Electrostatic Adsorption of Cytochromecon 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 ZrO 2 -gel 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 ZrO 2 -gel layer, as confirmed by scanning electron microscopy. Cyt. c showed weak adsorption at pH 4.0, since the ZrO 2 -gel layer had only a slight negative charge. When the ZrO 2 -gel layer was modified with octadecyltriethoxysilane, irreversible deposition of denatured Cyt. c was observed. In contrast, the ZrO 2 -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.