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

Abstract

Under acidic conditions, polycationic polymer coatings can serve as protective immobilization matrices that buffer local acidity and help preserve enzyme function. However, it remains unclear how polymer support design parameters, particularly film thickness and effective cationic charge density, govern that vital protonation process. Leveraging the nanometer-scale 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 normalized initial ONPG hydrolysis rate of immobilized β-galactosidase protein (LacZ protein). 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 methods, 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. Together, these results identify protonation depth and charge density as orthogonal, tunable design parameters and establish a thickness regime that maximizes protection without unnecessary film growth.