Tyrosinase–SWCNT Electrochemical Biosensor for Tyramine Detection: Experimental Development and Molecular Dynamics Insights

Abstract Tyramine (TYM) serves as a critical indicator of microbial spoilage and significant health risks at elevated concentrations. Herein, we report an integrated experimental and computational approach to develop a high-performance electrochemical biosensor for selective TYM detection. Tyrosinase (Tyro) was immobilized onto carboxylated single-walled carbon nanotube-modified screen-printed carbon electrodes (Tyro–COOH-SWCNT/SPCE) to construct a nanostructured bioelectrode. The resulting biosensor exhibits a detection limit of 1.15 × 10–6 M and a linear range from 9.98 × 10–6 to 1.08 × 10–4 M. Furthermore, the biosensor demonstrates high selectivity toward TYM, with minimal interference (<10%) from common biogenic amines, including histamine, putrescine, spermidine, and spermine. Practical applicability was validated using spiked chicken meat samples, achieving satisfactory recoveries between 89.10–108.59%. To provide a molecular-level interpretation of TYM recognition by Tyro, molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area (MMPBSA) analysis was performed. The atomistic simulations of Tyro reveal a copper-stabilized binding pocket that accommodates TYM through predominantly hydrophobic interactions. Molecular mechanics Poisson–Boltzmann surface area (MMPBSA) analysis yields a favorable binding free energy (ΔG = −22.46 ± 2.26 kcal mol–1), while per-residue decomposition identifies key histidine and valine residues near the binuclear copper center as major contributors to binding. By integrating experimental electrochemical performance with atomistics insights into enzyme–substrate recognition, this study provides a mechanism-informed framework for understanding Tyro-based biosensing and demonstrates its potential for rapid, selective, and on-site monitoring of food spoilage.