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Description
Quantifying molecular interactions is critical to the understanding of many biological processes and drug screening. To date, various detection techniques have been developed to determine the binding kinetics. However, because most of the mainstream detection technologies detect signals that scale with the mass of ligands bond to the sensor surface, it is still challenging to quantify the binding kinetics of small molecules. To address this problem, two different detection technologies, charge-sensitive optical detection (CSOD) and critical angle reflection (CAR), are developed for label-free detection of molecular interactions with the ability to detect a wide range of molecules including small molecules. In particular, CSOD technique detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here a technique capable of detecting molecular binding kinetics in normal ionic strength buffers is presented. An H-shaped sample well was developed to overcome this problem. With this new design, the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands were measured in normal buffer. To further improve the signal-to-noise ratio of CSOD and move it toward high-throughput detection, CSOD was implemented with a quadrant-cell detector to achieve detection in higher frequency range and decrease low-frequency noise.This improved CSOD technique is capable for direct quantification of binding kinetics of phage-displayed peptides to their target protein using the whole phages. CAR imaging can be performed on surface plasmon resonance (SPR) imaging setups. It was shown that CAR is capable of measuring molecular interactions including proteins, nucleic acids and cell-based detections. In addition, it was shown that CAR can detect small molecule bindings and intracellular signals beyond SPR sensing limit. CAR exhibits several distinct characteristics over SPR, including tunable sensitivity and dynamic range, deeper vertical sensing range, and fluorescence compatibility. CAR is anticipated to have the ability to expand SPR capability in small molecule detection, whole cell-based detection, simultaneous fluorescence imaging, and broader conjugation chemistry.
ContributorsLiang, Runli (Author) / Wang, Shaopeng (Thesis advisor) / Blain Christen, Jennifer (Thesis advisor) / Jing, Tianwei (Committee member) / Wang, Chao (Committee member) / Arizona State University (Publisher)
Created2021