We developed an impedance quartz crystal microbalance (QCM) approach with the ability to simultaneously record mass changes and calibrated energy dissipation with high sensitivity using an impedance analyzer. This impedance QCM measures frequency shifts and resistance changes of sensing quartz crystals very stable, accurately, and calibrated, thus yielding quantitative information on mass changes and dissipation. Resistance changes below 0.3 Ω were measured with corresponding dissipation values of 0.01 µU (micro dissipation units). The broadband impedance capabilities allow measurements between 20 Hz and 120 MHz including higher harmonic modes of up to 11th order for a 10 MHz fundamental resonance frequency quartz crystal. We demonstrate the adsorbed mass, calibrated resistance, and quantitative dissipation measurements on two biological systems including the high affinity based avidin-biotin interaction and nano-assemblies of polyelectrolyte layers. The binding affinity of a protein-antibody interaction was determined. The impedance QCM is a versatile and simple method for accurate and calibrated resistance and dissipation measurements with broadband measurement capabilities for higher harmonics measurements.

Biotinylated bait molecules can be immobilized on biotinylated sensor chips by formation of biotin–avidin–biotin bridges which are very stable when using wild-type (strept)avidin. Stable immobilization of biotinylated baits is important for monitoring reversible binding and dissociation of prey molecules. For measurements with another bait molecule, however, it is desirable to replace all immobilized proteins by fresh (strept)avidin and new biotinylated bait. In this study, five avidin mutants have been characterized with respect to their ability to form switchable biotin–avidin–biotin bridges on biotinylated chip surfaces, as needed for complete chip regeneration. All five mutants formed stable biotin–avidin–biotin bridges at pH 7, were more or less stable at pH 2–3, and required the combination of pH 2 with SDS for quantitative removal from the chip surface. Mutant #3 (“switchavidin”) showed the best combination of properties, i.e., low nonspecific adsorption of protein and nucleic acids, high binding capacity, and good stability at pH 2–3, as typically used for quantitative removal of prey molecules in repeated measurement cycles