Systematic investigation of biomolecular interactions using combined frequency and motional resistance measurements

Abstract

The resonance frequency of acoustic biosensors is today used as a label-free technique for detecting mass changes on sensor surfaces. In combination with an appropriate continuous flow system it has earlier been used for affinity and kinetic rate determination. Here, we assess the potential of a modified acoustic biosensor, monitoring also the real-time dissipation through the resistance of the sensor, to obtain additional kinetic information related to the structure and conformation of the molecules on the surface. Actual interaction studies, including an attempt to determine avidity, are presented along with thorough verification of the experimental setup utilizing true viscous load exposure together with protein and DNA immobilizations. True viscous loads show a linear relationship between resistance and frequency as expected. However, in the interaction studies between antibodies and proteins, as well as in the immobilization of DNA and proteins, higher surface concentrations of interacting molecules led to a decrease (i.e. deviation from the linear trend) in the differential resistance to frequency ratio. This is interpreted as increased surface rigidity at higher surface concentrations of immobilized molecules. Consequently, studies that aim at obtaining biological binding information, such as avidity, from real-time resistance and dissipation data should be conducted at low surface concentrations. In addition, the differential resistance to frequency relationship was found to be highly dependent on the rigidity of the preceding layer(s) of immobilized molecules. This dependence can be utilized to obtain a higher signal-to-noise ratio for resistance measurement by using low surface densities of immobilized interaction partners.