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

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 phospho-transferring reaction between ADP and phosphoenolpyruvate (PEP) to generate pyruvic acid; 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 μM–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.