The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

Abstract

JOSHA is a service that helps scholars, researchers, and students descover, use, and build upon a wide range of content DeclarationI hereby confirm that this master thesis at hand is solely my own written work and that no other sources, than those indicated in the references, were used.All text passages and diagrams, quoted or paraphrased, from these sources have been properly acknowledged as such and fully cited.This thesis has not been submitted in the same, similar or even partial version to another examination board and was not published elsewhere. In the past few years, this technology has developed a lot with the implementation of novel surface chemistries, detection techniques, and different assay formats. 3D-hydrogel microarrays were developed using unique immobilization techniques based on hydrophilic polymer networks. The 3D polymer network attaches and immobilizes the capture molecules onto the surface in form of a spot with molecules immobilized both on the surface and inside the gel matrix. This retains the molecule's natural confirmation and structure and enables higher immobilization efficiency, which allows for higher sensitivity. However, the achieved sensitivities are still far from the theoretical limit and in many cases, long incubation times are required. These limitations are caused by both the resolution of the used detection techniques and the assay kinetics. This hinders the transfer of the technology from the laboratory to routine diagnostics.The kinetics of a biochemical assay in a microarray can be controlled either by the transport of molecules to the spot or by the binding interaction itself. This work focuses on studying the assay kinetics in 3D-hydrogel microarrays in order to understand and characterize the limiting step for signal development. To simulate conditions of high affinity binding partners and therefore reduce the influence of the reaction kinetics, the biotin-streptavidin system was selected as the biological model for this work.A microarray to study the kinetic processes involved in the signal development was designed and the optimum working concentrations for kinetic characterization were defined. To confirm the mass transport limited kinetics, the measured kinetics was compared to the ideal reaction kinetics depending on the affinity parameters for biotin-streptavidin interaction. The ideal reaction kinetics was three orders of magnitude faster than measured kinetics. The two-compartment model, which is widely used to describe the assay kinetics in 2D microarrays, was used to fit the observed kinetics. However, a deviation from the model after the initial phase of signal development was observed. A hypothesis was made that this deviation is due to an additional diffusion step in the hydrogel. Therefore, a microarray model to study this diffusion step was designed. In this model, the microarrays were dip coated with hydrogel layers of various thickness and mesh sizes. This model simulated conditions where the signal development should depend only on the diffusion t...