Molecular Holograms - Design principles of robust biosensors at the example of focal molography

Abstract

Holograms have fascinated humans ever since their first creation nearly 70 years ago. On the other hand, label-free optical biosensors are an invaluable tool for molecular interaction analysis. This thesis is about applying holographic detection to biomolecular interaction analysis and by this overcoming many of the drawbacks of state-of-the-art biosensors. Over the past 30 years, refractometric biosensors, and in particular surface plasmon resonance, have matured to the de facto standard of this field. However, since their introduction no fundamental technological breakthrough that could address the major problems of refractometric transducers occured. Sensor equilibration, temperature drifts, buffer change artefacts and nonspecific binding are still significantly lowering throughput, limit the application scope and complicate the analysis of molecular binding experiments. Most importantly, the stabilization requirements and the cross-sensitivity of refractometric biosensors have impeded label-free (bio-)sensors to truly extend their scope beyond the controlled conditions of a laboratory environment. Molecular holograms or diffractometric biosensors should finally enable this step and create biosensors that can analyze molecular interactions in their natural habitat - the crowded environment of body fluids, tissues, cells and membranes. This thesis provides the physical explanation and the experimental evidence why this is not just a dream but actually very well possible. First, I introduce the spatial affinity lock-in and use signal processing to explain why diffractometric biosensors are finally solving the inherent stability problems of refractometric biosensors. Second, by simulation and experiments I show that molecular holograms achieve diffraction-limited focusing and derive mass quantification formulas and an optimization criterion for diffractometric biosensors. Third, I demonstrate that waveguide based diffractometric biosensors can function as a combined refractometric sensor. In addition, in a direct comparison of a state-of-the-art biosensor system to an unstabilized diffractometric biosensor I show that diffractometric biosensors surpass refractometric biosensors in terms of mass resolution and require less precise readout instrumentation. Lastly, I end with an in-depth noise analysis to identify the intrinsic noise limit of biosensors in general and the extrinsic noise limits of the setups developed in this thesis. In summary, this thesis provides the explanation why molecular holograms at optical frequencies are the physical principle to build robust and sensitive molecular sensors.