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A novel impedimetric biosensor was microfabricated for the selective and quantitative determination of urea by immobilizing urease onto the 1 cm × 2 cm × 0.5 mm PHB substrate which was patterned with gold interdigitated electrode arrays (IDAs) using a combination of photolithography, etching, and thermal evaporation processes. In this study, urease was covalently immobilized onto the gold IDAs functionalized with 10-carboxyl-1-decanethiol (CDT) and similarly, onto gold IDAs functionalized with CDT-polypropylenimine dotriacontaamine dendrimer (Generation 4.0, DAB-Am-32) for monitoring factors that affect urease immobilization. The response studies were carried out as a function of urea concentration with impedimetric measurements. These two immobilization techniques were introduced to assay for in vitro urea and evaluate the two immobilization technique in terms of their repeatability, linear dynamic range, temperature and pH stability. The CDT-urease biosensor displayed linearity for assayed urea up to 10 mM with a response time (t95) of 2 s and yielded a Km(app) of 0.7 mM, indicating the urease enzyme immobilized using CDT onto the PHB IDA surface had a high affinity to urea. The CDT-DAB-Urease biosensor exhibited a dynamic linear range of 0.10 - 300.0 mM, which represented a 30-fold increase in linear dynamic urea range compared to the biosensor with the CDT-urease. The CDT-DAB-Urease yielded a Km(app) of 2.2 mM, which is not significantly different from that for the native, solution-borne enzyme (3 mM), thereby indicating an affinity to urea. Preliminary results suggest that the coupling of the CDT-DAB immobilization technique with microfabrication technology can offer large scale production of PHB urea biosensor.