Orientational Analysis of an SHG-Active Dye Interacting with a DPPC Monolayer Using Vibrationally Resonant and Nonresonant Sum Frequency Generation Signals

Abstract

Ap3 is a nonfluorescent second-harmonic-generation (SHG)-active dye used to stain cellular and artificial membranes. In this study, the interaction between Ap3 and a Langmuir monolayer of the phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), at the air/aqueous interface was investigated by heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy. The second-order susceptibility (χ(2)) spectra at the air/aqueous Ap3 solution interface in the presence of the DPPC monolayer strongly depended on the visible probe wavelength, indicating that electronic resonances significantly contribute to the spectra. Two distinct VSFG bands at 1601 and 1638 cm–1, as well as a large vibrationally nonresonant (VNR) contribution, were detected in the χ(2) spectra, all of which are attributed to the Ap3 molecules interacting with the DPPC monolayer at the interface. We analyzed the molecular orientation of Ap3 using both vibrationally resonant and nonresonant SFG signals. The 1638 cm–1 VSFG band indicated that the azobenzene moiety is tilted approximately 55° from the interface normal, with the alkyl chains pointing toward the air phase. Analysis of the VNR signal using a simplified model of the electronic response gave a tilt angle of approximately 57°, in close agreement with the VSFG-band result.