Direct electron transfer between hemoglobin and pyrolytic graphite electrodes enhanced by Fe3O4 nanoparticles in their layer-by-layer self-assembly films

Alternate adsorption of negatively charged Fe3O4 nanoparticles from their pH 8.0 aqueous dispersions and positively charged hemoglobin (Hb) from its pH 5.5 buffers on solid substrates resulted in the assembly of {Fe3O4/Hb}n layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the film growth. A pair of well-defined, nearly reversible CV peaks for HbFe(III)/Fe(II) redox couples was observed for {Fe3O4/Hb}n films on pyrolytic graphite (PG) electrodes. Although the multilayered films grew linearly with the number of Fe3O4/Hb bilayers (n) and the amount of Hb adsorbed in each bilayer was generally the same, the electroactive Hb could only extend to 6 bilayers. This indicates that only those Hb molecules in the first few bilayers closest to the electrode surface are electroactive. The electrochemical parameters such as the apparent heterogeneous electron transfer rate constant (ks) were estimated by square wave voltammetry (SWV) and nonlinear regression. The Soret absorption band position of Hb in {Fe3O4/Hb}6 films showed that Hb in the films retained its near native structure in the medium pH range. The {Fe3O4/Hb}6 film electrodes also showed good biocatalytic activity toward reduction of oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite. The electrochemical reduction overpotentials of these substrates were lowered significantly by {Fe3O4/Hb}n films.