Colorimetric detection of methotrexate leveraging the halogen peroxidase-mimicking activity of BiWO nanoflowers.

This study explores BiWO nanoflowers as novel haloperoxidase (HPO) mimetics and their application in analytical science, aiming to develop an efficient colorimetric method for methotrexate (MTX) detection. BiWO nanoflowers were synthesized 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 () and maximum reaction velocity (), exceeded those of most of the reported HPO nanozymes. Investigation of the catalytic mechanism identified singlet oxygen (O) as a reactive intermediate. Leveraging the inhibitory effect of MTX on BiWO-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 BiWO nanoflowers as efficient HPO nanozymes and provides a reliable approach for the rapid and simple detection of MTX.