QCM OPERATION IN LIQUIDS - CONSTANT SENSITIVITY DURING FORMATION OF EXTENDED PROTEIN MULTILAYERS BY AFFINITY

The quartz crystal microbalance (QCM) is a well-established tool in ma ss-sensitive detection, Due to recent improvements in experimental pro cedures, QCMs are finding increasing attention for applications in liq uids, One important application is bioaffinity measurements for analyt ical or research purposes, The effect of the formation of solid films at a QCM surface, especially in gases or vacuum, is well understood. H owever, 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 hy drogel extending several hundred nanometers from the transducer surfac e as interacting matrix is common in bioaffinity applications and furt her increases complexity. Pure mass-related effects as wed as viscosit y-mediated effects may contribute to the overall frequency response ob served experimentally. To improve our understanding of the effects dur ing the formation of extended biological overlayers we have investigat ed systematically the formation of protein multilayers with a QCM in s itu, The attenuation of the QCM oscillation by the liquid leads to a b roadening of the resonance frequency. We have overcome this limitation by frequency-dependent admittance analysis and by curve fitting of th e 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 liqu ids, 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 f rom the Sauerbrey equation. However, in water, the corresponding frequ ency decrease was increased by a factor of 4, thereby indicating that significant amounts of water are embedded in the hydrated protein laye r, Unexpectedly a constant frequency decrease per layer was found duri ng the successive formation of up to 20 protein layers (similar to 400 nm). Neither noise nor drift increased with the number of protein lay ers, These results indicate that, despite the high hydration of the pr otein layers, viscosity-induced effects play a negligible role and tha t the frequency decrease reflects primarily mass changes at the surfac e.