Invesigating plasmonically driven DNA denaturation with single molecule fluorescence imaging

Plasmonic nanoparticles have been used in applications ranging from green chemistry to gene therapy due to their unique light-matter interactions, which enable them to convert incident light into other forms of energy, such as heat (thermal) and chemical reactivity (nonthermal). By conjugating DNA to nanoparticle surfaces, researchers have been able to trigger the release of genetic material into cells simply by exposing the particles to plasmon-resonant light. Initially, it was believed that heat was responsible for the release; however, research has shown that enhanced DNA denaturation near excited nanoparticles enables release at temperatures below expectations. The exact mechanism that enables plasmon-enhanced melting remains elusive, as both thermal and nonthermal decay pathways can occur simultaneously when plasmons are excited. In this dissertation, we aim to elucidate the roles that various plasmon decay pathways may play in enhanced DNA denaturation. In Chapter 1, we discuss the decay pathways of plasmons and the debated roles of thermal and nonthermal effects, particularly in relation to plasmonically enhanced DNA denaturation. We also highlight the fundamentals of super-resolution imaging necessary to understand the single-molecule strategies we employed, which are sensitive enough to detect single strands of DNA bound to nanoparticle surfaces. In Chapter 2, we give technical details on the sample preparation and methodologies used herein. In Chapter 3, we conducted a proof-of-concept study in which surface-tethered DNA was melted via controlled external heating. After a sample is exposed to thermal energy, we use DNA-PAINT single-molecule counting to determine how much DNA is released as a function of applied temperature, finding that our methodology could repeatably measure melting curves for DNA bound to surfaces across a range of DNA strands and salt conditions. This work demonstrated that we could reliably determine DNA thermodynamics at nanoscale interfaces during thermally driven denaturation. In Chapter 4, we transitioned to plasmon-induced denaturation, in which we drove DNA melting from gold nanotriangle arrays using quadrupole plasmon-resonant laser light. By directly comparing thermal to light-induced melting, we isolate the thermal and nonthermal contributions and find evidence for the hypothesis that chemical reactivity (hot carriers) destabilizes double-stranded DNA, causing it to melt at temperatures below expectations. We also found a complex interplay between the stability of a DNA strand (Tm) and its magnitude of nonthermal destabilization, leading all DNA strands to melt at nearly the same temperature, reproducing trends from earlier work in the field. In Chapter 5, we develop a dual super-resolution imaging technique that allows correlation of super-resolved DNA PAINT fluorescent events with nanoparticle structural features. This provided a method to directly visualize where single-stranded DNA was on the surface of nanoparticles, and by extension, where DNA melted. Lastly, in Chapter 6, we fully leverage the super-resolution capabilities of DNA-PAINT to study plasmon-enhanced melting at two length scales: across the array in response to a non-uniform light source, and at the individual triangle level. This work highlighted spatial differences in thermal and nonthermal effects, yielding a mechanistic picture that distinguishes DNA based on charge-transfer efficiency, while rejecting a near-field-mediated destabilization hypothesis. This dissertation bolsters the speculation that hot carriers can enhance DNA melting, leading us to believe we could design conditions (nanoparticle size, shape, and DNA conjugation linker) that leverage them to deliver genetic material to cells with minimal hyperthermic cellular damage.