Monitoring vibrational fingerprints of polypeptide monolayers by plasmonic nanoantennas-enhanced infrared absorption

Proteins and peptides play a central role in the biochemical processes of living cells. In such complex environments, their functionality depends not only on their molecular composition but also on their three-dimensional structure. Infrared vibrational spectroscopy is a powerful technique for studying protein conformations, but its inherent low sensitivity limits its applicability. This limitation can be overcome by Surface-Enhanced Infrared Absorption (SEIRA) spectroscopy, which amplifies molecular vibrational modes through coupling with the resonances of periodic plasmonic nanoantenna arrays. However, the SEIRA response depends on the orientation of the target molecule relative to the near-field distribution induced at the nanostructures, hindering the extraction of quantitative information and the identification of different vibrational modes. In this study, firstly the vibrational fingerprint of the TRP-cage miniprotein is studied by performing density functional theory (DFT) calculations of its Amide I and Amide II vibrational modes. In parallel, it is established a biofunctionalization protocol to bind the protein to the gold surface of gold nanoantennas with controlled orientation, minimizing steric hindrance and allowing the peptide to oscillate at its natural frequencies. The design and fabrication of gold plasmonic nanoresonators (Au nanoantennas) enable the detection of characteristic vibrational features of the TRP-cage, revealing both low- and high-frequency components of the Amide I band, as well as contributions from the Amide II region. Extending this strategy to other proteins could enable the investigation of different conformational variants in response to external stimuli such as pH, temperature, or chemical environment, ultimately contributing to the development of a comprehensive theoretical and experimental IR absorption database for the identification of vibrational modes within the Amide I and II bands of proteins.