Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

<p>Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission<br> cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the<br> conditions for fluorescent dyes are even more demanding inDNAorigami nanoantennas. Here, we<br> briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal<br> additional factors relevant in the context of plasmonic fluorescence enhancement. Weshow that<br> despite the improved photostability of single-molecule fluorophores byDNAorigami nanoantennas,<br> their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying<br> photophysical processes, such as formation of dim states and photoisomerization. These photophysical<br> processes limit the photon count rates, increase heterogeneity and aggravate quantification of<br> fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved<br> in biophysical single-molecule experiments. Finally, we show how the photophysics of aDNAhairpin<br> assay with a fluorophore-quencher pair can be influenced by plasmonicDNAorigami nanoantennas<br> leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that<br> such assays can produce false positive results by premature photobleaching of the dark quencher. Here we demonstrate the raw data on which our findings based on.</p>