The kinetics of adsorption and surface structure of adsorbed layers of the human fibrinogen on the gold surface, determined by Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM) analysis, was employed to probe the lateral distribution and preferred orientation of protein molecules within the monolayer. In this study, special sets of immunoassays are presented for fibrinogen adsorption/conformation analysis. The results show that kinetic parameters of antigen-antibody interactions are directly related to the interfacial conformation of fibrinogen molecules. Various interfacial structures of adsorbed fibrinogen aggregates, namely single, bi-and three-molecular aggregates, were obtained using a combination of AFM imaging and SPR analysis. Adsorption of fibrinogen onto the surface of polycrystalline gold is a complex process including surface-induced unfolding, local selfassembly and adsorption, occurring concurrently with and on the time scale of each other. This result confirmed the utility of the proposed approach for detecting the spatial distribution and biofunctional properties of specific proteins adsorbed from biological liquids in biosensors.

Reliability and precision of characterization of surface layers by SPR method was evaluated with relation to the experimental conditions and the strategy of extracting the film parameters. Consideration is bound up with sensor applications of SPR phenomenon and focused at problems of separate extraction of optical constants and thickness of the layer and determination of the total quantity of material constituting the surface coverage. Computational scheme for modeling the SPR resonance for multilayer assembly, based on the Abeles matrix formalism, is presented. It is demonstrated that improper choice of the angle range the measurements are taken over may result in ambiguity in determination of the real part of the refractive index n and the film thickness d. Nevertheless, the total quantity of material in the film can be estimated with reasonable accuracy even when correct separate extraction of n and d parameters is hampered by experimental errors and inadequacy of theoretical model of layered system.

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