Label Free and Immobilization Free Technology to Detect Protein and Ligand Interactions for Drug Discovery
- Author(s): Zhang, Tiantian
- Advisor(s): Lo, Yu-Hwa
- et al.
This thesis presents a new technique, Transient Induced Molecular Electronic Spectroscopy (TIMES), to study biomolecular interaction without protein engineering or chemical modification. The TIMES method incorporates a unique design of microfluidic platform and integrated sensing electrodes, and is designed to operate in a label-free and immobilization-free manner to provide crucial information for protein-ligand interactions under relevant physiological conditions.
We developed a physical model for the TIMES signal, and mathematically formulated the problem to attain physical insight of protein-ligand interactions without molecular probes, fluorescent labels, or immobilization of molecules. Using soft lithography and thin film technology, we designed and fabricated microfluidic based devices for the biomolecular detection. Incorporated with electronic amplification and signal processing, we achieved real time detection using the TIMES method. To demonstrate the functionality, accuracy and sensitivity of this method, we used the TIMES signals to find the dissociation constants for the affinity of reactions between several pairs of protein and ligand. Followed by experiments, we used simulations to prove the mechanism of TIMES method. Computational fluid dynamics (CFD) simulations were performed to demonstrate that the surface electric polarization signal originates from the induced image charges during the transition-state of surface mass transport, which is governed by the overall effects of protein concentration, hydraulic forces and surface fouling due to protein adsorption. Hybrid atomistic molecular dynamics (MD) simulations and free energy computations show ligand-binding affects protein structures and stability, producing different adsorption orientation and surface polarizations to generate characteristic TIMES signals.
As a unique tool, TIMES offers a simple and effective method to investigate fundamental protein chemistry and drug discovery. Although the current work is focused on protein-ligand interactions, the TIMES method is a general technique that may be applied to study signals from reactions between many kinds of molecules.