TY - THES A1 - Choi, Youngeun T1 - DNA origami structures as versatile platforms for nanophotonics T1 - DNA Origami Struktruen als Vielseitige Plattform für Nanophotonik N2 - Nanophotonics is the field of science and engineering aimed at studying the light-matter interactions on the nanoscale. One of the key aspects in studying such optics at the nanoscale is the ability to assemble the material components in a spatially controlled manner. In this work, DNA origami nanostructures were used to self-assemble dye molecules and DNA coated plasmonic nanoparticles. Optical properties of dye nanoarrays, where the dyes were arranged at distances where they can interact by Förster resonance energy transfer (FRET), were systematically studied according to the size and arrangement of the dyes using fluorescein (FAM) as the donor and cyanine 3 (Cy 3) as the acceptor. The optimized design, based on steady-state and time-resolved fluorometry, was utilized in developing a ratiometric pH sensor with pH-inert coumarin 343 (C343) as the donor and pH-sensitive FAM as the acceptor. This design was further applied in developing a ratiometric toxin sensor, where the donor C343 is unresponsive and FAM is responsive to thioacetamide (TAA) which is a well-known hepatotoxin. The results indicate that the sensitivity of the ratiometric sensor can be improved by simply arranging the dyes into a well-defined array. The ability to assemble multiple fluorophores without dye-dye aggregation also provides a strategy to amplify the signal measured from a fluorescent reporter, and was utilized here to develop a reporter for sensing oligonucleotides. By incorporating target capturing sequences and multiple fluorophores (ATTO 647N dye molecules), a reporter for microbead-based assay for non-amplified target oligonucleotide sensing was developed. Analysis of the assay using VideoScan, a fluorescence microscope-based technology capable of conducting multiplex analysis, showed the DNA origami nanostructure based reporter to have a lower limit of detection than a single stranded DNA reporter. Lastly, plasmonic nanostructures were assembled on DNA origami nanostructures as substrates to study interesting optical behaviors of molecules in the near-field. Specifically, DNA coated gold nanoparticles, silver nanoparticles, and gold nanorods, were placed on the DNA origami nanostructure aiming to study surface-enhanced fluorescence (SEF) and surface-enhanced Raman scattering (SERS) of molecules placed in the hotspot of coupled plasmonic structures. N2 - Nanophotonik bezeichnet die Untersuchung von Licht in Wechselwirkung mit Materie im Nanometermaßstab. Die exakte Kontrolle über den Aufbau und die räumliche Anordnung der beteiligten Komponenten ist ein entscheidender Faktor für die Erforschung der Optik nanoskalierter Systeme. Eine mögliche Lösung bietet die selbstorganisatorische Eigenschaft von DNA-Origami-Nanostrukturen, die im Rahmen dieser Dissertation, insbesondere zur Kopplung verschiedener Farbstoffe bzw. plasmonisch aktiver Nanopartikel, verwendet wurden. Im ersten Teil dieser Dissertation wurden unterschiedliche Förster-Resonanzenergietransfer- (FRET) Farbstoff-Matrizen, bestehend aus Fluorescein (FAM) als FRET-Donor und Cyanine 3 (Cy 3) als FRET-Akzeptor, hergestellt und nachfolgend hinsichtlich des Einflusses ihrer Gesamtgröße und ihrer Anordnung via statischer und zeitaufgelöster Fluoreszenzspektroskopie untersucht. Daraufhin erfolgte die Weiterentwicklung der ermittelten optimalen Anordnung der Farbstoffe in einen ratiometrischen pH-Sensor, bestehend aus dem pH stabilen Coumarin 343 (C343) als FRET-Donor und dem pH sensitiven FAM als FRET-Akzeptor. Die erhaltenen Ergebnisse zeigten, dass sich die Sensitivität ratiometrischer Sensoren, insbesondere durch die wohldefinierte Anordnung der beteiligten Farbstoffe in der Matrize, deutlich steigern lassen. Selbige Anordnung konnte auch erfolgreich zur Entwicklung eines Giftstoffsensors, zum Nachweis des Hepatoxins Thioacetamid (TAA), verwendet werden. Die Möglichkeit der Anordnung mehrerer Farbstoffe, unter Vermeidung ungewollter Farbstoff-Aggregation, ermöglicht außerdem die Verstärkung der Signale sogenannter Fluoreszenzreporter. Dies führte, im Rahmen dieser Arbeit, zur erfolgreichen Entwicklung eines auf Mikroperlen basierenden Oligonukleotid-Sensors, welcher ohne die Notwendigkeit einer vorherigen Zielverstärkung (z.B. durch Polymerase-Kettenreaktion) auskommt. Die anschließende Analyse mittels VideoScan, einer Multiplex-Analyse-Technik basierend auf der Fluoreszenzmikroskopie, ergab deutlich niedrigere Nachweisgrenzen für auf DNA-Origami basierende Reporter im Vergleich zu DNA-Einzelstrang basierenden Reportern. Abschließend erfolgte die Verwendung der DNA-Origamis als Substrat für die präzise räumliche Anordnung verschiedener plasmonisch aktiver Nanopartikel zur Untersuchung des optischen Verhaltens von Zielmolekülen im plasmonischen Nahfeld. Die Untersuchung der oberflächenverstärkten Fluoreszenz (SEF) und oberflächenverstärkten Raman-Streuung (SERS) von Molekülen im plasmonischer Hotspots erfolgte insbesondere mit Fokus auf den Einfluss der unterschiedlichen Anordnung von Gold-Nanostäbchen, Gold-Nanopartikel, und Silber-Nanopartikel. KW - DNA origami KW - Förster resonance energy transfer KW - plasmonics KW - DNA Origami KW - Förster-Resonanzenergietransfer KW - Plasmonik Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-421483 ER - TY - JOUR A1 - Choi, Youngeun A1 - Kotthoff, Lisa A1 - Olejko, Lydia A1 - Resch-Genger, Ute A1 - Bald, Ilko T1 - DNA origami-based forster resonance energy-transfer Nanoarrays and their application as ratiometric sensors JF - ACS applied materials & interfaces N2 - DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye-dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Forster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pH responsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences. KW - DNA origami KW - nanoarray KW - FRET KW - ratiometric sensing KW - pH sensing Y1 - 2018 U6 - https://doi.org/10.1021/acsami.8b03585 SN - 1944-8244 SN - 1944-8252 VL - 10 IS - 27 SP - 23295 EP - 23302 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Schmidt, Carsten A1 - Schierack, Peter A1 - Gerber, Ulrike A1 - Schroeder, Christian A1 - Choi, Youngeun A1 - Bald, Ilko A1 - Lehmann, Werner A1 - Rödiger, Stefan T1 - Streptavidin homologues for applications on solid surfaces at high temperatures JF - Langmuir N2 - One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 mu m in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 degrees C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability. Y1 - 2020 U6 - https://doi.org/10.1021/acs.langmuir.9b02339 SN - 0743-7463 VL - 36 IS - 2 SP - 628 EP - 636 PB - American Chemical Society CY - Washington ER -