Integrated optical waveguide attenuated total reflection spectrometry and resonance Raman spectroscopy of adsorbed cytochrome c

The heme group of cytochrome c (Cyt C) has resonance absorptions at 520 and 550 nm that arise from x-y degenerate in-plane electronic transitions of the heme moiety. In the present paper, horse heart Cyt C was adsorbed to the surface of a micron-thick silicon oxynitride integrated optical waveguide configurated into a liquid flow cell and prism-coupled with 514.5-nm laser light that was polarized horizontal (TE) and nearly vertical (TM) with respect to the waveguide surface. The adsorbed protein film absorbed light from the evanescent field at the waveguide surface, resulting in two measurable quantities: (1) an increased attenuation of the guided mode intensity in the waveguide and (2) excitation of Cyt C resonance Raman emission. Propagation losses owing to the adsorbed film revealed a Cyt C surface density indicating submonolayer coverage (50-75 ng/cm²). The dichroic ratio measured by guided mode attenuation was 1.23 ± 0.37, indicating the heme plane of Cyt C had an ensemble-averaged orientation angle of 48° with respect to the surface normal. The TE and TM polarized resonance Raman bands at 1578 cm^-1 of the adsorbed Cty C had an intensity ratio of 1.49 that is statistically indistinguishable from the dichroic ratio measured by attenuated total reflection spectrometry. The integrated optical waveguide resonance Raman results are believed to be the first reported Raman spectra of a protein film bound to a dielectric substrate.