Nanomechanical and Surface Potential Characterization of Protein Films via Atomic Force and Kelvin Probe Force Microscopy

Amplitude Modulation Atomic Force Microscopy (AM-AFM) is a powerful technique used for high-resolution imaging and mechanical characterization of surfaces at the nanoscale. It excels in analyzing weak intermolecular forces, such as van der Waals and electrostatic interactions, which are crucial for understanding protein films and biofunctionalized surfaces in biosensing. This study employed AM-AFM to analyze the mechanical properties of anti-IgM protein physisorbed on stiff gold films on solid substrates, a process driven by weak intermolecular forces. The research examined the mechanical properties, including Young’s modulus, stiffness, and adhesion forces, of both protein clusters and films. It is revealed that the anti-IgM protein films were more compact, and exhibited higher Young’s modulus, stiffness and less adhesion force compared to the protein clusters. Energy dissipation patterns observed in AM-AFM further supported these findings. Protein clusters exhibited higher energy dissipation in the attractive regime, indicating that they are softer and less ordered. In contrast, protein films demonstrated lower dissipation due to their more compact, ordered arrangement. These results suggest that protein films are more resistant to deformation, making them suitable for biosensor applications where mechanical stability is crucial. Additionally, Kelvin Probe Force Microscopy (KPFM) was utilized to measure surface potential changes resulting from antigen-antibody interactions, demonstrating its potential as a diagnostic tool for biosensing. KPFM is a non-invasive technique that measures the contact potential difference between a conducting cantilever tip and a sample surface, allowing simultaneous high-resolution topographic and surface potential imaging. The study demonstrated the effectiveness of KPFM in detecting surface potential shifts resulting from antigen-antibody binding, such as the interaction between anti-IgM biofunctionalized surfaces and IgM molecules. The formation of the immuno-complex caused a measurable decrease in surface potential, attributed to electrostatic interactions at the binding sites. The surface potential shift was detected across the entire functionalized area, even at extremely low IgM concentrations, highlighting KPFM’s sensitivity in detecting single-molecule binding events. These results suggest that KPFM can serve as a label-free, highly sensitive diagnostic tool for detecting biomolecular interactions in biosensing platforms.