Carboxyl-functionalized covalent organic framework with precisely matched pore size achieving effective loading of...

The immobilization of enzymes is crucial for enhancing their catalytic activity and stability. Covalent organic frameworks (COF), with abundant active sites and tunable pore structures, enable effective immobilization of enzymes. Here, we designed carboxyl-functionalized COF (COF-COOH) to immobilize Cytochrome C (Cyt C), aiming to regulate the perfect pairing of the COF pore (3.67 nm) and the Cyt C dimension (2.6 nm × 3.2 nm × 3.3 nm). Meanwhile, the large amount of -COOH can increase the electrostatic and hydrogen bonding forces between COF-COOH and Cyt C. Thus, the Cyt C was efficiently loaded into COF-COOH through the post-modification method (loading efficiency = 62.37 %). The catalytic activity (k_cat/K_m) of Cyt C@COF-COOH toward H₂O₂ was significantly enhanced to 309.96 s^-1 M^-1 as compared to free Cyt C of 105.55 s^-1 M^-1. The catalytic activity of Cyt C@COF-COOH toward H₂O₂ still exceeds 80 % in some harsh environments (acetonitrile, dimethyl sulfoxide, tetrahydrofuran and 60 °C). The detection range of electrochemical H₂O₂ biosensor based on Cyt C@COF-COOH is as wide as 2.0-80 μM, and the sensitivity is as high as 0.373 μA μM^-1 cm^-2.