Self-assembly of electroactive layer-by-layer films of heme proteins with anionic surfactant dihexadecyl phosphate

Anionic surfactant dihexadecyl phosphate (DHP) with two hydrocarbon chains can be self-assembled into a double-layer structure with tail-to-tail configuration and negatively charged head groups toward outside in its aqueous dispersion. Due to this unique biomembrane-like structure, the "charge reversal" in DHP adsorption on solid surface was realized, and the DHP was successfully assembled with positively charged myoglobin (Mb) or hemoglobin (Hb) into {DHP/protein}(n) layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor or confirm the film assembly process. The {DHP/protein}(n) films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about -0.35 V vs SCE in pH 7.0 buffers, characteristic of the protein heme Fe(III)/Fe(II) redox couples. Based on the direct electrochemistry of heme proteins, the {DHP/protein}(n) films could also be used to electrochemically catalyze reduction of oxygen, hydrogen peroxide and nitrite with significant lowering of reduction overpotentials. Scanning electron microscopy (SEM), UV-vis spectroscopy, and reflectance absorption infrared (RAIR) spectroscopy were employed to characterize the {DHP/protein}(n) films, suggesting that the proteins in the films retain their near-native structure.