Comparative kinetics of CuAAC and thiol–yne click chemistries for high-density streptavidin immobilization on alkyne-terminated glass surfaces

This study presents a systematic comparison of two prominent click chemistries, copper-catalyzed azide-alkyne cycloaddition (CuAAC) and thiol-yne coupling (TYC), for the controlled immobilization of biological macromolecules on glass surfaces. Two distinct alkyne-terminated surfaces were fabricated: a short-chain 4-pentynoic acid surface and a long-chain polyethylene glycol (PEG) surface prepared with Alkyne PEG Silane (ALK-PEG-Si, MW 3400). The efficiency of biotin immobilization, followed by subsequent binding of the protein streptavidin, was evaluated kinetically over 10, 20, and 40 min. The results indicate that the choice of surface chemistry profoundly influences reaction kinetics. CuAAC proceeded most efficiently on the flexible, hydrophilic ALK-PEGSi surface, achieving high streptavidin binding density at 20 min. In contrast, TYC demonstrated a faster initial reaction rate on the rigid, hydrophobic 4-pentynoic acid surface, reaching near-maximal intensity within 10 min. At longer reaction times (40 min), both chemistries and both surfaces converged to similar maximum immobilization levels. This work provides a fundamental understanding of how the interplay between linker design and click reaction mechanism can be strategically exploited to optimize the kinetics and density of macromolecular immobilization for applications such as biosensors and microarrays.