Developing luminescent nanoprobes for labeling focal adhesion complex proteins and performing combined AFM-TIRF imaging of these conjugates

Abstract

Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and made biocompatible. A process to coat the QDs with silk fibroin, a fibrous protein derived from the Bombyx mori silk worm, is described. Following the coating process, the characterization of size, optical properties and biocompatibility profile of these particle systems is described. In addition, conjugation of the silk fibroin coated QDs to different labeling proteins such as phalloidin and streptavidin is described. Proteins on the surface of ovarian cancer cells (HeyA8) and of cytoskeletal components participating in the formation of focal adhesion complex (FAC), such as F-actin in endothelial cells (HUVECS) were labeled using the bio-conjugated QDs. Various imaging techniques such as epi-fluorescence, TIRF and AFM were used to study the QD labeled cells. Overall the project has produced luminescent nanoprobes that enable the study of FAC formation dynamics and potentially a better in vivo fluorescent marker tool.