Real-time, label-free characterization of oligosaccharide-binding proteins using carbohydrate microarrays and an ellipsometry-based biosensor

Abstract

Carbohydrates present on cell surfaces mediate cell behavior through interactions with other biomolecules. Due to their structural complexity, diversity, and heterogeneity, it is difficult to fully characterize a variety of carbohydrates and their binding partners. As a result, novel technologies for glycomics applications have been developed, including carbohydrate microarrays and label-free detection methods. In this paper, we report using the combination of oligosaccharide microarrays and the label-free oblique-incidence reflectivity difference (OI-RD) microscopy for real-time characterization of oligosaccharide binding proteins. Aminated human milk oligosaccharides were immobilized on epoxy-coated glass substrates as microarrays for reactions with Family 1 of solute binding proteins from Bifidobacterium longum subsp. infantis (B. infantis). Binding affinities of these protein-oligosaccharide interactions showed preferences of Family 1 of solute binding proteins to host glycans, which helps in characterizing the complex process of human milk oligosaccharides foraging by B. infantis.

Reusable OIRD Microarray Chips Based on a Bienzyme-Immobilized Polyaniline Nanowire Forest for Multiplexed Detection of Biological Small Molecules

“…It is able to monitor surface/interface processes in a real-time and label-free form by measuring the phase and amplitude variations of the s and p components of the elliptically polarized reflected light caused by a change in the surface/interface dielectric constant . This technology has been employed to monitor oxide film growth, molecular adsorption at gas/solid interfaces, and the potential distribution at transparent electrode/solution interfaces, enabling label-free detection by microarray chips since its establishment. − …”

Non-fouling polymer brush grafted fluorine-doped tin oxide enabled optical and chemical enhancement for sensitive label-free antibody microarrays

“…Thus, OIRD provides a desirable method for label-free and real-time detection of high-throughput microarrays. 10–12…”

“…Thus, OIRD provides a desirable method for label-free and real-time detection of high-throughput microarrays. [10][11][12] Because of their limited sensitivity, however, OIRD-based label-free microarrays do not yet realize their full potential in medicine screening and clinical diagnosis to date. For instance, the limit of detection (LOD) of OIRD antibody microarrays towards some clinically important protein markers is several orders of magnitude higher than that of labelling based ones, e.g., fluorescent microarrays, and cannot meet the required cut/ off values for clinical disgnosis.…”

Label-free OIRD microarray chips with a nanostructured sensing interface: enhanced sensitivity and mechanism

“…, which is in sharp contrast to the well-established label-free SPR technique, thus providing a universal method for label-free detection of microarray chips. 25–28…”

“…More importantly, OIRD is applicable to microarrays printed on arbitrary substrates such as glass, silicon, etc., which is in sharp contrast to the well-established label-free SPR technique, thus providing a universal method for label-free detection of microarray chips. [25][26][27][28] At present, the relatively low sensitivity is the main factor limiting the broad application of OIRD in label-free microarray chips. Compared to fluorescence, OIRD shows much lower sensitivity and generally several orders of magnitude higher limit of detection (LOD) for protein microarrays.…”