Engineering <i>Bacillus</i> Spores to Display Nicotine Oxidase: In Situ Specific and Sensitive Nicotine Detection

Nicotine exposure poses a significant public health challenge, necessitating highly sensitive and real-time detection methods. This study presents a novel live electrochemical biosensor (EN@FeSe@MHCS/GCE) by integrating synthetic biology and materials engineering. A genetically engineered NOX mutant-with 9.5-fold enhanced activity and thermal stability (<i>T</i>_m = 75 °C) was displayed on <i>Bacillus subtilis</i> spores via genomic integration. The FeSe@MHCS composite electrode, features a hierarchical porous 3D nanoflower structure (specific surface area: 196.8 m²·g^-1, pore size: 8.46 nm) and hydrophilicity (contact angle ≈ 15°), enabling rapid electron transfer and enzyme stabilization. As expected, the biosensor achieved an ultralow detection limit (0.25 nM) for nicotine via H₂O₂-mediated signal amplification, with minimal matrix interference (recovery: 98 ± 1.36-107% ± 2.72 in blood/urine), exceptional stability (98% ± 1.78 current retention after 14 days) and a reproducibility RSD of 0.62% across different electrode batches, demonstrating its efficacy as a real-time nicotine monitoring tool. Electrochemical impedance spectroscopy (EIS) confirmed low charge transfer resistance (<125 Ω), while differential pulse voltammetry (DPV) demonstrated a linear response (<i>R</i>² > 0.97) across nicotine concentrations. The integration of spore-displayed NOX and FeSe@MHCS eliminates reliance on bacterial growth and complex genetic circuits, offering a robust platform for real-time nicotine monitoring. This work successfully validates the proof-of-concept for developing NOX into a live electrochemical sensor, advances enzyme-based electrochemical sensing and provides a blueprint for biosensors targeting diverse analytes.