OPTIMIZATION OF THE SELF-QUENCHING RESPONSE OF NITROBENZOXADIAZOLE DIPALMITOYLPHOSPHATIDYLETHANOLAMINE IN PHOSPHOLIPID-MEMBRANES FOR BIOSENSOR DEVELOPMENT

Incorporation of the lipid-conjugated fluorescent probe nitrobenzoxa-d iazole dipalmitoylphosphatidylethanolamine (NBD-PE) into bilayer lipid membranes (BLMs) provides a matrix wherein changes in the structure o f the membrane can be transduced into changes in fluorescence intensit y or lifetime. In the work reported here, a comparison was made betwee n an empirical model recently developed by our group to account for al terations in the fluorescence Lifetime and average fluorescence intens ity 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-quenchin g data obtained from Lipid membranes containing 1 to 50 mol % of NBD-P E. This model was used to determine the optimum initial surface concen tration of NBD-PE to be incorporated into phospholipid membranes for b iosensor development. Optimization was based on the magnitude of the c hange in fluorescence intensity as a function of changes in the local concentration of the probe. The presence of acidic headgroups in the m embrane results in negligible improvement in sensitivity, while a hete rogeneous membrane structure greatly enhances the signal magnitude. Ex perimental results did not provide accurate optimum concentrations, al though two NBD-PE surface concentrations were found to yield close agr eement with theoretically predicted optimum surface concentrations of 0.027 +/- 0.001 and 0.073 +/- 0.001 molecules NBD-PE nm(-2).