Electroactive cytochrome P450BM3P450BM3 cast polyion films on graphite electrodes

Films of electrochemically active cytochrome P450BM3P450BM3 were constructed on graphite electrodes using alternate assembly with polyethyleneimine (PEI). The original layer-by-layer adsorption method was slightly modified here to form so-called “cast polyion” films. The cast polyion films were elaborated by immobilizing two successive layers of PEI and protein in very large excess with respect to a monolayer, without any intermediate washing step. Following the immobilization steps by SEM showed that uniform films of a few micrometers were deposited on the graphite surface. The electrochemically activity of the immobilized cytP450 was tested with regard to the reduction of oxygen and the one-electron reduction of the heme. Cyclic voltammetry indicated surface concentration of electrochemically active cytP450 around 0.6 nmol/cm2, which corresponded to 5% of the total amount of protein that was consumed by the immobilisation process. Adapting the procedure to a graphite felt electrode with the view of scaling up porous electrodes for large scale synthesis increased the concentration to 0.9 nmol/cm2. Cast polyion films may represent a simple technique to immobilize high amount of electrochemically active protein, keeping the advantage of the electrostatic interactions of the regular layer-by-layer method.