Polyacrylamide (PAM) hydrogels are extensively used as extracellular matrix mimics to study specific cell-materials interactions. However, chemistries typically applied for biofunctionalization of PAM lack chemo-selectivity and control over ligand density, which undermine reproducibility of cellular behavior, which can lead to inconclusive experiments. In this work, we introduce firefly luciferin-inspired click ligation to enable controlled and tunable biofunctionalization of PAM hydrogels. A novel acrylamide-based co-monomer is synthesized and incorporated in PAM hydrogels using traditional protocols, which introduces cyanobenzothiazole (CBT) functional groups. CBT mediates biofunctionalization of PAM with N-Cys bearing biomolecules via luciferin click chemistry. Biofunctionalization takes place under mild conditions, with high efficiency within only a few minutes, and does not require light exposure. When compared to the current commercial gold standard for PAM biofunctionalization sulfo-SANPAH, hydrogels modified via luciferin click ligation show increased control in loading of cell-adhesive biochemical cues. This leads to increased cellular attachment, spreading and proliferation due to a more efficient, homogeneous, and functional biofunctionalization. Luciferin-inspired click ligation may become a new standard for reliable biofunctionalization of PAM hydrogels with increased control over the density and preserved function of the presented biological cues, thus allowing more robust platforms for 2D cell-materials interaction experimentation.
Additional materials and methods, and supplementary results.