Abstract

Optical biosensing with single-molecule sensitivity requires high performant fluorescent probes. Many different functionalities have to be combined into small chemical entities. In this thesis, the DNA origami technique was used to tailor single-molecule biosensors according to their diverse needs as it offers a modular probe development with straightforward iteration possibilities. The focus was on a nucleic acid detection assay for future in vitro diagnostic applications and on voltage sensors to be applied to cell membranes. Particularly, for the nucleic acid detection assay, a silver nanoparticle was bound to a DNA origami pillar yielding a so-called nanoantenna which enhances the fluorescence of a dye in close proximity through plasmonic field interaction. This phenomenon was used to increase the optical signal released from a single DNA hairpin equipped with a dye-quencher pair. In the presence of a specific target nucleic acid, the hairpin’s secondary structure was broken and a fluorescence signal was observed. Using a hairpin sensing Zika specific sequences, the assay was characterized in terms of hairpin opening yield and fluorescence enhancement as well as single-nucleotide variation sensitivity and multiplexing ability. Further, diagnostic conditions were imitated by enriching heat-inactivated human serum with target DNA and using RNA targets. The presented detection assay yielded promising results for further development and future application in in vitro diagnostic assays at the point-of-care. In addition, two single-molecule biosensors for electrical membrane potentials were developed; one sensor for transmembrane potentials and a second one for membrane surface charges of lipid head groups. Both sensors translated the voltage into Förster Resonance Energy Transfer (FRET) signals. A rectangular DNA origami was used as an assembly platform with different optional modifications, i.e. for membrane targeting, surface immobilization and voltage sensing. In both sensors, the sensing unit protruding from the origami plate, consisted of DNA and carried a FRET-compatible dye pair. The red dye anchored the sensing unit to the membrane and provided a stable FRET acceptor. A green dye was placed on DNA between the membrane and the DNA origami plate and flexibly changed its conformation in response to the voltages which resulted in the desired FRET change. For both, the transmembrane and the surface charge sensor, the sensing unit’s chemical structure was adapted to meet the different requirements. The functionality of the transmembrane potential sensor was tested using liposomes with defined electrical potentials. It was shown that changes in the transmembrane potential were translated into different single-molecule FRET signals. Further, by introducing small chemical variations in the molecular structure of the sensing unit, the biosensor’s sensitivity was changed to respond either to de- or hyperpolarized membranes. Also, the membrane charge sensor yielded promising results; changes in the amounts of anionic lipids in liposomes resulted in different FRET signals. These findings suggested a quantitative translation of membrane surface charges into optical signals and were read out on the level of single sensors. Both sensing mechanisms were further characterized with molecular dynamic (MD) simulations for the transmembrane potential sensor and with fluorescence correlation spectroscopy (FCS) for the membrane surface charge sensor. Overall, three different biosensors with optical single-molecule read-out were introduced in this thesis using DNA origami as an assembly platform. The sensors were examined for potential diagnostic applications and future in vivo voltage imaging. The presented results underline the potential of DNA origami for further single-molecule biosensors beyond the ones investigated within this thesis.