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-enzyme system of pyruvate oxidase (PyOx), pyruvate kinase (PK) and urea amidolyase (UA) on a polydimethylsiloxane (PDMS)-coated electrode. UA catalyzes the adenosine-5 ' -triphosphate (ATP)-dependent hydrolysis of urea to generate 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 PyOx-reaction product, hydrogen peroxide. Thus, the concentration of urea (5 muM - 0.35 mM) could be determined from the decrease in the cathodic current at -0.4 V versus Ag/AgCl in a test solution containing ATP and PEP. The cathodic detection with the tri-enzyme system provided the urea determination which was free from the error caused by coexisting species, such as acetaminophen, uric acid, phosphate in the sample to be measured. (C) 2001 Elsevier Science B.V. All rights reserved.