Copper sensing by urease in synthetic urine: application for Wilson's disease monitoring

Copper, Cu, is an essential transition metal whose concentration has to be carefully regulated to prevent high-level toxicity associated with its accumulation, as observed in Wilson's disease (WD). Conventional methods such as Atomic Absorption Spectroscopy (AAS) and Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) are widely used for metal detection and quantification. In recent years, researchers have focused on developing miniaturized and portable devices with alternative sensing approaches. Herein, we exploited the inhibitory effect of Cu(II) on urease, a well-known Ni-dependent enzyme, to develop a selective monitoring system for the presence of Cu(II) in Artificial Urine (AU). This reagent-exploiting biosensing-like platform enables the direct detection of Cu(II) by monitoring urease activity in a clinically relevant matrix without external substrate addition. An optimized in vitro spectrophotometric assay was carried out to follow the hydrolysis of urea, catalyzed by urease. A concentration-dependent inhibition of urease activity was observed, reaching an enzymatic inhibition of more than 90% under optimized conditions. In parallel, Cu(II)-mediated urease inhibition was applied to develop an electrochemical assay through voltammetric readout. Finally, the AU matrix was tested with the developed urease-based system, showing a significant inhibition starting from 25 ppb in the colorimetric assay and 50 ppb in the electrochemical assay. These findings highlight the viability of utilizing enzymes to engineer advanced biosensors for heavy metal monitoring, particularly forCu(II) detection, opening the way to the rapid and on-site detection of copper in biological fluids.