Nanoscale Protonation Limits and Charge Density in Polymer Films Govern the Activity of Immobilized LacZ under Acid Stress

Under acidic conditions, polycationic polymer coatings function as protective immobilization supports through protonation-mediated local pH buffering. However, it remains unclear how polymer support design parameters, such as film thickness and charge density, govern that vital protonation process. Leveraging the precise control of film thickness and copolymer composition enabled by initiated chemical vapor deposition (iCVD), we systematically investigated how these parameters govern the protonation behavior of poly[glycidyl methacrylate-co-2-(dimethylamino)ethyl methacrylate] (pGD) thin films and, in turn, the activity of immobilized {beta}-galactosidase (LacZ). Infrared spectroscopy suggests that proton penetration was capped at a depth of [~]250 nm in pGD with 65% DMAEMA, limiting the polycationic thickness in pGD films thicker than this value. Consistent with this limit, immobilized LacZ activity under acidic stress (pH 4) increased with protonated thickness up to [~]250 nm and then plateaued. Raising the polycationic monomer content from 25 to 65 mol% increased LacZ activity at pH 4 by up to 83%, consistent with a higher positive charge density providing stronger local pH buffering. To test whether this behavior depends on immobilization sites, we evaluated two approaches: random immobilization (via amine-epoxy ring-opening reactions) and site-directed immobilization (via SpyCatcher/SpyTag binding). Directed immobilization preserved higher LacZ activity than random immobilization, but the protonation-dependent protection trend remained consistent for both strategies. These findings establish protonation depth and charge density as tunable design parameters for polycationic immobilization supports that stabilize enzymes under acidic conditions.