The quartz crystal microbalance (QCM) is a well-established tool in mass-sensitive detection, Due to recent improvements in experimental procedures, QCMs are finding increasing attention for applications in liquids, One important application is bioaffinity measurements for analytical or research purposes, The effect of the formation of solid films at a QCM surface, especially in gases or vacuum, is well understood. However, the situation is more complex in bioaffinity applications due to the comparably high viscosity of the liquid and the softness of the biological overlayer. Typically frequency responses found for protein layers exceed the values expected from simple models, The use of a hydrogel extending several hundred nanometers from the transducer surface as interacting matrix is common in bioaffinity applications and further increases complexity. Pure mass-related effects as wed as viscosity-mediated effects may contribute to the overall frequency response observed experimentally. To improve our understanding of the effects during the formation of extended biological overlayers we have investigated systematically the formation of protein multilayers with a QCM in situ, The attenuation of the QCM oscillation by the liquid leads to a broadening of the resonance frequency. We have overcome this limitation by frequency-dependent admittance analysis and by curve fitting of the resulting admittance. A time resolution of 5 s and a noise of 0.2 Hz has been achieved with 6-MHz AT-cut quartz crystals operating in liquids, Protein multilayers were formed by successive incubations with a biotin-albumin conjugate and streptavidin. Frequency responses for dry protein layers in air were in agreement with mass changes estimated from the Sauerbrey equation. However, in water, the corresponding frequency decrease was increased by a factor of 4, thereby indicating that significant amounts of water are embedded in the hydrated protein layer, Unexpectedly a constant frequency decrease per layer was found during the successive formation of up to 20 protein layers (similar to 400 nm). Neither noise nor drift increased with the number of protein layers, These results indicate that, despite the high hydration of the protein layers, viscosity-induced effects play a negligible role and that the frequency decrease reflects primarily mass changes at the surface.
