Abstract

Förster resonance energy transfer (FRET) is a common tool to measure the distances between a donor and an acceptor fluorophore and is employed as a spectroscopic ruler. This non-radiative energy transfer is utilized to not only measure distances but also to observe dynamics in the field of biophysics and medicine. However, main limitations of FRET are the limited time resolution and working range between donor and acceptor molecules of 10 nm. To increase the application of FRET, this limitation can be circumvented by the introduction of different ma-terials in the close proximity. For characterization of the altered distance dependence, a precise distance control between the dyes and the applied material is required, which here is provided by the DNA origami technique. In DNA origami, DNA self-assembles into programmable, complex, and robust structures, which can be easily modified with dyes and other entities with nanometric control. DNA origami nanoantennas constructed of a pair of gold nanoparticles have recently been introduced to substantially increase the obtainable fluorescence signal that yields a higher time resolution in biophysical single-molecule FRET experiments. In this context, it is crucial to understand the influence of the gold nanoparticles on the FRET process itself. In this work, gold nanoparticles are placed next to FRET pairs using the DNA origami technique (see publication I). A measurement procedure to accurately determine energy transfer efficiencies is estab-lished and reveals that in the intermediate coupling regime, the energy transfer efficiency drops in the presence of nanoparticles whereas the energy transfer rate constant from the donor to the acceptor is not significantly altered. Next, graphene is introduced to increase the range of energy transfer. Graphene is a 2D carbon lattice, which can also be employed as an unbleachable broadband acceptor without labeling. To understand the principles of the energy transfer between a fluorophore and the graphene surface, the distance dependence of the energy transfer from a fluorophore to graphene is investigated (see publication II). As such experiments require high quality graphene surfac-es, a cleaning and transferring procedure to generate reproducible graphene-on-glass-coverslips is established (see publication III) and characterized by different spectroscopic methods. Finally, the full potential of graphene-on-glass coverslips as microscopy platforms are highlighted by adopting graphene in the fields of biosensing, biophysics and super-resolution microscopy (see publication IV). The designed biosensors are capable to detect a DNA target, a viscosity change, or the binding of a biomolecule. In addition, FRET between two dyes is expanded by additional graphene energy transfer (GET) that reveals the relative orientation of the FRET pairs to the graphene surface. Finally, GET is used in super-resolution experiments to reach isotopic nanometric 3D-resolution and track a single fluorophore that undergoes 6-nm jumps. The developed techniques and assays have the potential to become the basis for numerous new applications in single-molecule sensing, biophysics, and super-resolution microscopy.