2-Methyl-3-(omega-mercaptoalkyl)-1,4-naphthoquinones, containing 5, 7, and 9 carbon atoms in the alkyl chain, were synthesized, and the structural and electrochemical parameters of their self-assembled monolayers (SAMs) on gold were studied by in situ Fourier-transform surface-enhanced Raman spectroscopy (FT-SERS) and cyclic voltammetry (CV). We have demonstrated that the alkyl chains of SAM prepared from the concentrated (1 mM) substance solution contain a number of gauche defects. Nevertheless, it has been found that the 2-methyl-1.4-naphthoquinone (2-MeNQ) group does not interact directly with gold. In contrast, spectral changes for surface layers prepared from diluted (0.05 mu M) solutions have indicated flat ring orientation and direct involvement of 2-MeNQ group in the interaction with An. It has been found that the Au-S stretching band at 291 cm(-1) considerably increases in intensity and blue-shifts by similar to 10 cm(-1) in the surface spectra of monolayers prepared from low concentration coating solution indicating different Au-S bonding geometry and coordination of sulphur atom with Au. Up-shift in the C=O band from 1654 to 1660 cm(-1) has been detected for the mixed monolayer prepared using shorter alkyl chain diluent, heptanethiol, and indicates absence of stacking interaction between the 2-MeNQ rings as well as involvement of the C=O group in the hydrogen bonding with interfacial water. The electrochemical response of 2-MeNQ group buried in the long-chain thiols containing mixed monolayers was found to be controlled by the nature of the diluent terminal group. Thus, replacement of the CH3 group in dodecanethiol by COOH has induced electrochemical activity of 2-MeNQ group in the mixed SAM. (c) 2006 Elsevier B.V. All rights reserved.