Silanes to Silatranes: Robust Functionalization for Single-Molecule Force Spectroscopy.

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) experiments require stable and reproducible functionalization of cantilevers and surfaces. Conventional silane-based methods suffer from poor stability, drastically hindering their reproducibility and performance. Here, we present an ethanol-based aminopropyl-silatrane functionalization protocol that achieves superior performance in a similar process timeframe. In particular, combining this approach with bacterial adhesin coupling, we demonstrated a significant twofold improvement in the fraction of attempted SMFS experiments yielding usable single-molecule data. The method can be generally applied to silicon-nitride and glass cantilevers and surfaces, including mechanically modified cantilevers optimized for fast time response, low stiffness, and low drift. Furthermore, we demonstrate a simple and cost-efficient method to clean these functionalized cantilevers using common solvents and a benchtop UV-ozone cleaner, allowing for refunctionalization and prolonged cantilever lifespans. Together, this allows for a reliable, reusable, and versatile functionalization protocol for high-quality SMFS experiments.