Copper-based nanozymes as catalysts and reaction terminators for dual-channel colorimetric urea sensing

Urea is an important clinical biomarker for health monitoring and is also used as a nitrogen-containing adulterant in food products to artificially increase the apparent protein content, creating an important demand for portable and reliable detection methods. In this work, Cu₂O nanocubes were used as representative copper-based nanozymes to construct a dual-channel colorimetric platform for urea sensing by combining UV-vis absorbance with smartphone-assisted RGB analysis. In the sensing cascade, urease first catalyzes the hydrolysis of urea to generate NH₃, leading to an increase in solution pH. Since the peroxidase-like (POD-like) activity of Cu₂O is highly pH-dependent, this pH evolution is converted into a concentration-dependent colorimetric response through the H₂O₂-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). However, residual urease can continue to hydrolyze urea during the subsequent color-development step, causing further pH drift and unstable optical signals. Importantly, Cu₂O nanocubes function not only as catalytic nanozymes for TMB oxidation, but also as reaction terminators that suppress the residual urease activity, thereby stabilizing the system pH and improving the signal reliability. Based on this mechanism, a gelatin hydrogel sensor was further developed for portable urea monitoring. The absorbance channel showed a linear response over 0-2.2 mM with a detection limit of 5.75 μM, while the RGB channel provided smartphone-readable quantitative analysis. The proposed platform was successfully applied to urea determination in sweat and milk samples, demonstrating its potential for portable biosensing and food safety analysis.