Evaluation of Taylor Dispersion Injections: Determining Kinetic/Affinity Interaction Constants and Diffusion Coefficients in Label-Free Biosensing

Quinn JG, 421: 401-410, Anal biochem, 2012

Reported herein is the use of Taylor dispersion injection (TDi) to generate a continuous analyte titration that could be used in both steady-state and kinetic analyses of label-free biomolecular interactions. The biophysical characterizations that can be performed by this new TDi-based assay format and standard fixed concentration injection (FCI) format were compared using the following three model interactions: Antibody fragment-receptor interaction, furosemide-carbonic anhydrase II interactions, and warfarin-human serum albumin (HSA) interaction. All binding assays described in this article were performed by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer instrument. In TDi analysis, ethylene tetrafluoroethylene (ETFE) and polyether ether ketone (PEEK) tubing were cut into appropriate lengths and were used to connect the injector valve and the reaction flow cell in the Pioneer system. For the antibody fragment-receptor interaction study, a COOHV sensor chip was immobilized with Protein G using standard amine coupling chemistry. Subsequently, the fusion protein, rhErbB2/Fc was captured onto Protein G immobilized sensor chip, followed by the injection of a serial dilutions of scFv. For the analysis of furosemide-carbonic anhydrase II interactions, carbonic anhydrase II was immobilized onto the sensor chip by amine coupling. Binding data were obtained upon injection of serial dilutions of Furosemide. For the affinity analyses of warfarin-HSA interaction, COOHV chip surface was immobilized with HSA by using standard amine coupling. Subsequently, the serial dilutions of warfarin were injected. The association rate constant (ka), the dissociation rate constant (kd), the maximum biosensor response expected if all ligand sites are occupied (Rmax), the analyte diffusion coefficient (D), and the mass transport constant (km) were determined. Overall results suggest that the TDi-based assay format is capable of providing high-quality biophysical characterizations while reducing assay complexity and run time as compared to the standard FCI approach.

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