Monodentate, Bidentate and Photocrosslinkable Thiol Ligands for Improving Aqueous Biocompatible Quantum Dots

Water-soluble Quantum Dots (QDs) are highly sensitive fluorescent probes that are often used to study biological species. One of the most common ways to render QDs water-soluble for such applications is to apply hydrophilic thiolated ligands to the QD surface. However, these ligands are labile and can be easily exchanged on the QD surface, which can severely limit their application. As one way to overcome this limitation while maintaining a small colloidal size of QDs, we developed a method to stabilize hydrophilic thiolated ligands on the surface of QDs through the formation of a crosslinked shell using a photocrosslinking approach. This ligand is known to crosslink through ultraviolet (UV) light but, interestingly, our results showed that QD-mediated crosslinking by visible light led to enhanced colloidal stability of the QDs compared to UV light. This was confirmed through spectroscopic, photographic and fluorescence correlation spectroscopy measurements. In order to maximize the biological applications of QDs, it is important to thoroughly investigate the binding and exchange mechanisms of ligands, and especially how these mechanisms affect the ability to control non-specific adsorption of biomolecules. To investigate this, we modified a near-infrared dye to contain a single thiol group to act as a highly sensitive spectroscopic probe for the binding and exchange of thiol groups to monodentate or bidentate ligand-coated QDs. Differences in how monodentate and bidentate ligands control binding of thiolated target (bio)molecules were discovered by fitting the data to the Hill equation. The results highlight how both the coordination geometry and the ligand packing density on the surface of QDs control the binding and exchange mechanisms. The proposed mechanistic scheme was then successfully tested by exposure to a reduced (i.e. -SH containing) antibody. Finally, Förster Resonance Energy Transfer of QD-dye conjugates was studied. At the single molecule level three species were identified: QD without a dye bound, QD with 1 dye attached, and QD with 2 or more dyes attached. The unusual statistical distribution of these different species suggests a highly complex process at the microscopic level. These discoveries will contribute to improving the applications of QDs in biophysical and biomedical studies