Abstract
Nanoparticle surface engineering increasingly relies on mixed-ligand monolayers to introduce multiple chemical functionalities onto a single nanoscale interface. Previous studies have shown that nanoparticle size and morphology can be tuned through synthetic parameters such as ligand-to-metal and reducing agent-to-metal ratios, but these relationships have been investigated primarily in single-ligand systems. Separately, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) has been used to probe ligand-shell organization in mixed-ligand nanoparticles by comparing experimental metal-ligand fragment distributions with composition-matched binomial distributions representing random mixing, calculated from the measured ligand fractions. However, these approaches have largely focused on relatively simple, nonionizable ligand combinations, in which ligand solubility, metal binding, nanoparticle stabilization, and MALDI ionization behavior are comparatively straightforward. Extending these strategies to polar and ionizable mixed-ligand systems presents a substantially greater challenge because functional groups such as -COOH and -NH₂ can affect both nanoparticle formation and the subsequent mass-spectrometric response.
A central challenge addressed in this dissertation was the lack of a sufficiently uniform and well-dispersed mixed-ligand nanoparticle platform for quantitative ligand-shell analysis. Previous work in our group had established relatively well-dispersed single-ligand Ag nanoparticles, but mixed-ligand Ag systems showed poorer uniformity and dispersion. This variability introduced an important source of uncertainty for subsequent MALDI-MS measurements and comparisons with simulated ligand-shell distributions, because differences in particle size and aggregation state could complicate interpretation of fragment statistics. To overcome this limitation, the reducing-agent-to-metal ratio (R) and ligand-to-metal ratio (S) were systematically tuned to improve nucleation, growth, and surface stabilization. This optimization produced three mixed-ligand Ag nanoparticle systems with substantially improved dispersion and uniformity and core diameters of approximately 4-5 nm. A related strategy was then extended to Au nanoparticles, where R and S were first adjusted to produce uniform single-ligand Au cores, followed by post-synthetic ligand exchange to independently control mixed-ligand composition. Using this approach, eight mixed-ligand Au nanoparticle systems with core diameters of approximately 5 nm were prepared with improved uniformity and dispersion. These results established a reproducible synthetic platform for mixed-ligand Ag and Au nanoparticles and reduced particle-size and aggregation-related variability that could otherwise contribute to discrepancies between MALDI-MS experiments and simulated ligand-shell distributions.
A second major challenge addressed in this dissertation was extending MALDI-MS analysis from neutral mixed-ligand monolayers to polar and ionizable ligand systems. Previous studies of ligand organization by MALDI-MS were primarily based on neutral ligands, for which metal-thiolate fragment distributions could be interpreted more directly in terms of ligand-shell organization. In contrast, COOH- and NH₂-terminated ligands introduce additional ionization and fragmentation pathways that can strongly affect the detected fragment population. To determine whether these effects could be controlled, the MALDI ionization environment was systematically varied through changes in matrix, apparent pH, and positive- and negative-ion modes for both Ag systems. These experiments showed that COOH-containing Ag nanoparticles did not yield a continuous and quantitatively useful mixed-ligand fragment series across the conditions examined, whereas corresponding Au systems produced well-resolved mixed Au-thiolate fragment distributions that allowed polar-ligand incorporation to be followed quantitatively. This difference is consistent with the greater susceptibility of Ag to oxidation and Ag⁺ formation, which can promote carboxylate coordination and disperse the MALDI fragment signal across multiple adducts and charge states. In contrast, the more stable Au-thiolate interface better preserves well-defined metal-thiolate fragments, enabling the Au platform to be extended to multiple COOH- and NH₂-terminated ligand systems for quantitative analysis of polar mixed-ligand monolayers.
Configurational-bias Monte Carlo (CBMC) simulations were then used to generate three-dimensional ligand-shell configurations and corresponding simulated fragment distributions, which were compared with analogous random references calculated from the simulated ligand fractions. The combined results identify when ligand lengths were similar, favorable heteroligand backbone contact together with configurational entropy stabilized random-like mixing. As the mismatch increased, reduced contact between unlike ligands increased the enthalpic penalty of heteroligand packing, while association of similarly sized ligands recovered more favorable chain-chain interactions; this packing advantage progressively outweighed the entropy of mixing and promoted nanoscale segregation. CBMC simulations reproduced these experimental trends and further showed that longer ligands preferentially occupied facet-center regions, whereas shorter ligands were enriched near nanoparticle edges, consistent with stronger cohesive stabilization of longer chains on flatter facets. Solvent-dependent changes in the CH₃/COOH system and differences between the COOH- and NH₂-containing series further showed that the final organization reflects the combined contributions of packing enthalpy, configurational and conformational entropy, terminal-group interactions, and ligand-solvent interactions.
Finally, this framework is extended to KTLLPTPC-containing nanoparticles, demonstrating that peptide organization depends not only on overall peptide loading but also on the molecular environment created by the surrounding background ligands. These findings identify background-ligand chemistry as an additional design parameter for controlling biomolecular presentation at nanoparticle interfaces.
Overall, this dissertation extends mixed-ligand nanoparticle characterization beyond relatively simple ligand systems by establishing how chemically functional, polar ligands influence both nanoparticle synthesis and MALDI-MS interpretation. By separating synthesis-dependent effects and core- and terminalgroup-dependent MALDI behavior from genuine ligand-organization signatures, this work establishes a quantitative foundation for probing increasingly complex functional nanoparticle interfaces.
