Incorporation of model proteins into lipid layers: Aspects of biosensors: Dissertation

In this work different approaches of the Langmuir-Blodgett (LB) technique have been studied in order to obtain an oriented and sensitive protein layer onto a solid surface. LB films of Cd arachidate or self-assembled films of octadecylmercaptan have been used as supports for the artificial protein layers. It is essential that the support is defect-free in order to minimise non-specific binding. The quality of LB layers of Cd arachidate was noticed to be strongly influenced by how the solid slide was positioned in relation to the compressing barrier during vertical deposition. Atomic force microscopy, AFM, revealed that the layers were more homogeneous, if the slide was placed parallel to the compressing barrier. The amount of layer transferred could be determined by using either a surface acoustic wave device or a quartz crystal as a microbalance. Moreover, surface plasmon resonance, SPR, could be used to evaluate monolayer transfer. Unilamellar vesicles fused to form monolayers when spread onto the air-water interface. The layers could be transferred to hydrophobic supports as visualised by AFM. This could be a means to incorporate membrane proteins into lipid layers, as membrane proteins easily can be embedded into vesicles. Aged vesicles were transferred as large domains. Antibodies of the C-reactive protein, CRP, formed a monolayer, when spread directly onto the air-water interface. The antibodies seemed to take up a slanted orientation or have a random distribution in the monolayer. Antibodies were also adsorbed from the subphase onto various monolayers. Binding of anti-CRP was dependent on the packing of the film, on the antibody concentration in the aqueous subphase, but also on the monolayer matrix. The binding was highest to a monolayer of octadecylamine. The monolayer was, furthermore, stabilised by the antibodies. Evaluation of the specific interaction between the antigen-antibody complex with a quartz crystal microbalance indicated that the antigen could be determined in the concentration range of 0.1 - 5 µg/ml. In order to obtain site-directed immobilization, various amounts of a biosynthetically produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. Incorporation of the single-chain antibodies, transfer of the layer onto solid slides, amount of non-specific adsorption and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by AFM revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution. The binding of hapten, which was used as an antigen, could be detected with SPR in the concentration range of 0.1 - 100 µg/ml.