilizers. This has been applied to the monitoring of enzyme (both protein enzyme and DNA enzyme) cleavage of DNA molecules, and DNA aptamer binding event to its target, respectively.</p> <p> We also studied how DNA polymer conformational changes influence AuNP colloidal stability, which has been employed to monitor DNA aptamer folding events on the AuNP surfaces. We found that AuNPs to which folded aptamer/target complexes are attached are more stable towards salt induced aggregation than those tethered to unfolded aptamers. Experimental results suggested that the folded aptamers were more extended on the surface than the unfolded (but largely collapsed) aptamers in salt solution. The folded aptamers therefore provide higher stabilization effect on AuNPs from both the electrostatic and steric stabilization points of view.</p><p> Finally, we demonstrated the well-defined assembly of AuNPs using long (hundred nanometers to microns) single-stranded (ss) DNA molecules as template in a three-dimensional (3D) fashion. Specifically, these long ssDNA containing repeating units are generated by protein enzymatic reaction (DNA extension through rolling circle amplification) on AuNP surface. The resultant product provides a 3D-like scaffold that can be subsequently used for periodical assembly of complementary DNA-attached nanospecies. </p> <p> We also expect that the facile colorimetric biosensing assays developed in this thesis work provide an attractive means to study biomolecular behaviors (e.g, biorecognition and conformational changes) on the surface, and to investigate other common DNA (or RNA) structural (e.g., triplex, G-quadruplex, hairpin, i-motif) and protein structural transitions.</p> <p> Finally, this thesis work provides some novel and general strategies for the control of nanoassemblies by tuning surface charges and surface-tethered polymers. We expect these principles can also be applied in other AuNP-based sensing platforms that exploit interparticle interactions and in the construction of well-defined nanostructures which involves other types of nano-scaled materials (e.g., quantum dots, nanotubes, nanowires, etc).</p> / Thesis / Doctor of Philosophy (PhD)

Electrolyte interactions with ligand functionalized gold nanoparticles (AuNPs) have broad implication to a wide range of applications in nanoparticle research field. Among a wide range of electrolytes, halides, nitrates, borohydrides, and sulfides are used to study the AuNP interfacial interactions. Although there are many studies on AuNP interactions with anionic species (halides, nitrates, borohydrides, and sulphides), there is limited information on AuNP interactions with metallic cations. Therefore, studying the nanoparticle interfacial interactions with both anionic and metallic cation species is highly important. The research reported here is focused on deepening the understanding of electrolyte interactions with ligand functionalized AuNPs in aqueous solutions. The stability of citrate-residues on AuNPs against ligand displacement has been controversial. In the first study, we demonstrated the direct experimental evidence for the simultaneous adsorption of both citrate-residues and solution impurities onto citrate-reduced AuNPs by using AuNPs synthesized with deuterated citrate in combination with the surface-enhanced Raman spectroscopic (SERS) analysis. The citrate-residues can be readily displaced from AuNPs by a wide range of specific and non-specific ligands including organosulfur and electrolytes. In the second study, we investigated the charge state and the mechanism of silver ion binding onto organothiol functionalized AuNPs. Mechanistic study reveals that silver binding onto AuNPs proceeds predominantly through reactive pathways with proton generations providing the first direct experimental evidence that Ag+ can disrupt the Au-S binding and enhance the mobility of the organothiols on AuNPs. Ligand displacement from AuNPs is important in a wide range of applications. Complete and non-destructive removal of ligands from AuNPs is important and challenging due to the strong Au-S binding and the steric hindrance imposed by ligand overlayer on AuNPs. In the final study, we investigated hydrogen sulphide (HS-), an anionic thiol as an effective ligand to induce complete and non-destructive removal of ligands from aggregated AuNPs. The new insights and methodologies presented in this dissertation are important for studying the electrolyte interfacial interactions with ligand functionalized AuNPs which have a broad impact on nanoparticle surface chemistry.

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