Extracting Affinity and Kinetics of Protein-Ligand Interactions using Rigorous Theory and High-Performance Electronic Measurements

Biochemical measurements that determine both the affinity and kinetics of protein-ligand interactions using field-effect transistors (FETs) are demonstrated. The measurements were performed in two configurations – a droplet measurement where the diffusion of concentrated protein was observed as a function of time and with a microfluidic injection where the fluid cell was filled with a protein with the desired final concentration. We chose the high-affinity Biotin–Streptavidin pair as a model system, where thiol modified biotin was conjugated to a gold surface to form a self-assembled monolayer. Streptavidin was then introduced at different concentrations to measure its interaction with Biotin.  Each method was rigorously modeled by describing the diffusion-limited kinetics of the interactions and by establishing error bounds on the spatio-temporal accuracy of the numerical solutions  [1,2]. The model included a novel denoising kernel to drastically improve the signal-to-noise ratio of the measurements. This modeling effort, in turn allowed the extraction of kinetic constants of the reaction from a single real-time time-series measurement. The results were found to be consistent across three orders of magnitude change in the Streptavidin concentration from 100 pM to 100 nM and the kinetic constants were found to be invariant and in agreement with literature values.  The results our approach can be readily used to quantify numerous protein and antibody systems for impact in biophysics and biotechnology.