ORIGINAL ARTICLE Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode ANA RITA BARBOSA & AMIN KARMALI Chemical Engineering and Biotechnology Research Center and Department of Chemical Engineering, Instituto Superior de Engenharia de Lisboa, Lisbon, Portugal Abstract A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL –1 , 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incuba- tion time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition. Keywords: Ion-selective electrode, aliphatic amidase, urea biosensor Correspondence: A. Karmali, Chemical Engineering and Biotechnology Research Center and Department of Chemical Engineering of Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro 1, 1950-062 Lisboa, Portugal. Tel: +351-21-8317052. Fax: +351-21-8317267. E-mail: akarmali@deq.isel.ipl.pt (Received 19 October 2010; revised 13 April 2011; accepted 24 May 2011) Introduction The food processing industry requires analytical meth- ods for quality control which are fast, reliable, spe- cific and cost-effective, since current wet chemistry tests are time-consuming and may require highly skilled labor and/or expensive equipment (Verma & Singh 2003). This urgent need is due to increased regula- tory action and consumer concern about food com- position and safety (Luong et al. 1997). Urea in adulterated milk is a major health concern and is especially harmful to pregnant women, children and sick persons (Mishra et al. 2010). Moreover, high urea concentrations in milk may affect adversely the reproductive performance of dairy cows by reducing fertility rates (Butler et al. 1996). High urea concen- trations in milk are often due to excessive levels of crude protein in the diet of dairy cows (Kirchgessner et al. 1986; Kirchgessner & Kaufmann 1987; DePeters & Ferguson 1992). Some wine and other fermented and distilled bev- erages can also contain high levels of urea. Urea in wines is probably due to excessive fertilization of vineyards (Ough et al. 1989; Francis 2006). The presence of urea in alcoholic beverages is of great concern because it reacts with ethanol to produce ethyl carbamate which is highly genotoxic, since it binds to DNA both in vitro and in vivo (Schlatter & Lutz 1990; Zimmerli & Schlatter 1991). Therefore, there is a need for a fast, cheap, sensi- tive, portable and reliable biosensor to monitor urea levels in milk and wines. Several urea biosensors have been developed based on immobilization of urease and use of transducers such as selective electrodes for carbon dioxide, ammonia gas, pH, ammonium ions, miniaturized urea electrodes using ion-selective field effect transistors and optical fibers (Dhawan et al. 2009). Recently, a flow injection analysis (FIA) biosensor for urea analysis in milk using an enzyme Biocatalysis and Biotransformation, July-August 2011; 29(4): 1–11 ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd. DOI: 10.3109/10242422.2011.591926 Biocatal Biotransformation Downloaded from informahealthcare.com by 194.210.197.147 on 07/04/11 For personal use only.