Fourier Transform Infrared Spectrovoltammetry and Quantitative Modeling of Analytes in Kinetically Constrained Redox Mixtures.

Redox-active analytes that do not support direct electron transfer on the electrode, such as proteins with buried redox centers, pose challenges to characterization of their structural and thermodynamic properties. Investigations of indirect transitions in analytes supported by complex redox mixtures require a careful balance between kinetic limitations and spectral interference from the mediators. Using methylene green and thionine acetate as redox mediators and myoglobin as the analyte, we demonstrate that normal pulse spectrovoltammetry (NPSV) with Fourier transform infrared (FT-IR) detection and subsequent global spectral regression analysis can resolve structural and thermodynamic properties simultaneously with little a priori information. Both the E1/2 and unbiased redox difference FT-IR spectra of the Fe(II)/Fe(III) redox couple of myoglobin in reduction and oxidation NPSV modes were in good agreement with those reported earlier by independent techniques. The thermodynamic and kinetic limitations of mediators/analyte interactions were investigated using comprehensive semiempirical kinetic simulation models. This modeling effort yielded a flexible computational tool capable of quantitatively predicting the redox response in mediated electrochemical studies and defining its limitations, thus greatly expanding the range and precision of the formal mediator/analyte concentration ratio rule.