Investigating Biochemical Interactions Relevant To Human Health Using Backscattering Interferometry

Abstract

This project is concerned with the characterization of biomolecular interactions that are pertinent to human health using backscattering interferometry (BSI). In this dissertation, I used the unique ability of BSI to measure binding events in tethered and free-solution formats to quantify the effect that surface immobilization has on carbohydrate-lectin binding affinities. This work helps to explain the large discrepancy commonly observed when comparing free solution measurements such as isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). I also demonstrated the utility of BSI in measuring allosteric interactions of protein-aptamer binding systems as well as lipoparticle-based ligand-receptor binding. This work validates the hypothesis that the CXCR4 receptor, once thought to bind the CXCL12 ligand exclusively, binds non-native ligands as well. A better understanding of neuroreceptor function will no doubt aid in future drug therapy development. Finally, I used BSI to quantify unknown concentrations of disease-specific biomarkers in complex matrices such as serum (Cyfra 21-1 and Galectin-7) and cell lysate (respiratory syncytial virus). I was able to quantify lung cancer biomarkers at levels that are up to 40-fold lower than current commercially available technologies. This work validated BSI as a biomarker quantification tool and paves the way for eliminating the bottleneck in clinical validation. In the long term, rapid, low-volume, highly sensitive, label-free, immobilization-free biomarker validation will enable personalized medicine and improve clinical outcomes.