Abstract

The surface potential of biological membranes, largely governed by the concentration of protons and other charged species at the lipid-water interface, is a critical determinant of membrane protein function and cellular signaling. This study investigates the utility of two widely-used electrochromic styryl dyes, di-4-ANEPPS and RH-421, not merely as voltage-sensitive probes, but specifically as sensors for protons on the surface of model lipid membranes. The research objective was to characterize the pH-dependent changes in the optical properties of these dyes when adsorbed onto bilayer lipid membranes (BLMs) and liposomes. Using fluorescence spectroscopy and measurements of the boundary potential ($\Delta\varphi_b$), we demonstrate that the fluorescence intensity and spectral shift of both di-4-ANEPPS and RH-421 are highly sensitive to the local proton concentration. This sensitivity is attributed to the protonation/deprotonation of the dye molecules or nearby lipid headgroups, which alters the dipole potential at the membrane interface. The findings confirm that these styryl dyes can serve as effective, real-time optical reporters for proton binding and surface charge dynamics, providing a valuable tool for studying proton-coupled processes and local pH microenvironments in cell biology and biophysics.