The need for a robust, specific and high-throughput biosensing platform is of significant interest in analysis of glycans and oligosaccharides. However, current analytical technology requires long run times, sophisticated and expensive equipment (e.g., HPLC-MS, HPAEC-PED). In particular, the carbohydrate linkage is especially difficult to discern. Paper based biosensors provide alternative approaches for rapid and easy-to-use assays. We have developed a <b>linkage-specific assay with colorimetric readout</b> visible to the naked eye or analyzed using imaging software for quantitative measurements that are a function of the concentration of the target molecule. <b>The assay takes approximately 30 seconds and is amenable to high throughput via multichannel pipetting. </b>Glycolytic enzymes are used alongside glycosyl-dehydrogenase to generate a redox system capable of producing a colorimetric change in organic dyes. Heterologous expression of enzymes and immobilization on paper substrate increases the commercial feasibility of the assay in terms of cost and durability. This paper based analytical device platform demonstrates its utility in qualitative and quantitative assays with potential for expansion to other molecules and applications. In this work, we demonstrate the paper-based quantitation of α1-2, α1-3, and α1-6 fucose, and α2,3 in the context of human milk oligosaccharides, Lewis antigens, and core N-glycans. Finally, we discuss how this method can be applied to bacterial oligo- and polysaccharide synthesis (e.g., for prebiotic production) and glycosidase characterization and engineering to expand the glycobiology analytical toolbox.

Linkage-specific, paper-based assay for characterization of N-glycans, O-glycans, and oligosaccharides

ACS Spring 2022

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