The development of electroactive total internal reflection integrated optical waveguide and total internal reflection fluorescence devices for the characterization of metalloprotein films

Abstract

Orientation distributions of redox active films that correlate with an electron transfer behavior were reported for the first time using electroactive total internal reflection fluorescence spectroscopy (EA-TIRF) and electroactive integrated optical waveguide (EA-IOW) absorbance technology developed here. The mean tilt angle and the angular distribution about the mean were recovered using a Gaussian model. Previous anisotropy calculations were only possible using a theoretical approach (Hansen's model). In the work presented here, a novel method was developed that was based on experimental measurements. This method used Fresnel's equations and Snell's law to determine the relative polarized electric field intensity at the interface of the EA-TIRF substrate. Optically transparent semiconductor surfaces of indium tin oxide (ITO) were used as the adlayer for EA-TIRF and EA-IOW devices. The ITO surface morphology, optical and conduction properties were characterized. The ITO was found to have adequate conduction, optical and surface morphology properties for molecular orientation distribution measurements. The results indicated the ITO morphology contributes a minimal degree of error to the calculated distribution. Surface-bound films of model methylene blue were used to characterize the EA-TIRF and EA-IOW techniques prior to the investigation of horse heart cytochrome c. The studies demonstrated potential control of redox active adsorbed films. The mean tilt angle and the angular distribution about that mean were successfully determined. In addition, the studies of the methylene blue films indicated the possibility of orientation-dependent quenching (the loss of an electron from the excited state LUMO to the ITO semiconductor conduction band). Studies of the cytochrome c films indicated anisotropic submonolayers electrostatically bound to the negative ITO surface. Cyclic voltammetry measurements showed the films to be electroactive, exhibiting quasi-reversible electrochemistry. An average tilt angle and the orientation distribution about the angle, as a function of potential, were reported for horse heart cytochrome c. This potential-dependent orientation distribution of submonolayer films is reported for the first time.