Fluorescence transduction of an enzyme-substrate reaction by covalently immobilized monolayers of amphiphiles

The emission intensity of the fluorophore nitrobenzoxadiazoledipalmitoylphosphatidylethanolamine (NBD-PE) is sensitive to the local environmental structure when this species is present as a component of an amphiphilic membrane. The physical and electrostatic structure of a membrane may be modulated by selective reactions which induce electrostatic changes at the surface of the membrane. The resulting change in the fluorescence signal may then be used to monitor the concentration of an analyte which is present in solution. The membrane-associated enzyme acetylcholinesterase (AChE) hydrolyses acetylcholine (ACh) to produce acetic acid and choline. The reaction of ACh with AChE was detected using a monolayer consisting of fatty acids of C16 chain length which were covalently attached to quartz wafers and which contained a small amount of NBD-PE (partitioned from water into the membrane). The enzyme-substrate reaction produced decreases in fluorescence intensity from the monolayer, detection of 2 μM ACh. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric mehtods and fluorescence microscopy.