Unraveling the Reverse Hofmeister Effect of 3,3',5,5'-Tetramethylbenzidine for Anion-Responsive Color-Changing...

Ions are essential regulators of biomolecular stability and function, and their effects are classically summarized in the Hofmeister series. While extensive studies have focused on proteins and other biomacromolecules, whether small molecules exhibit similar ion-specific behaviors remains largely unexplored. Herein, we report the common blue oxidation product of 3,3',5,5'-tetramethylbenzidine (TMB _ox1 ) as a minimal molecular system that exhibits a reverse Hofmeister effect. In aqueous solution, TMB _ox1 mimics the key structural features of proteins in terms of terminal amino groups and π-π stacking motifs and undergoes aggregation in an anion-dependent manner. Combining experiments with the density functional theory and molecular dynamics simulations, we identified the terminal amino groups as key anion interaction sites and π-π stacking as the intermolecular aggregation interface. MD-based thermodynamic analyses further indicate that chaotropic anions (e.g., I^-) disrupt hydration and shorten π-π separations, shifting TMB _ox1 toward denser entropy-favored states. More interestingly, when TMB _ox1 is confined in an agarose hydrogel, the Hofmeister effect shifts from conventional aggregation to a distinct anion-responsive discoloration. This study demonstrates that small molecules can recapitulate the complex Hofmeister behavior traditionally associated with biomacromolecules. This study provides insights into the molecular determinants of ion-specific effects and highlights their implications for colorimetric sensing and hydrogel-based applications.