Separation of shifts in surface plasmon resonance into refractive index and thickness of deposited layers

Abstract

Routine Surface Plasmon Resonance (SPR) measurements, which are widely applied in affinity (bio)sensors and surface science, do not allow one to separate the obtained SPR signals into the change of the refractive index and thickness of deposited layers. For thin layers the signal is proportional to the product of the layer thickness and the difference in the refractive indices of this layer and of the aqueous media. In this study, we suggest an approach to separate these parameters. It is performed by subsequent measurements in the presence and in the absence of polyethylene glycol (PEG), an inert additive which modifies the refractive index of the liquid phase but do not penetrate into the adsorbed layer. Both parameters were successively determined for human serum albumin (HSA) as well as for anti-HSA IgG in three different settings: i) HSA adsorbed directly onto a gold surface, ii) HSA chemically immobilized on a gold surface coated with a self-assembled monolayer of 1,16 mercaptohexadecanoic acid and iii) chemically immobilized HSA with additional monomolecular layer of anti-HSA IgG antibodies bond to this protein layer. The suggested approach can be applied in most SPR devices with a flow cell for analysis of various adsorbed layers.