Yeast Cytochrome c Monolayer on Flat and Nanostructured Gold Films Studied by UV–Vis Spectroscopic Ellipsometry

Abstract

Spectroscopic ellipsometry (SE) has been used to investigate the adsorption of yeast cytochrome c (YCC) on gold films. YCC is a prototypical molecular system to evaluate the sensitivity and reliability of optical methods in probing conformational changes upon adsorption at surfaces. Measurements have been performed in situ in aqueous solutions at room temperature. We have applied to monolayer-thick YCC films the same scheme of analysis of SE spectra used in our recent works on thiolate self-assembled monolayers on gold, exploiting the so-called δ and δ difference spectra. A comparison between δ and δ spectra obtained from YCC adsorption on flat and cluster-assembled nanostructured gold films, obtained by supersonic cluster beam deposition is proposed. For both kinds of substrate δ and δ , difference spectra show well-defined features related to molecular optical absorptions typical of the so-called heme group, in particular the Soret band at about 410 nm. These features appear in the same position found for molecules in solution, suggesting that YCC native conformation is maintained upon adsorption also in the case of a substrate with typical roughness at the nanoscale.

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

“…Comparing the calculations in panels A and B it is evident that the optical constants of both the molecular film and the substrate contribute to the shape of the δ-spectra. As already pointed out in our previous work on nano-porous Au [18], substrate-related features are indeed often present in δ-spectra and might hide molecule-related features.…”

“…Ellipsometry and UV-vis spectroscopic ellipsometry (SE) have been often used to investigate adsorption dynamics at surfaces and to estimate the thickness of thin and ultrathin biological films [8][9][10][11] and other complex molecular systems [12][13][14][15][16]. In our previous works we demonstrated that SE is able to detect a monolayer-thick YCC film deposited on Au substrates [17,18]. In that case SE difference spectra were used to detect and monitor mainly the Soret band, while the weak signal of the Q-bands was hidden by spectral features related to the Au substrate.…”

“…We therefore chose SiO 2 /Si which was expected to be a better platform, since the corresponding difference spectra do not show substrate-related features neither in the Soret-band region nor in the Q-bands one. The same experimental approach we had previously used for monitoring YCC adsorption on flat and nanostructured gold [17,18] allowed us to detect not only the Soret band but also the Q-bands, whose low intensity usually makes their detection challenging. Difference spectra have been compared with calculations using a suitable optical model for both the protein layer and the substrate.…”

Orthogonal chemical functionalization of patterned gold on silica surfaces

“…Second, while biotinylated poly(ethylene glycol) [ 5 – 8 ] may be well suited to immobilize some biomolecules (avidin derivatives), it is worth considering other surface chemistries. For instance, carboxylic acid-based [ 10 – 21 ], amine-based [ 22 – 26 ] or other [ 27 – 28 ] self-assembled monolayers may provide a higher diversity of potential biomolecules to immobilize. Shorter spacer chains (e.g., short alkyl chains) may also be useful to immobilize the target as close to the metal surface (i.e., the maximum intensity of the evanescent field) as possible.…”