In Situ Quantitation of Protein Adsorption Density by Integrated Optical Waveguide Attenuated Total Reflection Spectrometry

The adsorption of hemoglobin to a polystyrene (PS) thin film has been examined by integrated optical waveguide attenuated total reflection (IOW-ATR) spectrometry. Protein adsorption densities were determined by measuring the evanescent attenuation of propagating modes that were prism coupled into and out of the PS film. Knowledge of the electric field intensity distribution of the waveguide structure permitted adsorbed protein to be quantitated in the presence of bulk dissolved protein. The Langmuir adsorption model was used to fit the isotherm data and indicates that Hb binds with high affinity (K_a > 10⁶ M⁻¹) to PS and forms a complete monolayer at bulk concentrations greater than 3 µM. The results demonstrate that the IOW-ATR technique is particularly well suited to in situ measurements of protein adsorption density at the polymer/solution interface.