Orientation of 4′-Dihexylamino-4-Sulfoazobenzene Interacting with Phospholipid Monolayers Probed by Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy

Understanding the molecular-level interactions between membrane-binding dyes and lipid membranes is critical for advancing cellular imaging techniques. In this study, we applied heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy to investigate the interaction between a water-soluble dye, 4'-dihexylamino-4-sulfoazobenzene (DHASA), and a Langmuir monolayer of the phospholipid, 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC) at air/aqueous solution interfaces. HD-VSFG revealed a characteristic vibrational band around 1600 cm^-1 from a phenyl ring stretching mode of DHASA when interacting with DPPC monolayers. This band was absent in both the spectrum of the air/DHASA aqueous solution interface without DPPC monolayers and that of DPPC monolayers on pure water, confirming its origin from the specific DHASA-DPPC interaction. Notably the 1600 cm^-1 band intensity decreased with increasing DPPC surface density, suggesting that DHASA is incorporated into the DPPC monolayers with a preferred orientation at low densities but is excluded as density increases─a finding with significant implications for optimizing membrane-staining dye performance. Through polarization-dependent measurement, we determined that DHASA adopts a preferred orientation where the azobenzene moiety tilts approximately 45° from the interface normal, with the alkyl chains pointing toward the air phase, providing structural insights that may be informative for the future development of membrane-staining dyes.