Monitoring Cellular Interactions during T Cell Activation at the Single Molecule Level Using Semiconductor Quantum-Dots

摘要

Enhanced peptide-coated quantum dots (with high brightness and high saturation intensity) were developed. Two high-affinity targeting 'velcro-pairs' based on avidin-biotin and fluorescine-antibody interactions were demonstrated and used to specifically target single proteins in membranes of live cells. Single molecule spectroscopy and imaging of individual quantum dot-labeled lipid rafts receptors were performed. Software tools were developed to analyze individual diffusion and trafficking trajectories. These studies provide strong support for the lipid raft hypothesis. Cloning and fusion of avidin to four immune synapse components were achieved. These mutants are being characterized by flow cytometry and fluorescence microscopy. This report provides information on the following new findings: (1) hybrid approach to the synthesis of highly luminescent infrared CdTe/ZnS and CdHgTe/ZnS quantum dots; (2) bioactivation and cell targeting of semiconductor CdSe/ZnS quantum dots with phytochelatin-related peptides; (3) development of bright Cd+ rich peptide-coated quantum dots; (4) comparison of the photophysical and colloidal properties of biocompatible quantum dots using fluorescence correlation spectroscopy (FCS); (5) testing the lipid raft hypothesis by single molecule imaging of targeted peptide-coated quantum dots; and (6) molecular cloning and fusion of avidin to immunological synapse (IS) components.