Redox-active self-assembled monolayers (SAMs) provide an excellent platform for investigating electron transfer kinetics. Using a well-defined bridge, a redox center can be positioned at a fixed distance from the electrode and electron transfer kinetics probed using a variety of electrochemical techniques. Cyclic voltammetry, AC voltammetry, electrochemical impedance spectroscopy, and chronoamperometry are most commonly used to determine the rate of electron transfer of redoxactivated SAMs. A variety of redox species have been attached to SAMs, and include transition metal complexes (e.g., ferrocene, ruthenium pentaammine, osmium bisbipyridine, metal clusters) and organic molecules (e.g., galvinol, C 60 ). SAMs offer an ideal environment to study the outer-sphere interactions of redox species. The composition and integrity of the monolayer and the electrode material influence the electron transfer kinetics and can be investigated using electrochemical methods. Theoretical models have been developed for investigating SAM structure. This review discusses methods and monolayer compositions for electrochemical measurements of redox-active SAMs.

Despite their popularity, electrochemical biosensors often suffer from low sensitivity. One possible approach to overcome low sensitivity in protein biosensors is to utilize multivalent ligand-receptor interactions. Controlling the spatial arrangement of ligands on surfaces is another crucial aspect of electrochemical biosensor design. We have synthesized and characterized five biotinylated trinuclear ruthenium clusters as potential new biosensor platforms : [Ru3O(OAc)6CO(4-BMP)(py)]0 (3), [Ru3O(OAc)6CO(4-BMP)2]0 (4), [Ru3O(OAc)6L(4-BMP)(py)]+ (8), [Ru3O(OAc)6L(4-BMP)2]+ (9), and [Ru3O(OAc)6L(py)2]+ (10) (OAc = acetate, 4-BMP = biotin aminomethylpyridine, py = pyridine, L = pyC16SH). HABA/avidin assays and isothermal titration calorimetry were used to evaluate the avidin binding properties of 3 and 4. The binding constants were found to range from 6.5 – 8.0 × 106 M−1. Intermolecular protein binding of 4 in solution was determined by native gel electrophoresis. QM, MM, and MD calculations show the capability for the bivalent cluster, 4, to intramolecularly bind to avidin. Electrochemical measurements in solution of 3a and 4a show shifts in E1/2 of −58 to −53 mV in the presence of avidin, respectively. Self-assembled monolayers formed with 8–10 were investigated as a model biosensor system. Diluent/cluster ratio and composition were found to have a significant effect on the ability of avidin to adequately bind to the cluster. Complexes 8 and 10 showed negligible changes in E1/2, while complex 9 showed a shift in E1/2 of −43 mV upon avidin addition. These results suggest that multivalent interactions can have a positive impact on the sensitivity of electrochemical protein biosensors.