Tailorable Biofunctionalization of Poly(acrylamide) Hydrogels via Firefly Luciferin-Bioinspired Click Ligation...

Polyacrylamide (PAM) hydrogels are extensively used as extracellular matrix mimics to study specific cell-material interactions. However, conventional biofunctionalization strategies lack chemo-selectivity and control over ligand density, compromising reproducibility and experimental reliability. In this work, we introduce firefly luciferin-inspired click ligation to enable efficient and tunable biofunctionalization of PAM hydrogels. A novel acrylamide-based comonomer containing cyanobenzothiazole (CBT) moieties is synthesized and incorporated into PAM hydrogels. CBT mediates biofunctionalization of PAM with N-Cys bearing biomolecules via luciferin click chemistry. Biofunctionalization occurs within only a few minutes, under mild conditions, with high efficiency, not requiring light exposure. Compared to the widely used sulfo-SANPAH (SS)-based approach, our method offers enhanced biofunctionalization efficiency, homogeneity, and control over biomolecule loading while preserving biochemical functionality. This translates into improved presentation of cell-adhesive cues, resulting in significantly increased cell attachment, spreading, and proliferation, as demonstrated by using label-free holotomography. The novel luciferin click ligation offers a robust, efficient, and reproducible alternative for PAM biofunctionalization, providing precise control over the ligand density while maintaining bioactivity. As PAM hydrogels continue to evolve into increasingly sophisticated mechanobiology tools, our approach may serve as a new standard for engineering the interfacial properties of these materials to achieve robust two-dimensional (2D) cell culture platforms for fundamental studies in cell-material interactions.