Real-time binding kinetics of ligand-receptor interactions : development and application of a label-free optical biosensor for living bacteria

In principle, a major goal in drug development is to design a high-affinity compound with an appropriate pharmacological response, which requires understanding of physical processes underlying the drug-target interaction, affinity and specificity. This drug key information can be obtained by measuring drug binding kinetics in real time as the quantity of a ligand binding to a receptor immobilized on a biosensor chip surface. Almost all drugs independently of their targets interact with a complex cell membrane that incorporate various transmembrane proteins whose impact cannot be disregarded. Therefore, it is highly desirable that preclinical drug development is performed under conditions that are as close as possible to a native cell environment. The above reasons enhance the need for alternative approaches, such as whole-cell assays that are able to examine in vitro potential drug compounds. However, nowadays fragments of cell membranes have to be routinely isolated from the native cell environment due to limitations of currently available label-free technologies. In recent years, attempts were made to measure binding kinetics on living bacteria by surface plasmon resonance-based biosensors. Nevertheless, the experiments were not fully successful because of an insufficient penetration depth of the sensing evanescent field into living bacteria. Label-free biosensors based on metal-free photonic crystals support electromagnetic surface waves with a deeper penetration depth into the sample medium offering a solution for measuring binding interactions with living bacterial and, possibly, also eukaryotic cells. The deeper penetration of the surface waves creates a significant advantage for this non-disruptive technology over conventional immunoassays such as ELISA. For these reasons, label-free biosensors have been attracting a growing interest from pharmaceutical companies and research institutions by avoiding any experimental artefacts induced by labeling and allowing for real-time kinetic measurements. In this work, I contributed to the development of a label-free optical biosensor based on a photonic crystal that can be exploited for living bacteria. Moreover, I designed an experimental protocol and demonstrated how binding kinetics of various ligands, including antibodies and bacteriophages can be measured by the biosensor in vitro. Furthermore, this study attempts to apply the biosensor technology for bacterial viability tests exploring the ways in which the biosensor can detect metabolic activity of bacteria. Additionally, we performed theoretical studies on application of the Hillâ s and Mathieuâ s equations to study the effect of the photonic crystal design on the wave equation describing electromagnetic field distribution in the photonic crystal. This Thesis work demonstrates capabilities of a versatile biophysical method based on light interaction with living matter. In particular, the device sensitivity allows for measurements on living