Transduction of the reaction between urea and covalently immobilized urease by fluorescent amphiphilic membranes

Amphiphiles with chain lengths of 12 and 16 carbons having a triethoxy chlorosilane group at one terminus were covalently immobilized by reaction with hydroxyl groups on the surfaces of planar quartz wafers and optical fibres. The enzyme urease was covalently immobilized onto either carboxylic acid or amine functionalities at the other terminus of the immobilized amphiphiles, resulting in a surface coverage of about 60% of a close-packed monolayer of protein. A small amount (2–3 mol.%) of the fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) was partitioned into the membranes from an aqueous suspension following immobilization of the urease. Addition of urea to coated wafers or optical fibers placed into aqueous solutions resulted in substantial changes of fluorescence intensity from both the carboxylic acid and amine functionalized membranes. A 20μM change in the concentration of urea could be detected, with a limit of detection of 40μM of urea. The sensitivity degraded ten-fold over a period of 7 days when the samples were stored in buffer at a temperature of 4°C. An investigation into the mechanism of the fluorescence response revealed that local alterations of pH at the surface of membranes due to enzymatic hydrolysis of urea resulted in changes in the extent of ionization of both the membrane and the urease. The resulting changes in the electrostatic interactions between the membrane and the enzyme produced alterations in the rotational mobility of the amphiphiles and fluorophores which affected the self-quenching of NBD-PE.