The adsorption of phosphate anions on Ag, Au, and Cu electrodes from H2O and D2O solutions has been studied by means of surface-enhanced Raman spectroscopy (SERS). The interpretation of the spectra based on the solution Raman data and frequency shifts upon solution H2O/D2O exchange are presented. The prominent band at 1070−1100 cm-1, observed from adsorbed PO4 3- and HPO4 2- ions, exhibits downshifts of about 10 cm-1 in D2O solutions and has been assigned to the asymmetric P−O stretching mode. The corresponding asymmetric deformation mode has been assigned to the band located at ∼570 cm-1 which shows an upshift of 9−15 cm-1 in D2O solutions. Monodentate surface coordination of the PO4 3- and HPO4 2- ions is proposed. The dependence of the relative intensity of the internal modes on electrode potential was interpreted in terms of the migration of P−O groups from the surface as potential became more negative. Spectroscopic evidence was found for chemisorption of H2PO4 - ion on the Cu electrodes, but no such evidence was found in the cases of the Au and Ag electrodes. The adsorbed H2PO4 - ion on Cu showed an intense band at ∼907 cm-1 which was assigned to the symmetric stretching mode of the P−OH groups, based primarily on the considerable frequency downshift (∼11 cm-1) and peak broadening (∼15 cm-1) in D2O solutions. The formation of a P−O−P bond in the adsorbed state on Cu electrodes in acidic solutions is suggested. The force constants derived from experimental metal−oxygen frequencies are compared for the three electrodes, and the following strength order has been estimated:  k(Cu−O‘) > k(Au−O‘) > k(Ag−O‘).

Cytochrome c (cyt c) was adsorbed on N-acetylcysteine (NAC)-modified gold electrodes via electrostatic interaction. The cyt c layer exhibited reversible and stable electrochemical redox transformation in 0.01 M phosphate butter, pH 7.4, where the heterogeneous electron transfer (ET) constant k‘het was measured by three techniques:  cyclic voltammetry at high sweep rates (CV), electrochemical impedance (EI), and electroreflectance (ER) spectroscopy. In addition, k‘het was also determined from combining sets of simultaneous electrochemical impedance (EI) and electroreflectance (ER) measurements in a new impedance model in which a constant-phase element was used. The negligible shift (−0.023 mV) in the formal potential from the solution value, and the close agreement of the measured distribution around the CV peaks (full-width voltage at half-peak-height, E fwhh = 97 mV) with the theoretical value of 90.6 mV, suggested that the immobilized cyt c is retained at the electrode in the native state. Apparent k‘het values, as determined by each method separately, were as follows:  (k‘CV = 920 ± 280 s-1 by CV, k‘EI = 660 ± 200 s-1 by EI, and k‘ER = 2100 ± 300 s-1 by ER as interpreted using previously published methods. ,, In the combined EI/ER measurements, k‘ER was found to increase linearly with the frequency of the ac modulating current and spanned a range from about 400 to 800 s-1, which is close to the interprotein electron-transfer rate constant (800 s-1) measured between cyt c and one of its natural redox partners, cytochrome c peroxidase. It is concluded that further attempts to reconcile these discrepancies in k‘het determinations will require more detailed descriptions of the interfacial elements in the impedance models.

Adsorption of putidaredoxin (Pdx) on gold electrodes was studied using dynamic spectroscopic ellipsometry and differential capacitance. A method to account for the metal surface optical perturbation during protein adsorption at constant potential is described. The method is based on the concept of the charged transition layer which develops between metal substrate and adsorbate. Transition-layer charge was determined from the differential capacitance and the ellipsometry measurements in buffer solutions. A three-phase (metal substrate/protein film/solution) reflecting surface model was amended with a charged metal transition layer as a fourth phase, which allowed the elucidation of the adsorbed Pdx complex index of refraction spectrum. On the basis of this spectrum, a time dependent protein surface concentration was calculated. For short times, this surface concentration was in good agreement with estimates based on mass transport limited adsorption.