Electrostatic adsorption of cytochrome c on ultrathin ZrO₂-Gel layers and preparation of alternate multilayers

Positively charged proteins are electrostatically adsorbed on ultrathin metal oxide gel layers that are prepared by the surface sol-gel process. This feature was applied to assembly of multilayer films of cytochrome c (Cyt.c) and zirconium oxide. Regular growth of the alternate multilayer was verified by UV-vis absorption spectroscopy and quartz crystal microbalance measurements. The ZrO₂gel layer was uniformly covered by the Cyt.c at pH 10.0 with a thickness of 2.4 nm for the protein layer. An excess amount of Cyt.c was adsorbed at pH 7.0 to produce globular aggregates on the ZrO₂-gel layer, as confirmed by scanning electron microscopy. Cyt.c showed weak adsorption at pH 4.0, since the ZrO₂gel layer had only a slight negative charge. When the ZrO₂-gel layer was modified with octadecyltriethoxysilane, irreversible deposition of denatured Cyt.c was observed. In contrast, the ZrO₂-gel covered by an ultrathin poly(vinyl alcohol) layer adsorbed Cyt.c without denaturation. Such Cyt.c molecules were readily desorbed by changing pH to above the isoelectric point. The surface sol-gel process provides a convenient method to immobilize water-soluble proteins through moderate electrostatic attraction.