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 (H₂O₂) 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 H₂O₂ 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 H₂O₂-dependent electroenzymatic reactions, offering a promising platform for diverse applications in bioelectrosynthesis.