Molecular hydrogel-stabilized enzyme with facilitated electron transfer for determination of H₂O₂ released from live cells

In this work, small molecular hydrogel was first employed as a surrounding matrix to stabilize an enzyme model, Cytochrome c (Cyt c), and more importantly to facilitate electron transfer between redox enzyme and electrode. Direct electron transfer of Cyt c was successfully achieved in the molecular hydrogel with redox formal potential (E^0′) of 100.7 ± 3.2 mV versus Ag|AgCl and heterogeneous electron transfer rate constant (k_s) up to 18.6 ± 2.3 s^-1. Experimental data demonstrated that Cyt c was stably immobilized into the molecular hydrogel and retained its inherent bioactive activity toward H₂O₂. The direct redox reaction of Cyt c, followed by the biochemical reaction between Cyt c and H ₂O₂, established a reliable approach to determine H ₂O₂ at an optimized potential with high selectivity over other reactive oxygen species (ROS), oxygen, metal ions, ascobic acid (AA), and so on. In addition, the present biosensor for H₂O₂ also exhibited wide linear range and low detection limit, which fulfills the requirements for detection of H₂O₂ in a biological system. The remarkable analytical performance of the present biosensor, as well as the long-term stability and good reproducibility ascribed to the molecular hydrogel-stabilized enzyme, provided a durable platform for real-time determination of H₂O₂ from live cells.