Direct electrochemistry of heme proteins in their layer-by-layer films with clay nanoparticles

Stable films of myoglobin (Mb) or horseradish peroxidase (HRP) were assembled layer-by-layer with clay nanoparticles on various solid substrates by alternate adsorption of negatively charged clay platelets from their aqueous dispersions and positively charged heme proteins from their buffers at the appropriate pH. Cyclic voltammetry (CV), the quartz crystal microbalance (QCM), and UV–vis spectroscopy were used to monitor the film growth. CV of {clay ∣ protein}n films on pyrolytic graphite (PG) electrodes showed a pair of well-defined, nearly reversible peaks at about −0.20 V vs Ag/AgCl at pH 5.5, characteristic of the heme Fe(III)/Fe(II) redox couples. Although the amount of Mb or HRP adsorbed in each bilayer was essentially the same, the fraction of electroactive proteins decreased with the number of clay ∣ protein bilayers. The electroactivity of Mb and HRP extended to six and seven clay ∣ protein bilayers, respectively. The microscopic FTIR and UV–vis spectroscopy showed that both Mb and HRP in the dry {clay ∣ protein}n films retained their secondary structure. The Soret adsorption band of Mb in {clay ∣ Mb}6 films showed that Mb kept the conformation similar to its native state in the medium pH range. {Clay ∣ protein}n film electrodes catalyzed the electrochemical reduction of oxygen and hydrogen peroxide with a significant lowering of overpotential.