Optimization of the Self-Quenching Response of Nitrobenzoxadiazole Dipalmitoylphosphatidylethanolamine in Phospholipid Membranes for Biosensor Development

Incorporation of the lipid-conjugated fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) into bilayer lipid membranes (BLMs) provides a matrix wherein changes in the structure of the membrane can be transduced into changes in fluorescence intensity or lifetime. In the work reported here, a comparison was made between an empirical model recently developed by our group to account for alterations in the fluorescence lifetime and average fluorescence intensity of NBD-PE as a result of self-quenching and an earlier alternative model which describes self-quenching of membrane-bound chlorophyll a. Our model showed the more satisfactory correlation with self-quenching data obtained from lipid membranes containing 1 to 50 mol % of NBD-PE. This model was used to determine the optimum initial surface concentration of NBD-PE to be incorporated into phospholipid membranes for biosensor development. Optimization was based on the magnitude of the change in fluorescence intensity as a function of changes in the local concentration of the probe. The presence of acidic headgroups in the membrane results in negligible improvement in sensitivity, while a heterogeneous membrane structure greatly enhances the signal magnitude. Experimental results did not provide accurate optimum concentrations, although two NBD-PE surface concentrations were found to yield close agreement with theoretically predicted optimum surface concentrations of 0.027 ± 0.001 and 0.073 ± 0.001 molecules NBD-PE nm −2 .