DNA origami-templated individual gold nanoclusters: probing their photophysical dynamics using single-molecule fluorescence spectroscopy

Single-molecule fluorescence microscopy demands ultrahigh stability of single fluorophores with less photobleaching and essentially no intensity fluctuations on experimentally relevant time scales. In this respect, there is a need to design fluorescent probes with excellent photostability and long-lived dark transient states. Herein, we report the site-specific synthesis of gold nanoclusters (Au NCs) on DNA origami. Rectangular DNA origami bearing a poly-cytosine sequence in the centre was used as a template to guide the in situ synthesis of Au NCs. The photophysical properties of DNA-templated AuNCs were extensively studied using single-molecule confocal fluorescence microscopy, which revealed the average on-time and off-time of 0.08 seconds and 0.05 seconds, respectively. Photon antibunching measurements confirmed the existence of single Au NCs in the confocal volume and fluorescence transient studies revealed their excellent brightness and remarkable photostability with long survival times and less blinking. Such DNA-templated Au NCs with ultrahigh photostability, intense brightness, and low toxicity have tremendous potential to be used as probes in single-molecule dynamics measurements on biologically relevant timescales. The precise addressability of DNA origami nanostructures provides the controlled attachment of various biomolecules specifically at a predesigned location on the origami template, allowing such platforms to be used for single nanocatalysis as well as performing imaging and sensing studies at the single-molecule level.