Water Contact Angle Reveals pH‐Conditioning‐Enabled Single Molecule at a Large Surface Sensing Mechanisms

The reliable detection of a single molecule at a large surface remains a central challenge in biosensing, with direct implications for early diagnostics and precision medicine. Potentiometric immunoassays offer ultrahigh sensitivity, yet electronic amplification alone cannot fully account for the detection of single or few molecules with a millimeter‐wide gate. Here, we investigate the role of a pH‐conditioning surface treatment of an antibody capture layer as a crucial step to trigger the biochemical amplification mechanism that enables a sufficiently high signal‐to‐noise ratio. Physisorbed anti‐immunoglobulin G (anti‐IgG) layers were subjected to controlled pH‐conditioning (both acidic and basic) surface treatment and characterized using water contact angle (WCA) measurements to monitor changes in surface wettability. Complementary force–distance spectroscopy by atomic force microscopy (AFM) provided nanoscale mechanical characterization by quantifying Young’s modulus and adhesion force. The pH‐conditioning induced significant conformational rearrangements within the capturing layer, which, upon interaction with a few IgG molecules, led to pronounced increases in hydrophobicity alongside reductions in stiffness and changes in adhesion. These results reveal a direct mechanistic link between pH‐driven protein structural transitions and the amplification of single‐molecule sensing responses. By correlating surface wettability and nanomechanical properties, this study establishes WCA as a simple yet powerful probe for monitoring functional and structural states of protein layers critical for ultra‐sensitive biosensing.