Quantitative Diffractometric Biosensing


9. March 2021

This paper provides formulas for the quantification of the amount of analyte that has bound to any two dimensional diffractometric biosensor. Prior to this publication the majority of published diffractometric biosensors were not quantitative. This severely impeded assay independent comparison to established refractometric biosensors such as surface plasmon resonance. With this publication one can now compare the sensitivity and resolution of any diffractometric biosensor to established refractometric devices (Frutiger et al. 2020). Read more about it in our latest paper.

Investigating Complex Samples with Molograms of Low-Affinity Binders


25. February 2021

This paper stresses the importance of affinity (binding strength) matching of the binding elements on the active and passive regions on a mologram for background molecules (molecules that we do not want to detect but are present in the sample). More importantly, it introduces a completely new concept for medical diagnostic. Unlike traditional diagnostic methods that rely on fluorophores, multiple steps and are limited to one kind of molecule, this concept uses real-time data without any labels. In addition, since an entire array of different molograms is measured, one can use machine learning and classification to detect not only one but multiple diseases with the same sensor in one step.

The Concept of a Spatial Affinity Lock-in Amplifier


11. January 2021

Focal molography – a new method for Biomolecular Interaction Analysis (BIA).
The paper introduces and demonstrated the concept of the “spatial affinity lock-in” as a novel design principle to overcome the drawbacks of established BIA methods.The spatial affinity lock-in is analogous to the 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. Focal molography applies this fundamental detection principle to BIA. Combined with the right surface chemistry and recognition elements on the sensor surface focal molography enables robust, sensitive and fundamentally new BIA assays such as the direct and label-free monitoring of biomolecular interaction on the cell membrane.
Read more about ultra-stable molecular sensors by sub-micron referencing and why they should be interrogated by optical diffraction in our latest paper.

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


22. December 2020

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.

Focal Molography – an optical method for label-free detection of biomolecular interactions


1. October 2020

Focal molography is a new method for label-free molecular interaction analysis in crude samples. In contrast to refractometric optical sensors, focal molography is insensitive to nonspecific molecular interactions. This unique property is achieved with a special 2D nanopattern of molecular binding sites on a chip, termed mologram. A mologram is designed such that molecules bound to it diffract light constructively into a focal spot. The intensity of the focused light is measured to quantify the amount of bound molecules. In biological samples, highly abundant off-target molecules readily adsorb to the surface of the sensor. Yet, this process is completely random and the off-target molecules do not bind to the ordered binding sites of the mologram. Thus, their scattering is uniform in all spatial directions and therefore they hardly contribute to the measured light intensity in the narrow solid angle of the focal spot.

Quantification of Molecular Interactions in Living Cells in Real Time using a Membrane Protein Nanopattern


11. June 2020

Molecular processes within cells have traditionally been studied with biochemical methods due to their high degree of specificity and ease of use. In recent years, cell-based assays have gained more and more popularity since they facilitate the extraction of mode of action, phenotypic, and toxicity information. However, to provide specificity, cellular assays rely heavily on biomolecular labels and tags while label-free cell-based assays only offer holistic information about a bulk property of the investigated cells. Here, we introduce a cell-based assay for protein–protein interaction analysis. We achieve specificity by spatially ordering a membrane protein of interest into a coherent pattern of fully functional membrane proteins on the surface of an optical sensor. Thereby, molecular interactions with the coherently ordered membrane proteins become visible in real time, while nonspecific interactions and holistic changes within the living cell remain invisible. Due to its unbiased nature, this new cell-based detection method presents itself as an invaluable tool for cell signaling research and drug discovery.

Principles for Sensitive and Robust Biomolecular Interaction Analysis


28. January 2019

Label-free biosensors enable the monitoring of biomolecular interactions in real time, which is key to the analysis of the binding characteristics of biomolecules. While refractometric optical biosensors such as surface plasmon resonance (SPR) are sensitive and well-established, they are susceptible to any change of the refractive index in the sensing volume caused by minute variations in composition of the sample buffer, temperature drifts, and most importantly nonspecific binding to the sensor surface in complex fluids such as blood. The limitations arise because refractometric sensors measure the refractive index of the entire sensing volume. Conversely, diffractometric biosensors–for example, focal molography–only detect the diffracted light from a coherent assembly of analyte molecules. Thus any refractive index distribution that is noncoherent with respect to this molecular assembly does not add to the coherent signal. This makes diffractometric biosensors inherently robust and enables sensitive measurements without reference channels or temperature stabilization. The coherent assembly is generated by selective binding of the analyte molecules to a synthetic binding pattern–the mologram. Focal molography has been introduced theoretically [C. Fattinger, Phys. Rev. X 4, 031024 (2014)] and verified experimentally [V. Gatterdam, A. Frutiger, K.-P. Stengele, D. Heindl, T. Lübbes, J. Vörös, and C. Fattinger, Nat. Nanotechnol. 12, 1089 (2017)] in previous papers. However, further understanding of the underlying physics and a diffraction-limited readout is needed to unveil its full potential. This paper introduces refined theoretical models, which can accurately quantify the amount of biological matter bound to the mologram from the diffracted intensity. In addition, it presents measurements of diffraction-limited molographic foci, i.e., Airy discs. These improvements enable us to demonstrate a resolution in real-time binding experiments comparable to the best SPR sensors without the need for temperature stabilization or drift correction and to detect low-molecular-weight compounds label free in an endpoint format. The presented experiments exemplify the robustness and sensitivity of the diffractometric sensor principle.

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


26. November 2018

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 bi ological assays can be improved.

Focal molography is a new method for the in situ analysis of molecular interactions in biological samples


25. September 2017

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the ‘mologram’. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.

Coherent Signal Picks Out Biomolecular Interactions


11. August 2014

Proteins rarely act alone: Their functioning requires that they establish contact with other proteins and molecules, mostly through noncovalent interactions (i.e., interactions that do not involve the sharing of electrons, such as hydrogen-bond and van der Waals interactions). The study of such interactions is key for the understanding of biology at the molecular level, and may have important implications for drug discovery or the development of diagnostic tests. It is thus crucial to develop measurement techniques that can selectively probe these interactions, characterizing, for instance, the formation rate and strength of a specific protein-ligand complex, while discriminating from other, nonspecific interactions (like those arising, for instance, between a protein and fluctuating solute molecules). Writing in Physical Review X, Christof Fattinger, of Roche Innovation Center, Basel, Switzerland, investigates theoretically a novel analytical method, called focal “molography” (molecular holography), which represents a potential breakthrough for the selective detection of molecular interactions.