Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Enam, Fatima; Mansell, Thomas J.

doi:10.3791/59253

Cited by 1 publication

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“…To couple reduction of colorimetric dye to the quantification of 2'-FL, we used fucosidases known in literature 33,34 to be specific to the carbohydrate linkage to fucosylated molecules. Free fucose can then be assayed using a redox-coupled reaction in which fucose dehydrogenase (FDH) oxidizes L-fucose to fuconolactone, creating NADPH, which reduces the formazan dye.…”

Section: Fucosidases Act As Linkage-specific Detection Elements For Fmentioning

confidence: 99%

Paper-based Sensing of Fucosylated Biological Compounds

Enam

Kramer

Robinson

et al. 2020

Preprint

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Advances in sensing technology have enabled rapid analysis of various biomolecules including complex carbohydrates. However, glycan analysis is limited by the throughput and complexity of assays for quantifying them. We describe a simple, low-cost enzymatic assay for the rapid analysis of fucosylation, down to linkage specificity, and its application to high-throughput screening of biologically relevant fucosylated compounds, to facilitate simple and straightforward analytical techniques. Paper-based devices integrate biosensor platforms and other diagnostic assays by fusing them with wax printing technology, making their fabrication even more inexpensive and simple. The specificity of the assay is established by linkage-specific glycosidic enzymes and the colorimetric output is visible to the naked eye, with costs that are lower than fluorescence/luminescence-based assays ($0.02/reaction). This platform was further improved by enhancing storage stability to retain analytical performance over time using desiccation and freeze-drying techniques. The assay platform allows analysis of hundreds of samples in minutes and we anticipate that this rapid and simple analytical method will be extended towards developing a universal glyco-barcoding platform for high throughput screening of glycosylation.

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Section: Fucosidases Act As Linkage-specific Detection Elements For Fmentioning

confidence: 99%