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.
Shrimp aquaculture contributes significantly to global economic growth, and the whiteleg shrimp, Penaeus vannamei, is a leading species in this industry. However, Vibrio parahaemolyticus infection poses a major challenge in ensuring the success of P. vannamei aquaculture. Despite its significance in this industry, the biological knowledge of its pathogenesis remains unclear. Hence, this study was conducted to identify the interaction sites and binding affinity between several immune-related proteins of P. vannamei with V. parahaemolyticus proteins associated with virulence factors. Potential interaction sites and the binding affinity between host and pathogen proteins were identified using molecular docking and dynamics (MD) simulation. The P. vannamei-V. parahaemolyticus protein-protein interaction of Complex 1 (Ferritin-HrpE/YscL family type III secretion apparatus protein), Complex 2 (Protein kinase domain-containing protein-Chemotaxis CheY protein), and Complex 3 (GPCR-Chemotaxis CheY protein) was found to interact with -4319.76, -5271.39, and -4725.57 of the docked score and the formation of intermolecular bonds at several interacting residues. The docked scores of Complex 1, Complex 2, and Complex 3 were validated using MD simulation analysis, which revealed these complexes greatly contribute to the interactions between P. vannamei and V. parahaemolyticus proteins, with binding free energies of -22.50 kJ/mol, -30.20 kJ/mol, and -26.27 kJ/mol, respectively. This finding illustrates the capability of computational approaches to search for molecular binding sites between host and pathogen, which could increase the knowledge of Vibrio spp. infection on shrimps, which then can be used to assist in the development of effective treatment.