Probing Molecular Binding Mechanisms between a Therapeutic Antibody and an Antigen Mimotope Peptide In Situ in Real Time at a Buried Interface

Abstract Bioanalytical platforms, such as biosensors and biochips, are widely used for detecting biomolecules including antibodies. Determining the conformation and orientation of antibodies captured by surface-immobilized recognition elements is essential for designing biosensors and biochips with improved performance. However, obtaining such structural information remains challenging because antibodies are located at buried solid/liquid interfaces that are difficult to characterize. Here, we investigated the binding interaction between Herceptin (trastuzumab), a therapeutic monoclonal antibody, and CH19, a HER2 mimotope peptide immobilized at a buried solid/liquid interface. Sum frequency generation (SFG) vibrational spectroscopy revealed specific binding of Herceptin to the surface-immobilized CH19 peptide, whereas no detectable interaction was observed for the non-target antibodies Cetuximab and Rituximab. By integrating SFG measurements with molecular dynamics (MD) simulations and one-exciton Hamiltonian spectral calculations, we determined the interfacial molecular structure of Herceptin and found that the antibody adopts a lying-down orientation on the CH19-functionalized surface. These combined experimental and computational results demonstrate that CH19−Herceptin recognition at the interface differs fundamentally from the corresponding interaction in solution, highlighting the unique influence of interfacial environment on antibody−antigen recognition. Our results support a surface-enabled multivalent binding model, in which multiple immobilized CH19 peptides interact with broader regions of Herceptin and generate a contact pattern distinct from that in solution. These results demonstrate that the combined SFG, MD, and spectral-calculation approach can determine the molecular structure of a full-length antibody at a buried interface, offering valuable insights for the rational design of biosensors, biochips, and other surface-based biorecognition analytical technologies.