Bimetallic oxide (CoNi2O4) nanozyme as ROS independent for ascorbic acid detection with computational study

Herein, spinel CoNi₂O₄ nanoflowers (NFs) were successfully synthesized by a two-step hydrothermal annealing process and testified to have intrinsic enzyme mimic activities. The CoNi₂O₄ NFs can effectively oxidize the chromogenic substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) to ox-TMB showing catalytic behavior that follows enzyme kinetics of Michaelis-Menten equation with a low constant (K_m = 0.0143 mM). Innovatively, experimental data and Density Functional Theory (DFT) calculations disclosed the stable structure and mechanism of spinal CoNi₂O₄NFs, which demonstrated oxidase-like activity and reactive oxygen species independence across a wide temperature range. Accordingly, a colorimetric biosensor for rapid and sensitive detection of ascorbic acid (AA) was successfully developed, displaying excellent stability, selectivity, sensitivity and low limit of detection (0.44 μM). This biosensor was applied to vitamin C capsules and fresh lemon fruit, showing favorable reproducibility and feasibility. DFT and molecular modeling (MD) calculations indicate a cobalt atom as the optimal site for catalytic conversion, while the amine group in the TMB molecule is the optimal nucleophilic attack site.