Covalent immobilization of amphiphilic monolayers containing urease onto optical fibers for fluorimetric detection of urea

Abstract

Bifunctional 11 or 12 carbon chain length amphiphiles with a triethoxy chlorosilane group at one terminus and an amine functionality at the other terminus are covalently immobilized onto planar quartz wafers and optical fibers. A small amount of the fluorescent probe nitrobenzaoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) is partitioned into the membranes from an aqueous suspension. For coated wafers or fibers placed into aqueous solutions, alterations of pH change the physical and electrostatic structure of the membranes, which in turn alters the emission intensity of the NBD-PE owing to changes in self-quenching. The fluorescence intensity decreases as the degree of ionization of headgroups within the membrane decreases, consistent with an increase in self-quenching. Samples which are immobilized onto optical fibers have better sensitivity to changes of pH than those immobilized onto planar wafers. The enzyme urease is covalently linked onto the functional groups at the surface of some membranes to investigate immobilized membranes that are chemically selective. A small amount of NBD-PE is partitioned from water into the membrane either before or after immobilization of urease. Addition of urea to this system produces ammonia and carbonic acid, and results in changes in fluorescence intensity from the immobilized layer owing to alteration of surface charge at the membrane. The detection system is sensitive to changes in the bulk concentration of urea as small as 20 μM, with a limit of detection of 40 μM of urea.