Optical waveguide light mode spectroscopy (OWLS) is a new technique that is particularly well suited to the in situ study of biomolecule adsorption kinetics on surfaces. Here we describe improvements to a commercial OWLS instrument in order to allow for easy combination with other ex situ surface-characterization methods, such as x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and atomic force microscopy. Further, the problem of contamination of the waveguide surface arising from the use of silicone in the flow-through cuvette with which biomolecules are brought into contact with the adsorbing surface had to be resolved, as it greatly altered the wetting and adsorption properties of the waveguide. Finally, through physical vapor deposition of thin, nanosized layers of titanium oxide onto the waveguide layer, it is possible to simulate the surface properties of oxide-covered titanium implant surfaces. However, scanning angle constraints set by the mechanics of the commercial instrument must be borne in mind.
A sterically conservative, neutralizing mutation (glutamic acid to glutamine) in either of two different positions (15 or 48) of the soluble core tryptic fragment of cytochrome b5 results in two proteins with vastly different adsorption propenies. The kinetics of adsorption were measured under well-defined hydrodynamic conditions on a variety of different surfaces, of controlled electrostatic potential. prepared by modifying planar optical waveguides. Repeated measurement of the guided mode spectrum in the presence of protein solution allowed the temporal evolution of the number of adsorbed molecules to be determined. A highly positively charged surface acted as a perfect sink. Le.. adsorption was only limited by transport, adsorption to a highly negatively charged surface was fully reversible, and adsorption to a neuual phospholipid bilayer was very slow and practically irreversible. The macroscopic adsorption behavior can in large part be interpreted in terms of molecular-scale interactions between the protein and the adsorbent surface.
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