Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes

The principle of electrochromic modulation of excited-state intram olecular proton-transfer reaction was applied for the design of fluorescence probes with high two-color sensitivity to dipole potential, psi(d), in phospholipid bilayers. We report on the effect of psi(d) variation on excitation and fluorescence spectra of two new 3-hydroxyflavone probes, which possess opposite orientations of the fluorescent moiety in the lipid bilayer. The dipole potential in the bilayer was modulated by the addition of 6-ketocholestano or phloretin and by substitution of dimyristoyl phosphatidylcholine lipid with its ether analog 1,2-di-o-tetradecylsn-glycero-3-phosphocholine, and its value was estimated by the reference styryl dye 1-(3-sulfonatopropyl)-4-{beta[2-(di-noctylamino)-6-naphthyl]vinyl}pyridinium betaine. We demonstrate that after psi(d) changes, the probe orienting in the bilayer similirly to the reference dye shows similar shifts in the excitation spectra, whereas the probe with the opposite orientation shows the opposite shifts. The new observation is that the response of 3-hydroxyflavone probes to psi(d) in excitation spectra is accompanied by and quantitatively correlated with dramatic changes of relative intensities of the two well separated emission bands that belong to the initial normal and the product tautomer forms of the excited-state intramolecular proton-transfer reaction. This provide! a strong response to psi(d) by change in emission color.