Investigating Complex Samples with Molograms of Low-Affinity Binders

Abstract

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.

Advanced Materials and Sensors for Microphysiological Systems: Focus on Electronic and Electrooptical Interfaces

“…Reproduced with permission. [ 144 ] Copyright 2021, American Chemical Society. https://pubs.acs.org/doi/10.1021/acssensors.0c02346.…”

“…showed that diffractometric measurements are insensitive to unsought nonspecific bindings and, therefore, allow the detection of unprocessed, complex samples such as plasma. [ 144 ] They used focal molography, a technique used to visualize molecular binding on a coherent pattern of binding sites (molograms) on a single‐mode optical waveguide (Figure 10b). The molographic designs are a combination of ridges and grooves containing high‐affinity probes for extracellular protein domains and a passivated layer, respectively.…”

“…In contrast, b-ii-2) affinity-matched molograms with very similar chemistry on ridges and grooves do not support coherent nonspecific bindings. Reproduced with permission [144]. Copyright 2021, American Chemical Society.…”

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

“…Here, we introduce a generalizable, quantitative live-cell assay for the analysis of cytosolic PPIs, which is based on the working principle of focal molography. − Focal molography is an optical system for molecular interaction analysis that is insensitive to nonspecific molecular interactions . This unique property is achieved by the arrangement of binding sites into a regular ( i.e.…”

“…31−33 Focal molography is an optical system for molecular interaction analysis that is insensitive to nonspecific molecular interactions. 34 This unique property is achieved by the arrangement of binding sites into a regular (i.e., coherent) pattern, termed the "mologram" (Figure 1a). The molographic pattern alternates ridges, which are usually made to contain high-affinity probes, and nonfunctional grooves, which act as an intrinsic reference.…”