UC Merced

Monolayer-protected gold nanoclusters (AuNCs) are atomically precise assemblies less than 2 nanometers in diameter, giving them size-specific physical and chemical properties. This small size lends them to biosensing, electronics, optics, and biomedicine applications. The ligand monolayer contributes to the physical and chemical properties. Many nanomedicine applications require the conjugation of biomolecules into this ligand monolayer to interface with biological systems. While many bioconjugation strategies, DNA-based conjugations offer stability, functionality, and specificity. Thus, specific molecular recognition by DNA can be leveraged to target cell types and tissues.Moreover, this programmable specificity of base-pairing allows for the precise self-assembly of multiple nanomaterials. Despite the advantages of AuNC-DNA conjugates, a detailed study has not been undertaken into the experimental conditions that lead to successful and predictable ligand exchange outcomes. The ligand monolayer significantly contributes to the physiochemical properties of AuNCs and has an even greater impact on how AuNCs interface with biological systems. Thus for AuNC-DNA conjugates, a purification technique must be able to isolate AuNC-(DNA)n from AuNC-(DNA)n±1. By isolating each conjugate species, it is possible to study how each conjugate interfaces with a biological system. Finally, an emerging area of interest is how AuNCs couple with other nanoscale components. For instance, the distance-dependent energy transfer between an AuNC and fluorophore would lend itself to future applications in biosensing and imaging. Coupling between AuNCs and other materials is sensitive to their relative position from each other. Thus, self-assembly methods capable of predefined geometrical arrangements and precise nanometer separation are needed. No publications meet these conjugation, isolation, and self-assembly needs in the AuNC field. The projects detailed within this dissertation hope to address these gaps. In Chapter 2, we leverage the small size of monolayer-protected Au25 NCs to enable polyacrylamide gel electrophoresis (PAGE) and densitometric analysis to characterize the distribution of AuNCs with discrete numbers of DNA ligands under different reaction conditions. We found that both AuNC and salt concentrations affect ligand exchange products and that the noncovalent interactions of DNA are a nontrivial and complex variable in these reactions. The DNA ligands' length and sequence affect the final conjugation products of such reactions. The complexity of noncovalent DNA interactions and their effect on final products is not inconsequential. In Chapter 3, we sought to develop a method that could isolate AuNC-(DNA)n from AuNC-(DNA)n±1 to aid in characterizing individual conjugate assemblies for downstream in vivo applications. We found ion-paired reversed-phased HPLC methods suitable for isolating and purifying the individual AuNC-DNA conjugates. After purification, the steady-state fluorescence studies that characterized the AuNC-DNA conjugates illustrated how differently an AuNC-(DNA)n can behave from an AuNC-(DNA)n±1. Chapter 4 demonstrates the first successful AuNC self-assembly with precisely controlled positions on a DNA origami template. These results demonstrate that by functionalizing the AuNCs with the oligonucleotides via the ligand exchange reaction and then using the purified mono-functionalized products, we can program the self-assembly of the AuNCs with nanometer precision. It is our hope that the work amassed here will further the field of AuNC applications. The specificity of DNA base-pairing combined with the stability of water-soluble thiolate monolayer-protected AuNC has significant potentialities. We have demonstrated the suitability of purified AuNC-DNA conjugates for self-assemblies.