Interference-free, amperometric measurement of urea in biological samples using an electrode coated with tri-enzyme/polydimethylsiloxane-bilayer membrane

An amperometric urea-sensing electrode was prepared by immobilizing a tri-e nzyme system of pyruvate oxidase (PyOx), pyruvate kinase (PK) and urea amid olyase (UA) on a polydimethylsiloxane (PDMS)-coated electrode. UA catalyzes the adenosine-5 ' -triphosphate (ATP)-dependent hydrolysis of urea to gene rate adenosine-5 ' -diphosphate (ADP); PK, the phosphotransferring reaction between ADP and phosphoenolpyruvate (PEP) to generate pyruvic acids PyOx, the oxidation of pyruvic acid with oxygen. The oxygen consumption could be monitored by using a PDMS-coated electrode without interference from the Py Ox-reaction product, hydrogen peroxide. Thus, the concentration of urea (5 muM - 0.35 mM) could be determined from the decrease in the cathodic curren t at -0.4 V versus Ag/AgCl in a test solution containing ATP and PEP. The c athodic detection with the tri-enzyme system provided the urea determinatio n which was free from the error caused by coexisting species, such as aceta minophen, uric acid, phosphate in the sample to be measured. (C) 2001 Elsev ier Science B.V. All rights reserved.