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An automated solution exchange (SE) mechanism has been introduced to slab optical waveguide spectroscopy to estimate the protein immobilizing ability of a slab optical waveguide (SOWG) surface. In each SE process, SOWG spectral change in absorbance at peak position of cytochrome c (cyt.c) Soret band at 409 nm was observed to analyze the desorption ratio of cyt.c adsorbed on SOWG surface. Continuous SE processes for 100 times have successfully brought us a kind of master desorption curve of cyt.c, which was well fitted by a double exponential equation, indicating the existence of three kinds of adsorbed states, including weakly adsorbed, strongly adsorbed, and immobilized cyt.c. The present results showed that around 30 times SE processes were enough to anticipate the ratio of desorbed and immobilized amounts of cyt.c adsorbed on SOWG surface.

In situ Observation of Desorption Reaction of Cytochrome c from Solid/Liquid Interfaces with Slab Optical Waveguide Spectroscopy