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

Frutiger, Andreas; Gatterdam, Karl; Blickenstorfer, Yves; Reichmuth, Andreas M.; Fattinger, Christof; Vörös, János

doi:10.3390/s21010009

Cited by 9 publications

(9 citation statements)

References 87 publications

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“…However, macroscopic references fail to capture micron-scale differences, such as surface composition or temperature, in sensing and referencing channels. Diffractometric biosensors, − in particular focal molography, , provide the ability to address this challenge by employing a molecular diffraction grating, which may serve the double purpose of readout and nanoscale referencing. , However, diffractometric biosensors can also be affected by nonspecific binding if they are designed carelessly as has been pointed out previously . To estimate the fraction of occupied binding sites due to coherently, nonspecifically bound molecules, let us start with the general case of affinity biosensors.…”

Section: Resultsmentioning

confidence: 99%

“…However, macroscopic references fail to capture micron-scale differences, such as surface composition or temperature, in sensing and referencing channels. Diffractometric biosensors, 33−40 in particular focal molography, 39,40 provide the ability to address this challenge by employing a molecular diffraction grating, which may serve the double purpose of readout and nanoscale referencing. 39,40 However, diffractometric biosensors can also be affected by nonspecific binding if they are designed carelessly as has been pointed out previously.…”

Section: ■ Resultsmentioning

confidence: 99%

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Investigating Complex Samples with Molograms of Low-Affinity Binders

et al. 2021

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In vitro diagnostics relies on the quantification of minute amounts of a specific biomolecule, called biomarker, from a biological sample. The majority of clinically relevant biomarkers for conditions beyond infectious diseases are detected by means of binding assays, where target biomarkers bind to a solid phase and are detected by biochemical or physical means. Nonspecifically bound biomolecules, the main source of variation in such assays, need to be washed away in a laborious process, restricting the development of widespread point-of-care diagnostics. Here, we show that a diffractometric assay provides a new, label-free possibility to investigate complex samples, such as blood plasma. A coherently arranged sub-micron pattern, that is, a peptide mologram, is created to demonstrate the insensitivity of this diffractometric assay to the unwanted masking effect of nonspecific interactions. In addition, using an array of low-affinity binders, we also demonstrate the feasibility of molecular profiling of blood plasma in real time and show that individual patients can be differentiated based on the binding kinetics of circulating proteins.

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Section: Resultsmentioning

confidence: 99%

“…However, macroscopic references fail to capture micron-scale differences, such as surface composition or temperature, in sensing and referencing channels. Diffractometric biosensors, 33−40 in particular focal molography, 39,40 provide the ability to address this challenge by employing a molecular diffraction grating, which may serve the double purpose of readout and nanoscale referencing. 39,40 However, diffractometric biosensors can also be affected by nonspecific binding if they are designed carelessly as has been pointed out previously.…”

Section: ■ Resultsmentioning

confidence: 99%

Investigating Complex Samples with Molograms of Low-Affinity Binders

et al. 2021

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“…20 min) without any drift correction. If a proper noise characterization is required, the power spectral density should be computed and stated [9,62]. State-of-the-art diffractometric biosen-sors have resolutions of coh below 100 fg/mm 2 over 20 min [7].…”

Section: Characterization Of Diffractometric Biosensorsmentioning

confidence: 99%

“…It is important to state already here that a diffractometric biosensor is fundamentally different and more stable than a double referenced refractometric biosensor. This was explained in detail in two previously published papers [9,10].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Diffractometric Biosensing

et al. 2021

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Diffractometric biosensing is a promising technology to overcome critical limitations of refractometric biosensors, the dominant class of label-free optical transducers. These limitations manifest themselves by higher noise and drifts due to insufficient rejection of refractive index fluctuations caused by variation in temperature, solvent concentration, and, most prominently, nonspecific binding. Diffractometric biosensors overcome these limitations with inherent self-referencing on the submicron scale with no compromise on resolution. Despite this highly promising attribute, the field of diffractometric biosensors has only received limited recognition. A major reason is the lack of a general quantitative analysis. This hinders comparison to other techniques and amongst different diffractometric biosensors. For refractometric biosensors, on the other hand, such a comparison is possible by means of the refractive index unit (RIU). In this paper, we suggest the coherent surface mass density, coh , as a quantity for label-free diffractometric biosensors with the same purpose as the RIU in refractometric sensors. It is easy to translate coh to the total surface mass density tot , which is an important parameter for many assays. We provide a generalized framework to determine coh for various diffractometric biosensing arrangements that enables quantitative comparison. Additionally, the formalism can be used to estimate background scattering in order to further optimize sensor configurations. Finally, a practical guide with important experimental considerations is given to enable readers of any background to apply the theory. Therefore, this paper provides a powerful tool for the development of diffractometric biosensors and will help the field to mature and unveil its full potential.

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“…, coherent) pattern, termed the “mologram” (Figure a). The molographic pattern alternates ridges, which are usually made to contain high-affinity probes, and nonfunctional grooves, which act as an intrinsic reference. , Binding of target analytes to the probes on the ridges results in the physical manifestation of a diffraction grating in the shape of the mologram. The mologram is designed such that the light of a guided laser beam is scattered at specifically bound molecules and constructively interferes in a diffraction-limited focal spot. , The light intensity in the focal spot is measured to quantify molecular binding in real time .…”

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confidence: 99%

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

et al. 2021

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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.

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

Cited by 9 publications

References 87 publications

Investigating Complex Samples with Molograms of Low-Affinity Binders

Investigating Complex Samples with Molograms of Low-Affinity Binders

Quantitative Diffractometric Biosensing

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

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