Real-time microsecond dynamics of single biomolecules probed by plasmon-enhanced fluorescence

Biomolecules exhibit dynamics on widely varying timescales, which are critical to their functioning in living systems. Particularly sub-millisecond dynamics, such as low-affinity ligand binding and interactions of individual binding sites within multivalent complexes, remain elusive. These dynamics are typically probed in real-time using single-molecule fluorescence, but photophysics limits it to millisecond timescales. Here, we exploit and optimize the strong plasmonic fluorescence enhancement provided by monocrystalline gold nanoparticles to detect $>10^7$ photons/s from single fluorophores. The ultrahigh signals uniquely enable real-time single-molecule fluorescence studies with 1-10 $\mu$s temporal resolution, and single-molecule correlation spectroscopy within $<1$ s. We use this method to reveal molecular diffusion and ultralow-affinity DNA interactions on microsecond timescales. We further exploit the field gradient around the nanoparticles to reveal dynamic binding transitions of multivalently binding Holliday junctions. Our results pave the way towards real-time microsecond studies of biomolecular complexes using an implementation compatible with existing single-molecule fluorescence methods.