Nile Blue Microsolvation Reveals Ion-Specific Lipid Binding

Nile blue is a cationic, red-emitting fluorophore widely used in chemistry and biology, whose fluorescence reliability critically depends on microenvironment tuning and incubation time. Without proper control, the dye undergoes gradual spectral shifts driven by microsolvation-mediated microstructural changes. Here, we investigate ion-specific microsolvation of Nile blue and leverage it as a molecular tool to report on multivalent cation-lipid interactions by examining how physiologically relevant modern versions of Hofmeister salts modulate Nile blue's electronic properties in aqueous solutions. Salts containing strong kosmotropic anions (SO₄^2-, PO₄^3-) with Na⁺ tend to stabilize the dye microenvironment and suppress time-dependent spectral shifts. In contrast, salts with strong chaotropic anions (I^-, SCN^-) typically destabilize the microenvironment and enhance spectral variability. Salts with moderate chaotropic (NO₃^-) to mild kosmotropic anions (CH₃COO^-), strong kosmotropic multivalent cations (Mg²⁺), or strong chaotropic-kosmotropic cation-anion pairs also broadly reduce spectral stability. Mechanistic analysis reveals that ion-specific microsolvation leads to proton-transfer reaction, which is further promoted by salts containing strong chaotropic anions or strong kosmotropic cations, as well as by elevated temperatures. Extending these insights to lipid membranes, we find that salts containing kosmotropic multivalent cations substantially enhance membrane order relative to other salts, providing a robust basis for distinguishing their specific effects on lipid organization.