Design of a Peroxidase‐Based Gas Diffusion Electrode for Bioelectroenzymatic Applications

We report here the design and application of an all‐in‐one gas diffusion electrode (GDE) combined with surface immobilized horseradish peroxidase (HRP) capable of in situ generation of hydrogen peroxide (H 2 O 2 ) from air and simultaneous oxidation of a model substrate (ABTS) within a single‐cell electrochemical reactor. Carboxyl‐functionalized multiwalled carbon nanotubes (MWCNT‐COOH) were employed as an electrocatalyst for oxygen reduction reaction producing 719 ± 97 µM h −1 H 2 O 2 at −0.2 V ( vs Ag/AgCl) as an electron acceptor for HRP. We investigated two immobilization strategies to obtain HRP‐modified biocathodes using covalent amide conjugation between primary amine groups of HRP and carboxyl groups of MWCNT‐COOH (GDE/MWCNT‐COOH/HRP) and entrapment into a cross‐linked pyrene‐modified linear poly(ethylenimine) matrix (GDE/MWCNT‐COOH/Py‐LPEI/HRP). Fourier transform infrared spectroscopy (FT‐IR) and scanning electron microscopy (SEM) were used to characterize such surface modifications. The apparent catalytic activity achieved by HRP‐modified biocathodes via either covalent conjugation or entrapment into a polymer film was 311 ± 31 U mg −1 and 174 ± 17 U mg −1 , respectively, as compared to the activity of freely diffusing 188 ± 23 U mg −1 . The interfaces were reused showing 55% and 82% residual activity after 5 consecutive cycles for GDE/MWCNT‐COOH/HRP and GDE/MWCNT‐COOH/Py‐LPEI/HRP, respectively. Our findings illustrate prospects for integrating GDE and surface‐bound peroxidases for H 2 O 2 ‐dependent electroenzymatic reactions, offering a promising platform for diverse applications in bioelectrosynthesis.