Stability enhancement of uric acid biosensor via enzyme mutagenesis and nanocomposite integration

Urate oxidase (UOx) shows potential for developing uric acid (UA) sensors, but its low catalytic efficiency and environmental fragility limit broader application. This study engineered Bacillus mojavensis XH1 UOx using a semi-rational design approach, resulting in mutant UOx_Q170K with enhanced catalytic efficiency (2.84-fold increase in specific activity), thermal stability (melting temperature change +7.54 °C), and operational stability (1.94-fold extension in half-life). Structural analysis revealed that the Q170K mutation stabilized substrate binding via optimized hydrogen bonding and hydrophobic interactions, thereby reducing conformational flexibility while maintaining catalytic accessibility. To leverage these improvements, we have integrated zeolitic imidazolate framework-8 (ZIF-8) for enzyme immobilization, carbon nanotubes (CNTs) for enhanced electron transfer, and horseradish peroxidase (HRP) for H₂O₂ signal amplification to synthesise a multifunctional HRP@ZIF-8/CNT-UOx_Q170K nanohybrid. This design minimized intermediate diffusion distances and amplified electrochemical responses, achieving a limit of detection of 0.031 μM and sensitivity of 32.26 μA μM^-1 cm^-2 for UA on a glassy carbon electrode. The sensor exhibited robust anti-interference capability against common metabolites and retained >85 % signal stability over 14 days. This work establishes a synergistic approach combining enzyme engineering with nanomaterial design to advance electrochemical biosensing platforms, providing a paradigm for developing robust enzymatic detection systems with amplified signal transduction.