In Situ Investigation of Coadsorption of Myoglobin and Methylene Blue to Hydrophilic Glass by Broadband Time-Resolved Optical Waveguide Spectroscopy

Abstract

Recently, we have developed a broadband optical waveguide (OWG) spectrometer by using commercially available glass plates of tens of micrometers in thickness as the substrate-free multimode waveguides (Qi et al. Opt. Lett. 2002, 27, 2001-2003). The spectrometer having a bandwidth from 360 to 800 nm is capable of simultaneously detecting the Soret-band absorption of heme proteins and the visible absorption of organic dyes. In this article, the spectrometer was used to in situ investigate coadsorption of methylene blue (MB) and myoglobin from the mixed aqueous solution onto bare glass. Both MB and myoglobin in the mixed solution are positively charged, which makes them not only avoid the chemical interaction between each other but also easy to adsorb to hydrophilic glass. It was found that the coadsorption of MB and myoglobin occurred just in the early stage and the glass surface was finally occupied by myoglobin. The OWG spectroscopic investigation into the respective MB and myoglobin adsorptions shows that MB adsorption is reversible to some degree but that of myoglobin is irreversible. It reveals that the electrostatic binding of myoglobin to bare glass is stronger than the case of MB. Therefore, the adsorbed MB can be substituted by myoglobin. Moreover, via the electrostatic repulsion the tightly immobilized myoglobin prevents bulk MB from occupying the empty surface sites. It is the reason MB is absent from the hydrophilic glass coated with a submonolayer of myoglobin. In the article, we explained both the strong dimerization of MB at the interface and a slow decrease with time of the Soret-band absorbance after its maximum was reached. We also estimated the myoglobin coverage based on the waveguide theory. The study shows the distinguished applicability of the broadband OWG spectroscopy for in situ, real-time monitoring of the dye-protein coadsorption to silica from the mixed solution.