Challenges in the functionalization of nanoparticles for plasmon ruler-based single-protein studies
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Abstract
Proteins are an important building block of life. In this context, single-molecule methods are a key tool for deeper understanding of their function. One such method is the plasmon ruler: It combines the advantages of multiplexing, long-time measurement and good signal-to-noise ratio. My work investigates the chemical challenges of plasmon ruler assembly with aim to increase the robustness and yield of the dimer constructs, and therefore enable broader future application. For this, I systematically explore the theoretical constraints of the assembly, predicting optimal assembly conditions, the requirements for particle stabilization and expected assembly time. With these constraints in mind, I evaluate the quality of the particles and improve the particle functionalization protocol. Additionally, I quantify the particle functionalization, uncovering discrepancies between reality and previous theoretical assumptions, leading to a deeper understanding of the assembly. To complete the understanding of key factors leading to assembly success or failure, I consider the role of individual components such as the protein, the particle chemistry and the assembly concept. I single out the particle reactivity as a key bottleneck, showing the necessary steps for assembly improvement. With this, I ensure future assembly success. With the knowledge from my investigations, I revisit a novel and previously not understood dynamic signal in the plasmon ruler experiment. Combining the insights gained on particle quality and particle-substrate interactions, I explain the origin of this signal as an artifact. With this I highlight the importance of particle quality control in the dimer experiment. The total sum of my work is a route for successful plasmon ruler assembly: I provide a recipe for optimal particle functionalization and assembly concept, ensuring reproducibility and robustness. This work serves as basis for future scientists, bringing the plasmon ruler method closer to a streamlined application in the single-molecule community. The improved robustness and reproducibility of the plasmon ruler will help uncover the remaining unknows of proteins such as Hsp90, closing the knowledge gaps in the areas such as e.g. protein memory effects and long-term behavior.