Elucidating the Correlation Between Nanozyme–Substrate Binding Constants and Catalytic Efficiency by Affinity Capillary Electrophoresis

The correlation between the binding constants of nanozymes with their substrates and the catalytic efficiency of these nanozymes was investigated for the first time by using affinity capillary electrophoresis (ACE). As a demonstration, two types of oxidase-like nanoceria-based nanozymes (CeO₂@PAA (PAA, poly(acrylic acid)) and CeO₂@PEI (PEI, polyethylenimine)) with different surface charges were employed as models, and partially filled affinity capillary electrophoresis (PFACE) was chosen for determining the binding constants (<i>K</i>_b) between these nanozymes and their substrate 3,3',5,5'-tetramethylbenzidine (TMB). In PFACE, by variation of the filling length of the nanozyme in the capillary column, <i>K</i>_b was calculated to be 152.23 and 117.74 L/mol for CeO₂@PAA and CeO₂@PEI, respectively. Moreover, with increasing concentration of the effector F^-, the <i>K</i>_b values between CeO₂@PEI and TMB exhibited a linear increase. Interestingly, by exploring the relationship between <i>K</i>_b and the catalytic kinetic parameters of the nanozyme, a good linear correlation between <i>K</i>_b and the catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) (<i>r</i> = 0.909) was observed. This work provides a new strategy for studying the binding interaction between nanozymes and their substrates, thereby offering insights into the elucidation of the catalytic performance of nanozymes through the understanding of the binding process.