Yeast Cytochrome c adsorption on SiO2/Si substrates studied by in situ spectroscopic ellipsometry

In situ spectroscopic ellipsometry is employed to investigate the adsorption of Yeast Cytochrome c (YCC) on SiO2/Si substrates. In order to highlight the slight variations induced by protein adsorption, difference spectra (delta Delta and delta Psi) have been considered, following the approach introduced in our previous studies on self-assembled monolayers. The difference spectra show a sharp dip at about 410 nm, the Soret or B band, related to the heme optical absorption, whose fine position is sensitive to the protein environment and to the oxidation state of the iron ion within the heme. Remarkably, the analysis of the difference spectra allowed us to detect two lower intensity dips in the 590-650 nm range, the Q bands, whose position and lineshape provide additional information on protein conformation and redox state. Quantitative reproduction of experimental data obtained by using a simple isotropic optical model, accounting for the molecular absorption spectrum, is presented. Estimates of the film thickness and determination of the position and shape of the heme-related features were obtained from calculations. The results are compared with those previously obtained in a study on YCC adsorption on Au substrates. Complementary ex-situ atomic force microscopy measurements are also presented. (C) 2013 Elsevier B.V. All rights reserved.