This work was performed in the framework of an interdisciplinary graduate program that focuses on the establishment and extension of innovative compounds for the packaging of electronic systems. Such chemically or biotechnologically tailored compounds can be used for the direct patterning of optically, magnetically or biologically functional structures in nano- and biotechnical products. In order to organize matter at the nanometer scale, imprinting litho-graphy techniques or self-organization processes are appropriate. Fine-tuning of numerous engineering processes requires continuous and high precision monitoring as well as control of diverse parameters. These demands are only partially met by physical or chemical components since they use surrogate parameters, measure off-line, or provide insufficient performances. Biological compounds, in particular protein-based feedback systems, fulfill certain system requirements to a considerable degree.
Hydrophobins and S-layer proteins are surface active proteins, produced by filamentous fungi or bacteria. In nature, these (self )assembly proteins form highly ordered and robust structures. In addition, their tolerance for different sequence manipulations and chemical modifications allows extensive functionalization of these nanometer-sized proteins. Hence, these surface active proteins can also be fused with other protein domains to create chimera, which retain function of both original proteins. In conclusion, both hydrophobins and S-layer proteins represent a versatile tool in numerous fields of applied biotechnology, medicine or diagnostics. But until now, efficient in vitro operation in molecular designed protein coatings is strongly restricted due to their complex assembly mechanism.
In the first phase of this work, it was demonstrated, that representatives of class I and class II hydrophobins tend to form multilayered structures on solid surfaces. It was found that only two protein orientations seems to be preferentially formed. In the process of assembly, the orientation of the first hydrophobin layer strictly depends on the substrate wettability. Consequently, each of the following hydrophobin layers is inverse oriented to the layer before. This alternating assembly mechanism has to be taken into account, when working with functionalized hydrophobins, because a hydrophobin-fused functional protein domain is exclusively located on one side of the protein. Due to the densely packed structure of surface active proteins, a fused functional domain, embedded between two hydrophobins is barely available for external reagents. Basically, the simultaneous existence of a broad spectrum of ordered and disordered assembly structures, demonstrated the need of an uniform protein film assembly for applications in fine-diagnostics or biomedicine.
With regard to molecular designed protein coatings, this work further aimed at establishing conditions to develop a method for a ‘layer-by-layer’ assembly of protein chimeras. Based on their amphiphilic character, self-assembly behavior of surface active proteins can be influenced by conventional ionic surfactants. In order to study the effect of surfactants on the composition and morphology of adsorbed protein films, contact angle measurements, nulling ellipsometry, SEM, AFM and AFAM were performed. It was found that the layer thickness of assembled protein films is strictly dependent on the amount of added surfactant. At certain threshold surfactant concentrations, hydrophobins and S-layer proteins assemble in uniform layers, which are as thick as expected for a protein monolayer or a bilayer. Assembled protein films are covered by a smooth surfactant layer, which prevents further protein assembly. AFAM measurements reveal the formation of well defined lattice structures under the coverage of surfactants. Even the removal of the surfactant layer is possible without inter-fering with protein specific secondary structures. Solvent accessibility and functionality of protein-fused domains was successfully demonstrated. As compared to conventional assembly techniques, this novel protein deposition method offers a possibility for a ‘directed’ protein coating on solid surfaces. In addition, it guarantees broadly ranged homogeneous assembly of protein chimeras on non-planar or even porous surfaces independent of their position.
Finally, a prototype for an interfacial FRET was developed in a close collaboration with the Institute of Physical Chemistry (TUD). This innovative FRET between semiconducting nano-particles and illuminating protein chimeras takes place across an oil/water interface. Hydro-phobins were used to stabilize artificial oil droplets in aqueous solution. These small proteins possess the ability to attach fused functional domains very close to an oil/water interface. When, in addition to this, an optically active nanostructure directly docks to the hydrophobin, the distance of a protein-fused domain and the nanostructure are in the range of the FÖRSTER radius. It was successfully demonstrated that quantum dots and fluorescent proteins fulfill the spectroscopic requirements of such a donor/acceptor pair. The FRET performance of these excitable oil droplets was examined as a ‘proof of concept’. Due to its modular design, this signal amplification setup could be exploited in numerous fields of technical application ranging from quantification of micronutrient to photothermal cancer therapy.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-101569 |
| Date | 18 December 2017 |
| Creators | Gruner, Leopold Joachim |
| Contributors | Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, Prof. Dr. Gerhard Rödel, Prof. Dr. Malte Gather |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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