An amperometric urea biosensor based on covalently immobilized urease on an electrode made of hyperbranched polyester functionalized gold nanoparticles

An amperometric biosensor was fabricated for the quantitative determination of urea in aqueous medium using hematein, a pH-sensitive natural dye. The urease (Urs) was covalently immobilized onto an electrode made of gold nanoparticles functionalized with hyperbranched polyester-Boltron® H40 (H40–Au) coated onto an indium–tin oxide (ITO) covered glass substrate. The covalent linkage between the Urs enzyme and H40–Au nanoparticles provided the resulting enzyme electrode (Urs/H40–Au/ITO) with a high level of enzyme immobilization and excellent lifetime stability. The response studies were carried out as a function of urea concentration with amperometric and photometric measurements. The biosensor based on Urs/H40–Au/ITO as the working electrode showed a linear current response to the urea concentration ranging from 0.01 to 35 mM. The urea biosensor exhibited a sensitivity of 7.48 nA/mM with a response time of 3 s. The Michaelis–Menten constant for the Urs/H40–Au/ITO biosensor was calculated to be 0.96 mM, indicating the Urs enzyme immobilized on the electrode surface had a high affinity to urea.