Silane-Based Methyl- and Carboxyl-Functionalized Mica Substrates for High-Speed Atomic Force Microscopy

High-speed atomic force microscopy (HS-AFM) enables visualization of biomolecular dynamics in solution with high spatiotemporal resolution but requires substrates that support stable adsorption without perturbing their structure or dynamics. Mica is widely used as an HS-AFM substrate because it provides an atomically flat surface. Several methods have been proposed to functionalize mica surfaces, but they are not suitable for all biomolecules. Here, we expand the HS-AFM substrate toolbox by introducing methyl- and carboxyl (COOH)-functionalized mica surfaces based on a common silanization strategy. In particular, a COOH-functionalized mica substrate was prepared via a mild thiol-maleimide coupling reaction, enabling efficient surface modification under aqueous conditions. Using proteins with different charges and shapes, we evaluated adsorption and diffusion behaviors on these substrates by HS-AFM. Distinct differences in molecular mobility and binding were observed depending on surface properties. These results provide practical guidelines for substrate selection and broaden the applicability of HS-AFM to diverse biomolecular systems.