INTEGRATED OPTICAL WAVE-GUIDE ATTENUATED TOTAL-REFLECTION SPECTROMETRY AND RESONANCE RAMAN-SPECTROSCOPY OF ADSORBED CYTOCHROME-C

The heme group of cytochrome c (Cyt C) has resonance absorptions at 52 0 and 550 nm that arise from x-y degenerate in-plane electronic transi tions of the heme moiety. In the present paper, horse heart Cyt C was adsorbed to the surface of a micron, thick silicon oxynitride integrat ed optical waveguide configurated into a liquid flow cell and prism-co upled with 514.5-nm laser light that was polarized horizontal (TE) and nearly vertical (TM) with respect to the waveguide surface. The adsor bed protein film absorbed light from the evanescent field at the waveg uide surface, resulting in two measurable quantities: (1) an increased attenuation of the guided mode intensity in the waveguide and (2) exc itation of Cyt C resonance Raman emission. Propagation losses owing to the adsorbed film revealed a Cyt C surface density indicating submono layer coverage (50-75 ng/cm2). The dichroic ratio measured by guided m ode attenuation was 1.23 +/- 0.37, indicating the heme plane of Cyt C had an ensemble-averaged orientation angle of 48-degrees with respect to the surface normal. The TE and TM polarized resonance Raman bands a t 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 wave guide resonance Raman results are believed to be the first reported Ra man spectra of a protein film bound to a dielectric substrate.