Characterizations of protein-ligand reaction kinetics by transistor-microfluidic integrated sensors

Understanding the binding affinities and kinetics of protein-ligand interactions using a label-free method is crucial for identifying therapeutic candidates in clinical diagnostics and drug development. In this work, the IGZO-TFT (thin-film transistor) biosensor integrated with a tailored microfluidic chip was developed to explore binding kinetics of protein-ligand biochemical interactions in the real-time manner. The IGZO-TFT sensor extracts the binding characteristics through sensing biomolecules by their electrical charges. Using lysozyme and tri-N-acetyl-D-glucosamine (NAG₃) as an example, we established a procedure to obtain the parameters, such as the dissociation constant, K_d, and association rate constant, k_a, that are critical to biochemical reactions. The correlation between the lysozyme concentration and TFT drain current signal was first constructed. Next, solutions of lysozyme and NAG₃ of different mixing ratios were prepared. They were pre-mixed for various periods of reaction time before applying to the TFT sensor to extract signals of lysozyme molecules and the concentration remaining. With the knowledge of drain current changes at different reaction times, k_a and K_d can be obtained. The values from our experiment are comparable to other methods, which suggests the proposed approach can be employed to explore protein-ligand interaction kinetics in the massively parallel manner if the TFT array is considered.