Effect of the coupling reagent on the metal inhibition of immobilized urease in an optical biosensor

Abstract

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 bound on 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 (CU~+). 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.

Simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions

“…The sensing scheme of enzymatic determination of heavy metal ions was similar to that previous reported [1,2], which was designed in two steps. Firstly, the hydrolysis of urea into ammonia and carbon dioxide catalysed by urease, with the corresponding net increase in pH.…”

“…In development of the optical biosensors, the utilisation of immobilised enzymes is useful and perspective, due to their stability and easiness to coupling with the transducers [1,2,3,4]. The use of biosensors based on immobilised enzyme provides sensitivity as well as selectivity in determination of various pollutants (e.g.…”

“…In this case the immobilisation of enzyme often causes loss in enzyme activity and poor reproducibility [16,17,18]. Moreover, the immobilised enzymes, which involve coupling of reagents, often protect the enzyme from inactivation by metal ions [2].…”

“…This is due to enzyme activities are frequently inhibited specifically by low concentration of certain metal ions [9] and therefore enzymatic methods are commonly used for metal ion determinations [2,10]. Many of these systems have been proposed for heavy metal ions determination (such as Hg(II)) using xanthine oxidase [11], peroxidase [12], and urease [2,13,14,15] as enzymes. However, most of the sensors have been constructed using complex designs either in terms of their reaction schemes or their immobilisation of enzyme and transduction procedures.…”

A Dip-and-Read Test Strip for the Determination of Mercury(II) Ion in Aqueous Samples Based on Urease Activity Inhibition

“…Then, the enzyme substrate is introduced and the activity of enzyme is detected with electrochemical or optical transducers. Because obtaining the obvious enzyme inhibition is usually time consuming and the enzyme-catalyzed reaction is fast, 11,16,23 it is difficult to detect inhibition if the analytes and enzyme substrate are introduced to the enzyme reactors simultaneously. The dry reagent test strip method is one of the one-step analytical methods that has been widely used in clinical analysis.…”