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

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

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part II. Experimental Demonstration

Label-free optical biosensors are an invaluable tool for molecular interaction analysis. Over the past 30 years, refractometric biosensors and, in particular, surface plasmon resonance have matured to the de facto standard of this field despite a significant cross reactivity to environmental and experimental noise sources. In this paper, we demonstrate that sensors that apply the spatial affinity lock-in principle (part I) and perform readout by diffraction overcome the drawbacks of established refractometric biosensors. We show this with a direct comparison of the cover refractive index jump sensitivity as well as the surface mass resolution of an unstabilized diffractometric biosensor with a state-of-the-art Biacore 8k. A combined refractometric diffractometric biosensor demonstrates that a refractometric sensor requires a much higher measurement precision than the diffractometric to achieve the same resolution. In a conceptual and quantitative discussion, we elucidate the physical reasons behind and define the figure of merit of diffractometric biosensors. Because low-precision unstabilized diffractometric devices achieve the same resolution as bulky stabilized refractometric sensors, we believe that label-free optical sensors might soon move beyond the drug discovery lab as miniaturized, mass-produced environmental/medical sensors. In fact, combined with the right surface chemistry and recognition element, they might even bring the senses of smell/taste to our smart devices

Ultra-stable molecular sensors by sub-micron referencing and why they should be interrogated by optical diffraction—part i. The concept of a spatial affinity lock-in amplifier

Label-free optical biosensors, such as surface plasmon resonance, are sensitive and well-established for the characterization of molecular interactions. Yet, these sensors require stabilization and constant conditions even with the use of reference channels. In this paper, we use tools from signal processing to show why these sensors are so cross-sensitive and how to overcome their drawbacks. In particular, we conceptualize the spatial affinity lock-in as a universal design principle for sensitive molecular sensors even in the complete absence of stabilization. The spatial affinity lock-in is analogous to the well-established time-domain lock-in. Instead of a time-domain signal, it modulates the binding signal at a high spatial frequency to separate it from the low spatial frequency environmental noise in Fourier space. In addition, direct sampling of the locked-in sensor’s response in Fourier space enabled by diffraction has advantages over sampling in real space as done by surface plasmon resonance sensors using the distributed reference principle. This paper and part II hint at the potential of spatially locked-in diffractometric biosensors to surpass state-of-the-art temperature-stabilized refractometric biosensors. Even simple, miniaturized and non-stabilized sensors might achieve the performance of bulky lab instruments. This may enable new applications in label-free analysis of molecular binding and point-of-care diagnostics.ISSN:1424-822

Ultra stable molecular sensors by submicron referencing and why they should be interrogated by optical diffraction—part ii. Experimental demonstration

Label-free optical biosensors are an invaluable tool for molecular interaction analysis. Over the past 30 years, refractometric biosensors and, in particular, surface plasmon resonance have matured to the de facto standard of this field despite a significant cross reactivity to environmental and experimental noise sources. In this paper, we demonstrate that sensors that apply the spatial affinity lock-in principle (part I) and perform readout by diffraction overcome the drawbacks of established refractometric biosensors. We show this with a direct comparison of the cover refractive index jump sensitivity as well as the surface mass resolution of an unstabilized diffractometric biosensor with a state-of-the-art Biacore 8k. A combined refractometric diffractometric biosensor demonstrates that a refractometric sensor requires a much higher measurement precision than the diffractometric to achieve the same resolution. In a conceptual and quantitative discussion, we elucidate the physical reasons behind and define the figure of merit of diffractometric biosensors. Because low-precision unstabilized diffractometric devices achieve the same resolution as bulky stabilized refractometric sensors, we believe that label-free optical sensors might soon move beyond the drug discovery lab as miniaturized, mass-produced environmental/medical sensors. In fact, combined with the right surface chemistry and recognition element, they might even bring the senses of smell/taste to our smart devices.ISSN:1424-822

Total internal reflection focal molography (TIR-M)

Focal molography (“molography” in short) is a sensitive implementation of a diffractometric biosensor and has emerged as a new platform technology to study biomolecular interactions label-free in complex fluids and living cells. In contrast to established refractometric biosensors, in particular surface plasmon resonance, molography is almost insensitive to environmental noise, i.e. temperature gradients and nonspecific binding. Molography achieves this by modulating the analyte binding at a high spatial frequency and reads it out in Fourier space via diffraction of light at the bound molecules, i.e. molography applies the spatial lock-in principle for discrimination of the binding signal from disturbing effects on the sensor surface. In previous implementations of focal molography, the sensor was illuminated by a waveguide mode. While this arrangements has an outstanding resolution, it suffers from several drawbacks such as wavefront instabilities of the guided mode, the relatively high refractive index contrast at the waveguide interfaces and the manufacturing cost of waveguide and grating couplers. In this paper, we propose a simpler and more robust configuration for focal molography. Instead of a waveguide mode, it is based on darkfield illumination by total internal reflection (TIR) of a free space mode. We derive the coherent binding pattern, describe the fabrication process, show that its intensity distribution is as expected, derive the quantitative readout formula and perform a background and noise analysis. Real-time binding curves of streptavidin in buffer and concentrated bovine serum albumin solution show that TIR molography exhibits excellent resolution and robustness.ISSN:0925-400

Image reversal reactive immersion lithography improves the detection limit of focal molography

Focal molography is a label-free optical biosensing method that relies on a coherent pattern of binding sites for biomolecular interaction analysis. Reactive immersion lithography (RIL) is central to the patterning of molographic chips but has potential for improvements. Here, we show that applying the idea of image reversal to RIL enables the fabrication of coherent binding patterns of increased quality (i.e., higher analyte efficiency). Thereby the detection limit of focal molography in biological assays can be improved

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells