Preparation Method Determines Valinomycin Organization and Phase Behavior in Charged Langmuir Monolayers

Abstract

Amphiphilic molecules are typically incorporated into lipid Langmuir monolayers by two distinct methods: co-spreading, where lipid and amphiphile are deposited simultaneously, and injection, where the amphiphile is introduced into the aqueous subphase beneath a preformed monolayer. Whether these methods produce equivalent molecular arrangements remains an open question. Using valinomycin as a model ionophore, we investigate this question across lipid monolayers bearing anionic, zwitterionic, and cationic headgroups. Surface pressure–area isotherms, fluorescence imaging, and vibrational sum-frequency generation spectroscopy together reveal that the two preparation methods produce fundamentally different molecular organizations. Cospreading yields a uniform, well-incorporated monolayer, while injection produces a kinetically trapped, spatially heterogeneous state in which valinomycin resides predominantly at the headgroup–water interface. The magnitude of these differences scaled with lipid headgroup charge, implicating specific interactions between lipid headgroups and the carbonyl-lined cavity of valinomycin as key determinants of the incorporation pathway. These findings establish that preparation history is a critical parameter governing molecular organization in mixed amphiphile-lipid monolayers.