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In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors.
Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123.
Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal.
Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields.
Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins.
In this Thesis, the properties of aqueous hemicellulose polysaccharides are investigated using computer simulations. The high swelling capacity of materials composed of these molecules allows the generation of directed motion in plant materials entirely controlled by water uptake.
To explore the molecular origin of this swelling capacity, a computational model with atomistic resolution for hemicellulose polysaccharides is build and validated in comparison with experiments. Using this model, simulations of small polysaccharides are employed to gain an understanding of the interactions of these molecules with water, the influence of water on their conformational freedom, and the swelling capacity quantified in terms of osmotic pressure. It is revealed that the branched hemicellulose polysaccharides show different hydration characteristics compared to linear polysaccharides.
To study swelling properties on length and time scales that exceed the limitations imposed by atomistic simulations, a procedure to obtain transferable coarse-grain models is developed. The transferability of the coarse-grain models over both different degrees of polymerization as well as different solute concentrations is demonstrated. Therefore, the procedure allows the construction of large coarse-grained systems based on small atomistic reference systems. Finally, the coarse-grain model is applied to demonstrate that linear and branched polysaccharides show a different swelling behavior when coupled to a water bath.
In freshwater sciences, nitrogen gained increasing attention in the past as an important resource potentially influencing phytoplankton growth and thus eutrophication. Most studies and all management approaches, however, are still restricted to dissolved inorganic nitrogen (DIN = nitrate + nitrite + ammonium) since dissolved organic nitrogen (DON) was considered to be refractory for most of the photoautotrophs. In the meantime this assumption has been disproved for all aquatic systems. While research on DON in marine ecosystems substantially increased, in freshwater a surprisingly small number of investigations has been carried out on DON utilization by phytoplankton or even the occurrence and seasonal development of total DON or its compounds in lakes. Therefore, our present knowledge on DON utilization by phytoplankton is often based on single species experiments using a sole, usually low molecular weight DON component, often in unnaturally high amounts mainly carried out with marine phytoplankton species. Thus, we know that some phytoplankton species can take up different DON fractions if they are available in high concentrations and as sole nitrogen source. This does not necessarily imply that phytoplankton would perform likewise in natural environments. In addition, it will be difficult to draw conclusions on the behavior of freshwater phytoplankton from experiments with marine phytoplankton since the nutrient regime in marine environments differs from that of freshwater. In the light of the parallel availability of inorganic and organic nitrogen species in natural freshwater ecosystems, several questions must be raised: "If inorganic nitrogen is available, would phytoplankton really rely on an organic nitrogen source? Could a connection be detected between the seasonal development of DON and changes in the phytoplankton community composition as found for inorganic nitrogen? And if we reduce the input of inorganic nitrogen in lakes and rivers would the importance of DON as nitrogen source for phytoplankton increase, counteracting all management efforts or even leading to undesired effects due to changes in phytoplankton physiology and biodiversity?" I experimentally addressed the questions whether those DON compounds differentially influence growth, physiology and composition of phytoplankton both as sole available nitrogen source and in combination with other nitrogen compounds. I hypothesized that all offered DON - compounds (urea, natural organic matter (NOM), dissolved free and combined amino acids (DFAA, DCAA)) could be utilized by phytoplankton at natural concentrations. However, I assumed that the availability would decrease with increasing compound complexity. I furthermore hypothesized that the occurrence of low DIN concentrations would not affect the utilization of DON negatively. The nitrogen source, whatsoever, would have an impact on phytoplankton physiology as well as community composition. To investigate these questions and assumptions I conducted bioassays with algae monocultures as well as phytoplankton communities testing the utilization of various DON compounds by several freshwater phytoplankton species. Especially the potential utilization of NOM, a complex DON compound mainly consisting of humic substances is of interest, since it is usually regarded to be refractory. In order to be able to use natural concentrations of DON - compounds for my experiments the concentration of total DON and some DON - compounds (urea, humic substances, heigh molecular weight substances) was assessed in Lake Müggelsee. All compounds were able to support algae growth in the low natural concentrations supplied. However, I found that the offered DON compounds differ in their availability to various algae species, both, as sole nitrogen source or in combination with low DIN concentrations. As expected, the availability decreased with increasing complexity of the nitrogen compound. Furthermore, I could show that changes in algal physiology (nitrogen storage, metabolism) occur depending on the utilized nitrogen source. Especially the secondary photosynthetic pigment composition, heterocyst frequency and C:N - ratio of the algae were affected. The uptake and usage of certain nitrogen compounds might be more costly, potentially resulting in those physiology changes. Whereas laboratory experiments with single species revealed strong effects of DON, algal responses to DON in a multi-species situation remain unclear. Experiments with phytoplankton communities from Lake Müggelsee revealed that the nitrogen pool composition does influence the phytoplankton community structure. The findings furthermore show that several species combined might utilize the supplied nitrogen completely different than monocultures in the laboratory. Thus, besides the actual ability of algae to use the offered nitrogen sources other factors, such as interspecific competition, may be of importance. I further investigated, if the results of the laboratory experiments, can be verified in the field. Here, I surveyed the seasonal development of several dissolved organic matter (DOM) components (urea, high molecular weight substances (HMWS), humic substances (HS)) and associated parameters (Specific UV-absorption (SUVA), C:N - ratio) in Lake Müggelsee between 2011 and 2013. Furthermore, data from the long term measurements series of Lake Müggelsee such as physical (temperature, light, pH, O2) and chemical parameters (nitrogen, phosphorous, silica, inorganic carbon), zooplankton and phytoplankton data were used to investigate how much of the variability of the phytoplankton composition in Lake Müggelsee can be explained by DON/DOM concentration and composition, relative to the other groups of explanatory variables. The results show that DON mainly consists of rather complex compounds such as humic substances and biopolymers (80 %) and that only slight seasonal trends are detectable. Using variance partitioning I could show, that the usually investigated nutrients (DIN, silica, inorganic carbon, phosphorous) and abiotic factors together explain most of the algae composition as was to be expected (57.1 % of modeled variance). However, DOM and the associated parameters uniquely explain 10.3 % of the variance and thus slightly more than zooplankton with 9.3 %. I could therefore prove, that the composition of DOM (nitrogen and carbon) is connected to the algae composition in an eutrophic lake such as Lake Müggelsee. DON - compounds such as urea, however, could not be correlated with the occurrence of specific phytoplankton species. Overall, the results of this study imply that DON can be a valuable nitrogen source for freshwater phytoplankton. DON is used by various species even when DIN is available in low concentrations. Through the reduction of DIN in lakes and rivers, the DON:DIN ratio might be changed, resulting even in an increased importance of DON as phytoplankton nitrogen source. My work suggests that not only N2-fixation but also DON utilization might compensate for reduced N - input. Changes from DIN to DON as main nitrogen source might also promote certain, potentially undesired algae species and influence the biodiversity of a limnic ecosystem through changes in the phytoplankton community structure. Thus, DON, especially urea, should be included in calculations concerning total available nitrogen and when determining nitrogen threshold values. Furthermore, the input-reduction of DON, for example from waste-water treatment plants should also be evaluated and the results of my thesis should find consideration when planning to reduce the nitrogen input in freshwater.