Site-Specific\nImmobilization Boosts the Performance\nof a Galectin‑1 Biosensor

The analysis of protein-bound\nglycans has gained significant\nattention\ndue to their pivotal roles in physiological and pathological processes\nlike cell–cell recognition, immune response, and disease progression.\nRoutine methods for glycan analysis are challenged by the very similar\nphysicochemical properties of their carbohydrate components. As an\nalternative, lectins, which are proteins that specifically bind to\nglycans, have been integrated into biosensors for glycan detection.\nHowever, the effectiveness of protein-based biosensors depends heavily\non the immobilization of proteins on the sensor surface. To enhance\nthe sensitivity and/or selectivity of lectin biosensors, it is crucial\nto immobilize the lectin in an optimal orientation for ligand binding\nwithout compromising its function. Random immobilization methods often\nresult in arbitrary orientation and reduced sensitivity. To address\nthis, we explored a directed immobilization strategy relying on a\nreactive noncanonical amino acid (ncAA) and bioorthogonal chemistry.\nIn this study, we site-specifically incorporated the reactive noncanonical\nlysine derivative, N<sup>ε</sup>-((2-azidoethoxy)carbonyl)-l-lysine, into a cysteine-less single-chain variant of human\ngalectin-1 (scCSGal-1). The reactive bioorthogonal azide group allowed\nthe directed immobilization of the lectin on a biosensor surface using\nstrain-promoted azide–alkyne cycloaddition. Biolayer interferometry\ndata demonstrated that the controlled, directed attachment of scCSGal-1\nto the biosensor surface enhanced the binding sensitivity to glycosylated\nvon Willebrand factor by about 12-fold compared to random immobilization.\nThese findings emphasize the importance of controlled protein orientation\nin biosensor design. They also highlight the power of single site-specific\ngenetic encoding of reactive ncAAs and bioorthogonal chemistry to\nimprove the performance of lectin-based diagnostic tools.