Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Non-parametric estimation of the diffusion coefficient of a branching diffusion with immigration

Wir betrachten Systeme von endlich vielen Partikeln, wobei die Partikel sich unabhängig voneinander gemäß eindimensionaler Diffusionen [dX_t = b(X_t),dt + sigma(X_t),dW_t] bewegen. Die Partikel sterben mit positionsabhängigen Raten und hinterlassen eine zufällige Anzahl an Nachkommen, die sich gemäß eines Übergangskerns im Raum verteilen. Zudem immigrieren neue Partikel mit einer konstanten Rate. Ein Prozess mit diesen Eigenschaften wird Verzweigungsprozess mit Immigration genannt. Beobachten wir einen solchen Prozess zu diskreten Zeitpunkten, so ist zunächst nicht offensichtlich, welche diskret beobachteten Punkte zu welchem Pfad gehören. Daher entwickeln wir einen Algorithmus, um den zugrundeliegenden Pfad zu rekonstruieren. Mit Hilfe dieses Algorithmus konstruieren wir einen nichtparametrischen Schätzer für den quadrierten Diffusionskoeffizienten $sigma^2(cdot),$ wobei die Konstruktion im Wesentlichen auf dem Auffüllen eines klassischen Regressionsschemas beruht. Wir beweisen Konsistenz und einen zentralen Grenzwertsatz.