Publication I September 4, 2012

Quantitation of Affinity, Avidity, and Binding Kinetics of Protein Analytes with a Dynamically Switchable Biosurface

​A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers’ dynamic motion. We demonstrate the quantitation of binding kinetics (konkoff rate constants), dissociation constants (KD in the pM regime), and the influence of competitive binders (EC50 values). Moreover, the “switchSENSE” method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA3) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins’ individual chemical environments significantly influence the His6–NTA3 interaction.



Journal of the American Chemical Society