Colorimetric detection of methotrexate leveraging the halogen peroxidase-mimicking activity of Bi ₂ WO ₆ nanoflowers

This study explores Bi₂WO₆ nanoflowers as novel haloperoxidase (HPO) mimetics and their application in analytical science, aiming to develop an efficient colorimetric method for methotrexate (MTX) detection. Bi₂WO₆ nanoflowers were synthesized <i>via</i> a modified hydrothermal method and exhibited bromoperoxidase- and iodoperoxidase-like activities, catalyzing the bromination of phenol red (PR) and iodination of thymol blue (TB). After optimizing the reaction conditions, the kinetic parameters, including the Michaelis-Menten constant (<i>K</i>_m) and maximum reaction velocity (<i>V</i>_max), exceeded those of most of the reported HPO nanozymes. Investigation of the catalytic mechanism identified singlet oxygen (¹O₂) as a reactive intermediate. Leveraging the inhibitory effect of MTX on Bi₂WO₆-based nanozymes, a colorimetric assay for MTX was developed, demonstrating excellent detection performance in terms of a wide linear range and a low detection limit. Furthermore, the developed assay exhibited reliable performance in detecting actual samples. This study validates Bi₂WO₆ nanoflowers as efficient HPO nanozymes and provides a reliable approach for the rapid and simple detection of MTX.