EFFECT OF THE COUPLING REAGENT ON THE METAL INHIBITION OF IMMOBILIZED UREASE IN AN OPTICAL BIOSENSOR

Urease was immobilized on aminopropyl glass through different bifunctional coupling reagents (cyanuric chloride, glutaraldehyde, hexamethylene diisocyanate, and phenylene diisothiocyanate) and their metal inhibition characteristics were compared. The inhibition studies were performed using a fibre-optic. biosensor configuration, wherein the pH change resulting from the biocatalytic hydrolysis of urea was:compared before and after the exposure to the metal samples. The different immobilized urease preparations exhibited varying metal inhibition behaviour: urease bounden cyanuric chloride- (1) and glutaraldehyde-activated (2) support were generally the most sensitive to the inhibition of seven test metal solutions (Hg2+, Ag+, Cu2+, Zn2+, Pb2+, Cr3+, and Co2+), urease bonded through hexamethylene diisocyanate (3) coupling reagent suffered slightly lesser inhibition, while the enzyme bonded through phenylene diisothiocyanate (4) displayed excellent metal inhibition resistance. A similar trend was observed when the different urease reagents were subjected to long term exposure to a test metal ion (Cu2+), Reagent 4 exhibited only a very slight decrease in its enzyme activity even after a 17 h continuous inhibition with the metal solution while the response of the other three enzyme reagents deteriorated significantly within 3-4 h, The unique inhibition property of urease bound on the diisothiocyanate glass was attributed to the metal binding property of the thiourea linkage formed during the immobilization process and to a more stabilized conformation of the immobilized urease in the said support. The usefulness of 4 was demonstrated in the biosensing of urea in the presence of an inhibiting metal ion, while 1 was applied to the biosensing of metal ion (Hg2+) based on enzyme inhibition measurements.