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Designing single-molecule assays towards directed emission with DNA origami nanoantennas
Designing single-molecule assays towards directed emission with DNA origami nanoantennas
Nanoscale light-control requires the precise positioning of nanooptical elements such as single quantum emitters and different kinds of nanoparticles. In this context, DNA origami nanostructures have proven a versatile scaffold to control positions and stoichiometry in an efficient self-assembly process. Besides spatial control, the close environment of organic fluorescent dyes often used as quantum emitters plays an important role. Changes in the environment can impact the properties of exposed fluorophores and DNA origami structures. In this thesis, DNA origami nanostructures are used to assemble a gap nanoantenna with directed emission properties using gold nanoparticles. Different single-molecule assays are developed to detail environmental effects relevant for the assembly of nanoantennas and other complex assemblies based on DNA origami structures. For optical antennas, single quantum emitters have to be placed at the best coupling position between the emitter and the nanophotonic structure, which is the plasmonic hotspot. Besides a precise placement, the orientation of the fluorophore’s transition dipole moment is a critical parameter for optimal coupling. Studying the importance of transition dipole moment orientations in an optical antenna is the subject of the first part of this thesis. A freely rotating dye is compared to a dye coupled to an optical antenna. The data shows that it is not only the emission transition dipole moment that has a defined orientation but also the absorption transition dipole moment. In addition, an alignment of both transition dipole moments is disclosed, revealing that the antenna’s main resonance mode dominates the absorption as well as the emission. Conclusively, this study suggests that controlling the transition dipole orientations of fluorophores can create highly efficient antennas with the ability to control light at the nanoscale, such as complex routers or directors. As the alignment of fluorophores is not straight forward the second part of this thesis deals with the development of an assay to report on the relative orientation of a single fluorophore in a DNA origami structure. By a unique combination of super-resolution microscopy and polarization-resolved excitation microscopy, the orientations of structurally different dyes in different DNA origami nano-environments are determined. Supplementary molecular dynamic simulations help to rationalize the measured orientations and to assign possible conformational states. It is shown that the immediate surrounding such as missing nucleotides but also the molecular structures of the fluorophores play an important role for preferred dye-DNA interactions. All studies presented in this thesis are carried out in aqueous buffers with additive salts to stabilize the DNA origami structures. However, the concentration and identity of added salts can be crucial for DNA origami stability and functionality. In this context, super-resolution imaging reveals the fortuitous finding that changes in the concentrations of bivalent salts yield structural changes in a DNA origami rectangle. An energy transfer assay employing a gold nanoparticle as acceptor even reveals dynamical changes and indicates rolling-up of the structure along the diagonal axis that cannot easily be detected by common microscopy techniques. Furthermore, it is proven that dynamic structures do not need to be built with complex motifs like hinges, joints or catenanes or even hybridization locks to be functional. To gain as many insights on the single-entity level, one main focus of this work is placed on the development of single-molecule assays. Developed single-molecule fluorescent microscopy techniques include a combination of polarization-resolved wide-field imaging and defocused imaging to report on the orientations of the absorption and emission transition dipole moments. With combined DNA-PAINT and polarization-resolved wide-filed measurements the orientations of DNA origami rectangles and related fluorophore orientations in the DNA origami structures can be revealed. Finally, by a combination of DNA-PAINT and scanning confocal fluorescence lifetime microscopy, high structural and temporal resolution in a dynamically switching DNA origami structure is gained. The developed assays have the potential to be useful for answering other scientific questions, in particular on the single-entity level.
DNA origami, single-molecule microscopy, plasmonics, nanophotonics, metallic nanoparticles, optical antenna, super-resolution microscopy, DNA-PAINT
Hübner, Kristina Anna
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hübner, Kristina Anna (2022): Designing single-molecule assays towards directed emission with DNA origami nanoantennas. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Nanoscale light-control requires the precise positioning of nanooptical elements such as single quantum emitters and different kinds of nanoparticles. In this context, DNA origami nanostructures have proven a versatile scaffold to control positions and stoichiometry in an efficient self-assembly process. Besides spatial control, the close environment of organic fluorescent dyes often used as quantum emitters plays an important role. Changes in the environment can impact the properties of exposed fluorophores and DNA origami structures. In this thesis, DNA origami nanostructures are used to assemble a gap nanoantenna with directed emission properties using gold nanoparticles. Different single-molecule assays are developed to detail environmental effects relevant for the assembly of nanoantennas and other complex assemblies based on DNA origami structures. For optical antennas, single quantum emitters have to be placed at the best coupling position between the emitter and the nanophotonic structure, which is the plasmonic hotspot. Besides a precise placement, the orientation of the fluorophore’s transition dipole moment is a critical parameter for optimal coupling. Studying the importance of transition dipole moment orientations in an optical antenna is the subject of the first part of this thesis. A freely rotating dye is compared to a dye coupled to an optical antenna. The data shows that it is not only the emission transition dipole moment that has a defined orientation but also the absorption transition dipole moment. In addition, an alignment of both transition dipole moments is disclosed, revealing that the antenna’s main resonance mode dominates the absorption as well as the emission. Conclusively, this study suggests that controlling the transition dipole orientations of fluorophores can create highly efficient antennas with the ability to control light at the nanoscale, such as complex routers or directors. As the alignment of fluorophores is not straight forward the second part of this thesis deals with the development of an assay to report on the relative orientation of a single fluorophore in a DNA origami structure. By a unique combination of super-resolution microscopy and polarization-resolved excitation microscopy, the orientations of structurally different dyes in different DNA origami nano-environments are determined. Supplementary molecular dynamic simulations help to rationalize the measured orientations and to assign possible conformational states. It is shown that the immediate surrounding such as missing nucleotides but also the molecular structures of the fluorophores play an important role for preferred dye-DNA interactions. All studies presented in this thesis are carried out in aqueous buffers with additive salts to stabilize the DNA origami structures. However, the concentration and identity of added salts can be crucial for DNA origami stability and functionality. In this context, super-resolution imaging reveals the fortuitous finding that changes in the concentrations of bivalent salts yield structural changes in a DNA origami rectangle. An energy transfer assay employing a gold nanoparticle as acceptor even reveals dynamical changes and indicates rolling-up of the structure along the diagonal axis that cannot easily be detected by common microscopy techniques. Furthermore, it is proven that dynamic structures do not need to be built with complex motifs like hinges, joints or catenanes or even hybridization locks to be functional. To gain as many insights on the single-entity level, one main focus of this work is placed on the development of single-molecule assays. Developed single-molecule fluorescent microscopy techniques include a combination of polarization-resolved wide-field imaging and defocused imaging to report on the orientations of the absorption and emission transition dipole moments. With combined DNA-PAINT and polarization-resolved wide-filed measurements the orientations of DNA origami rectangles and related fluorophore orientations in the DNA origami structures can be revealed. Finally, by a combination of DNA-PAINT and scanning confocal fluorescence lifetime microscopy, high structural and temporal resolution in a dynamically switching DNA origami structure is gained. The developed assays have the potential to be useful for answering other scientific questions, in particular on the single-entity level.