Monofunctional Quantum Dot Probes for Single-Molecule Imaging

Abstract

Recently, it has been shown that the optical properties of quantum dot (QDs) nanoparticles enable novel experiments at the single molecule level in live cells, thereby opening new prospects for the understanding of cellular processes. One difficulty with these experiments is that the complex biological environment imposes stringent design requirements on QD probes, necessitating the development of smaller, low valency and more biocompatible QDs. In this work, we present our efforts towards minimizing the size and controlling the surface functionality of QDs. We show that an engineered peptide surface coating and a purification method based on gel electrophoresis are sufficient to produce compact monofunctional QDs covalently conjugated to streptavidin (SAV), biotin and antibodies. To prove the monofunctionality of the QD-SAV probes, we apply novel single-molecule assay following complexation of the QDs with a fluorescent dye biotin derivative. Counting the photobleaching steps of the fluorescent dye gives us direct access to the number of binding sites present on the QD surface. We then apply these QD probes to the targeting and tracking of individual biotinylated membrane proteins expressed in living HeLa cells. We analyze the diffusion properties of these membrane proteins and compare the measurements to those obtained using commercially available QD probes. Overall, these monofunctional QD probes should be useful for studying a wide-range of biophysical phenomena, down to the single molecule level in live cells.