Impact of the size effect on enzymatic electrochemical detection based on metal-organic frameworks

Metal-organic frameworks (MOFs) hold a great promise as immobilization carriers for enzymes and other biomolecules, owing to their enhanced stability, selectivity and controllability. However, enzyme-MOF complexes usually lead to a decrease in the apparent enzyme activity and apparent substrate affinity as a result of the constrained structure of the enzyme and the mass transfer limitation of the substrate, respectively. These results consequently impede the applications of enzyme-MOF complexes in biocatalysis and biosensing. In this study, zeolitic imidazolate framework-8 (ZIF-8) was synthesized to immobilize cytochrome c (Cyt c) via a one-step co-precipitation process under mild conditions. By adjusting the molar ratio of precursors, enzyme-MOF composites with different sizes from 100 nm to 1.3 μm were prepared. The decreased size of the prepared MOFs generated an increase in substrate affinity (with an over 50% decrease in the Michaelis constant K_m) and a 6.4-fold improvement in the apparent enzyme activity with a 6.26-fold increase in the enzymatic electrochemical detection sensitivity compared with native Cyt c. The enzyme-MOF composites were coated on a screen-printed electrode for the sensitive and fast detection of H₂O₂, which is the most common representative of reactive oxygen species in cellular environments, showing the potential for the construction of efficient biosensors with applications in biomedicine.