Figure 3. The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.

Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C₁₄-HSL (thick dashed line), 200 µM 3-oxo-C₁₂-HSL (solid black line) and 200 µM 3-oxo-C₁₀-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C_14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio R(460/520) was calculated (shown). All experiments n = 3.