Plasmonic gold bowtie nanoantennas for dual infrared and Raman detection of a cytochrome bd-type oxidase.

Plasmonic nanoantennas offer powerful opportunities for enhancing vibrational spectroscopic signals of biomolecules at surfaces, yet their application to structurally fragile membrane proteins remains challenging. Here, lithographically defined gold bowtie nanoantennas are investigated as multifunctional plasmonic substrates for the surface-enhanced vibrational spectroscopy detection of a membrane protein. Three bowtie geometries were rationally designed to provide strong and localized electromagnetic field confinement in spectroscopically relevant regions, as supported by electromagnetic simulations and experimental characterization. Using a site-specific Ni-NTA immobilization linker, the membrane protein cytochrome bd-I from Escherichia coli was immobilized at micromolar concentration and probed by both infrared and Raman spectroscopy. Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) analysis of the amide I band demonstrated the preservation of the predominantly α-helical conformation upon immobilization. The direct comparison of data obtained on gold nanoparticle substrates, on silicon and on the bowtie nanoantennas revealed substantially higher signal intensities of the amide I band. Surface-Enhanced Raman Spectroscopy (SERS) demonstrated a pronounced increase of the signal intensity of the protein- and heme-specific vibrational markers on the bow tie substrate. Together, these results establish gold bowtie nanoantennas as a robust and reproducible plasmonic platform enabling sensitive and selective, label-free infrared and Raman detection of membrane proteins at micromolar concentration, with potential for optical biosensing platforms targeting complex membrane protein systems.