Time-resolved evanescent wave absorption spectroscopy for real-time monitoring of heme protein adsorption to glass

Evanescent wave has been recognized as a highly sensitive optical probe for surface monitoring. By use of slab optical waveguides, time-resolved evanescent wave absorption spectroscopy was developed for the investigation of the interfacial behavior of biomolecules with a chromophore. In this study, 30-μm thick glass sheets served as freestanding multimode waveguides that were combined with a simple fiber-coupling method to lead to a broadband evanescent wave absorption spectrometer. With such a homemade instrument, adsorption of heme proteins onto glass slides from static aqueous solution was monitored in situ. The experimental results reveal that the interfacial behavior of myoglobin (Mb) is different from that of hemoglobin (Hb) and cytochrome c (Cc). Formation of dynamic equilibrium for Mb adsorption at both hydrophilic and hydrophobic surfaces always is behind the occurrence of the maximum coverage. However, simultaneous formation of the dynamic equilibrium and the maximum coverage was observed for Hb and Cc adsorptions.