Time-Resolved “Barbecue” Förster Resonance Energy Transfer for Sensitive and Homogenous Detection of Alginate

The development of glycoscience has been impeded by the inherent structural complexity of carbohydrates, the lack of template encoding, and the limitations of current methodologies for their identification and quantification. Carbohydrate-binding modules (CBMs) and carbohydrate-active enzymes (CAZymes) are two protein families that have evolved to interact with distinct carbohydrates, offering the potential to function as specific carbohydrate recognition elements. Here, we present the first homogeneous assays for alginate as a representative polysaccharide, leveraging the unique properties of CBMs and CAZymes (e.g., polysaccharide lyases). In contrast to conventional antibody-antigen-antibody "sandwich" immunoassays, our approach takes advantage of the linear and repetitive nature of polysaccharides, allowing for simultaneous binding to multiple CBMs and lyases. We exploit this feature in our novel "Barbecue" assay design, utilizing the combination of CBMs/lyases that are orthogonally labeled with the Tb-based donor fluorophore CoraFluor-1 (CRF) and Cy5 as an acceptor to establish a time-resolved Förster resonance energy transfer (TR-FRET) detection system. The homogeneous single-step assay uses a straightforward protocol without wash steps, enabling the sensitive quantification of alginate. The lower limit of quantification was determined to be as low as 0.4 ng·mL^-1 (0.01 ng) with CBMs and 1.6 ng·mL^-1 (0.03 ng) with alginate lyases, representing a ≥100-fold improvement compared to that of conventional microarray-based assays. The broad applicability of the analytical method was rigorously validated across biomedical, food, and cosmetics samples. The "Barbecue" FRET concept is generalizable and can be applied to develop similar rapid and simple quantification assays for other polysaccharides beyond alginate.