Engineering a Hollow Carbon Sphere‐Based Triphase Microenvironment for Enhanced Enzymatic Reaction Kinetics and Bioassay Performance

Abstract Electrochemical bioassays based on oxidase reactions are frequently used in biological sciences and medical industries. However, the enzymatic reaction kinetics are severely restricted by the poor solubility and slow diffusion rate of oxygen in conventional solid‒liquid diphase reaction systems, which inevitably compromises the detection accuracy, linearity, and reliability of the oxidase‐based bioassay. Herein, an effective solid‒liquid‒air triphase bioassay system is provided that uses hydrophobic hollow carbon spheres (HCSs) as oxygen nanocarriers. The oxygen stored in the cavity of HCS can rapidly diffuse to the oxidase active sites through the mesoporous carbon shell, providing sufficient oxygen for oxidase‐based enzymatic reactions. As a result, the triphase system can significantly improve the enzymatic reaction kinetics and obtain a 20‐fold higher linear detection range than the normal diphase system. Other biomolecules can also be determined using this triphase technique, and the triphase design strategy offers a new route to address the gas deficiency problem in catalytic reactions that involve gas consumption.