Freestanding HRP-GOx redox buckypaper as an oxygen-reducing biocathode for biofuel cell applications

Horseradish peroxidase (HRP) was immobilized on redox buckypapers followed by electropolymerization of pyrrole-modified concanavalin A enabling the subsequent additional immobilization of the glycoprotein glucose oxidase (GOx). Biocatalytic buckypapers were formed using pyrene-modified 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or bis-Pyr-ABTS, a redox mediator, as a cross-linker. ABTS-functionalized buckypaper enhances the electron transfer of the bioelectrocatalytic reduction of H₂O₂ by HRP. Since H₂O₂ is produced during glucose oxidation by GOx in the presence of oxygen, the bienzymatic GOx-HRP biocathode achieves the complete reduction of oxygen into water. A clearly improved performance of the biocathode was obtained by using an improved biocompatible immobilization strategy, enabling the prevention of enzyme loss while ensuring both diffusion of glucose and O₂ and the local production of H₂O₂. These freestanding flexible oxygen-reducing biocathodes can operate under physiological conditions and show a high onset potential at 0.60 (±0.01) V. In the presence of glucose (5 mM), such biocathodes exhibit a stable current density output of 1.1 (±0.1) mA cm^-2 at 0.1 V under continuous one-hour discharge. Furthermore, a marked increase in lifetime was observed, the biocathode displaying 64% of its initial electrocatalytic activity after 15 days.