Everett, William Neil (2007-08). Evanescent wave and video microscopy methods for directly measuring interactions between surface-immobilized biomolecules. Doctoral Dissertation.
Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions.