Distance and Orientation Dependence of Heterogeneous Electron Transfer:  A Surface-Enhanced Resonance Raman Scattering Study of Cytochrome c Bound to Carboxylic Acid Terminated Alkanethiols Adsorbed on Silver Electrodes

Abstract

The distance and orientation dependence of the heterogeneous electron-transfer reaction between ferrocytochrome c (Fe 2+ Cc) and a silver film over nanosphere (AgFON) electrode is examined in detail using electrochemical surface-enhanced resonance Raman spectroscopy (SERRS) as a molecularly specific and structurally sensitive probe. The distance between the Fe 2+ redox center and the electrode surface is controlled by varying the chain length x of an intervening carboxylic acid terminated alkanethiol, HS(CH 2 ) x COOH, self-assembled monolayer (SAM). The orientation of the heme in Fe 2+ Cc with respect to the AgFON/S(CH 2 ) x -COOH electrode surface is controlled by its binding motif. Electrostatic binding of Fe 2+ Cc to AgFON/S(CH 2 ) x -COOH yields a highly oriented redox system with the heme edge directed toward the electrode surface. The binding constants were determined to be K ) 5.0 × 10 6 M -1 and 1.1 × 10 6 M -1 , respectively, for the x ) 5 and x ) 10 SAMs. In contrast, covalent binding of Fe 2+ Cc yields a randomly oriented redox system with no preferred direction between the heme edge and the electrode surface. SERRS detected electrochemistry demonstrates that Fe 2+ Cc electrostatically bound to the x ) 5 AgFON/S(CH 2 ) x COOH surface exhibits reversible oxidation to ferricytochrome c, whereas Fe 2+ Cc electrostatically bound to the x ) 10 surface exhibits irreversible oxidation. In comparison, Fe 2+ Cc covalently bound to the x ) 5 and x ) 10 surfaces both exhibit oxidation with an intermediate degree of reversibility. In addition to these primary results, the work presented here shows that AgFON/S(CH 2 ) x COOH surfaces (1) are biocompatible -Fe 2+ Cc is observed in its native state and (2) are stable to supporting electrolyte changes spanning a wide range of ionic strength and pH thus enabling, for the first time, SERRS studies of these variables in a manner not accessible with either the widely used colloid or electrochemically roughened SERS-active surfaces.

Surface-Enhanced Raman Spectroscopy

“…The EM predicts that SERS does not require the adsorbate to be in direct contact with the surface but within a certain sensing volume. From a practical perspective, there are certain experiments, such as those involving surface-immobilized biological molecules (51), in which direct contact between the adsorbate of interest and the surface is not possible because the surface is modified with a capture layer for specificity or biocompatibility. Because the field enhancement around a small metal sphere decays with r −3 , using the E 4 approximation, the overall distance dependence should scale with r −12 .…”

Controlled Plasmonic Nanostructures for Surface-Enhanced Spectroscopy and Sensing

“…This results because the enhanced electric fields extend beyond the surface creating a sensing volume within a few nanometers of the surface. The ability to enhance Raman scattering from molecules not directly attached to the surface is of great importance for systems such as surface-immobilized biological molecules, 33 where direct contact between the adsorbate of interest and the surface is not possible because the surface is modified with a capture layer for specificity or biocompatibility.…”

Reproducible SERRS from structured gold surfaces

“…This makes it ideal for the study of biomolecules. Thus SERRS has been used to study heme proteins, 9 to study the mechanism of electron transfer in cytochromes, 10 and more recently for trace detection of DNA 11,12 and enzyme activity. 13 The sensitivity of SERRS exceeds that of fluorescence, which is currently the most prolific technique employed for studying biomolecules, as proven theoretically 14 and experimentally.…”