Development of a Novel QCM Technique for Protein Adsorption Studies
Sammanfattning: The first part of this thesis (Papers I and II) is concentrated on the development of a novel quartz crystal microbalance (QCM) technique to study protein adsorption. The second part (Papers III-V) is focused on the application of the technique on protein adsorption using well characterized surface-solution-protein systems.A QCM consists of a piezoelectric quartz crystal that can be used to measure very small masses. The quartz crystal is a thin piezoelectric quartz plate with metal electrodes deposited on each side. When an AC-voltage is applied over the electrodes, the crystal can be made to oscillate at its resonant frequency, f. If a mass is adsorbed on the electrodes, and if the adsorbed mass is small compared to the mass of the crystal, evenly distributed and rigidly attached, with no slip or deformation due to the oscillatory motion, the resonant frequency decreases proportionally to the mass of the film. However, protein adlayers adsorbed from aqueous solutions are non-rigid and it has been suggested that they may slip on the QCM sensor surface. This therefore represents a situation when all these assumptions are not valid. In Paper I is described how the absolute dissipation factor, D, of the QCM can be obtained with high accuracy and repetition rate. The principle behind the measurement is to drive the crystal to oscillation with a signal generator, disconnect the generator and then record how the crystal oscillation decays on a digitizing oscilloscope. The recorded decay curve is numerically fitted to a exponentially damped sinusoidal whereby the resonant frequency and dissipation factor, D, of the crystal are simultaneously obtained. In Paper II is explained the origin of conductivity effects, which can seriously disturb measurements that are carried out in conductive liquids (such as buffers containing proteins). In Paper II it is also shown how to minimize these effects. A number of proteins, which differ in mass size and rigidity, were investigated, in Paper III. Each protein caused a system specific response in both f and D, which must ultimately originate from differences in the viscoelastic properties of the adsorbed protein layers. No evidence of slip was found. In Paper IV, two structurally similar forms of the oxygen transporting protein hemoglobin, with different iron ligands (met-Hb and HbCO) were investigated. The results show that the D-shift measurements give unique information about the adsorption process. By conversion of pre-adsorbed met-Hb to HbCO, it was shown that the adsorbed protein molecules were in part native. The adsorption of ferritin at different salt concentrations were investigated in the study described in Paper V. The results show that upon increased salt concentration there is a threshold concentration (20-50 mM) after which the adsorption occurs significantly faster and result in larger coverage. It was also shown how the D-shift provides information about differences with respect to the structure of the adlayer.
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