@phdthesis{Hildebrandt2006,
  author    = {Hildebrandt, Niko},
  title     = {Lanthanides and quantum dots : time-resolved laser spectroscopy of biochemical F{\"o}rster Resonance Energy Transfer (FRET) systems},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-12686},
  school      = {Universit{\"a}t Potsdam},
  year      = {2006},
  abstract  = {F{\"o}rster Resonance Energy Transfer (FRET) plays an important role for biochemical applications such as DNA sequencing, intracellular protein-protein interactions, molecular binding studies, in vitro diagnostics and many others. For qualitative and quantitative analysis, FRET systems are usually assembled through molecular recognition of biomolecules conjugated with donor and acceptor luminophores. Lanthanide (Ln) complexes, as well as semiconductor quantum dot nanocrystals (QD), possess unique photophysical properties that make them especially suitable for applied FRET. In this work the possibility of using QD as very efficient FRET acceptors in combination with Ln complexes as donors in biochemical systems is demonstrated. The necessary theoretical and practical background of FRET, Ln complexes, QD and the applied biochemical models is outlined. In addition, scientific as well as commercial applications are presented. FRET can be used to measure structural changes or dynamics at distances ranging from approximately 1 to 10 nm. The very strong and well characterized binding process between streptavidin (Strep) and biotin (Biot) is used as a biomolecular model system. A FRET system is established by Strep conjugation with the Ln complexes and QD biotinylation. Three Ln complexes (one with Tb3+ and two with Eu3+ as central ion) are used as FRET donors. Besides the QD two further acceptors, the luminescent crosslinked protein allophycocyanin (APC) and a commercial fluorescence dye (DY633), are investigated for direct comparison. FRET is demonstrated for all donor-acceptor pairs by acceptor emission sensitization and a more than 1000-fold increase of the luminescence decay time in the case of QD reaching the hundred microsecond regime. Detailed photophysical characterization of donors and acceptors permits analysis of the bioconjugates and calculation of the FRET parameters. Extremely large F{\"o}rster radii of more than 100 {\AA} are achieved for QD as acceptors, considerably larger than for APC and DY633 (ca. 80 and 60 {\AA}). Special attention is paid to interactions with different additives in aqueous solutions, namely borate buffer, bovine serum albumin (BSA), sodium azide and potassium fluoride (KF). A more than 10-fold limit of detection (LOD) decrease compared to the extensively characterized and frequently used donor-acceptor pair of Europium tris(bipyridine) (Eu-TBP) and APC is demonstrated for the FRET system, consisting of the Tb complex and QD. A sub-picomolar LOD for QD is achieved with this system in azide free borate buffer (pH 8.3) containing 2 \% BSA and 0.5 M KF. In order to transfer the Strep-Biot model system to a real-life in vitro diagnostic application, two kinds of imunnoassays are investigated using human chorionic gonadotropin (HCG) as analyte. HCG itself, as well as two monoclonal anti-HCG mouse-IgG (immunoglobulin G) antibodies are labeled with the Tb complex and QD, respectively. Although no sufficient evidence for FRET can be found for a sandwich assay, FRET becomes obvious in a direct HCG-IgG assay showing the feasibility of using the Ln-QD donor-acceptor pair as highly sensitive analytical tool for in vitro diagnostics.},
  language  = {en}
}
@phdthesis{Riedel2018,
  author    = {Riedel, Marc},
  title     = {Photonic wiring of enzymatic reactions to photoactive entities for the construction of biohybrid electrodes},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-417280},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VIII, 168},
  year      = {2018},
  abstract  = {In this work, different strategies for the construction of biohybrid photoelectrodes are investigated and have been evaluated according to their intrinsic catalytic activity for the oxidation of the cofactor NADH or for the connection with the enzymes PQQ glucose dehydrogenase (PQQ-GDH), FAD-dependent glucose dehydrogenase (FAD-GDH) and fructose dehydrogenase (FDH). The light-controlled oxidation of NADH has been analyzed with InGaN/GaN nanowire-modified electrodes. Upon illumination with visible light the InGaN/GaN nanowires generate an anodic photocurrent, which increases in a concentration-dependent manner in the presence of NADH, thus allowing determination of the cofactor. Furthermore, different approaches for the connection of enzymes to quantum dot (QD)-modified electrodes via small redox molecules or redox polymers have been analyzed and discussed. First, interaction studies with diffusible redox mediators such as hexacyanoferrate(II) and ferrocenecarboxylic acid have been performed with CdSe/ZnS QD-modified gold electrodes to build up photoelectrochemical signal chains between QDs and the enzymes FDH and PQQ-GDH. In the presence of substrate and under illumination of the electrode, electrons are transferred from the enzyme via the redox mediators to the QDs. The resulting photocurrent is dependent on the substrate concentration and allows a quantification of the fructose and glucose content in solution. A first attempt with immobilized redox mediator, i.e. ferrocenecarboxylic acid chemically coupled to PQQ-GDH and attached to QD-modified gold electrodes, reveal the potential to build up photoelectrochemical signal chains even without diffusible redox mediators in solution. However, this approach results in a significant deteriorated photocurrent response compared to the situation with diffusing mediators. In order to improve the photoelectrochemical performance of such redox mediator-based, light-switchable signal chains, an osmium complex-containing redox polymer has been evaluated as electron relay for the electronic linkage between QDs and enzymes. The redox polymer allows the stable immobilization of the enzyme and the efficient wiring with the QD-modified electrode. In addition, a 3D inverse opal TiO2 (IO-TiO2) electrode has been used for the integration of PbS QDs, redox polymer and FAD-GDH in order to increase the electrode surface. This results in a significantly improved photocurrent response, a quite low onset potential for the substrate oxidation and a broader glucose detection range as compared to the approach with ferrocenecarboxylic acid and PQQ-GDH immobilized on CdSe/ZnS QD-modified gold electrodes. Furthermore, IO-TiO2 electrodes are used to integrate sulfonated polyanilines (PMSA1) and PQQ-GDH, and to investigate the direct interaction between the polymer and the enzyme for the light-switchable detection of glucose. While PMSA1 provides visible light excitation and ensures the efficient connection between the IO-TiO2 electrode and the biocatalytic entity, PQQ-GDH enables the oxidation of glucose. Here, the IO-TiO2 electrodes with pores of approximately 650 nm provide a suitable interface and morphology, which is required for a stable and functional assembly of the polymer and enzyme. The successful integration of the polymer and the enzyme can be confirmed by the formation of a glucose-dependent anodic photocurrent. In conclusion, this work provides insights into the design of photoelectrodes and presents different strategies for the efficient coupling of redox enzymes to photoactive entities, which allows for light-directed sensing and provides the basis for the generation of power from sun light and energy-rich compounds.},
  language  = {en}
}