A biosensor based on urate oxidase  /peroxidase coupled enzyme system for uric acid determination in urine Erol Akyilmaz *, M. Kemal Sezgintu ¨ rk, Erhan Dinc ¸kaya Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Received 24 October 2002; received in revised form 21 February 2003; accepted 25 February 2003 Abstract A new amperometric biosensor based on urate oxidase  /peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H 2 O 2 from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 mM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 8C were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9 /10 3 U cm 2 for urate oxidase and 512.7 U cm 2 for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Uric acid; Urate oxidase; Peroxidase; Coupled enzyme systems; Biosensors 1. Introduction The development of biosensors is most promis- ing in the progress of analysis of biologically active compounds. In the past two decades biosensors had emerged from laboratories and some cases became conventional devices for routine analysis [1  /3] owing to the advantages biosensors possess, such as simple measurement procedure, short response time, sensitivity and selectivity. As a result of these important factors biosensors can be widely used for the analysis of many metabo- lites [4  /6]. Traditional monitoring methods are often slower and call for expensive equipment which makes them unsuitable for real time control and this also makes biosensors more attractive [7]. Uric acid is the primary end-product of purine metabolism and its excreted in the urine is derived from purines arising from the catabolism of diet- ary and endogenous nucleic acid stem from increased catabolism dysfunction of one of the shunt pathways which leads to increased urate production. Excessive production of uric acid may lead to precipitation in the kidney and lower extremities [8]. Moreover, one of the biggest problems about the uric acid metabolism is the * Corresponding author. Fax:  /90-232-388-8264. E-mail address: akyilmaz@sci.ege.edu.tr (E. Akyilmaz). Talanta 61 (2003) 73  /79 www.elsevier.com/locate/talanta 0039-9140/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0039-9140(03)00239-X