In this paper we report our work on the development of a human serotonin transporter (hSERT)
antagonist that can be conjugated to quantum dots. This approach has been used to target and visualize the human
serotonin transporter protein (hSERT). We demonstrate that labeling is blocked by the addition of high affinity hSERT
antagonists such as paroxetine. This approach may be useful for the development of fluorescent assays to study the
location and temporal dynamics of biogenic amine transporters and also holds promise for the development of plate-based
high throughput assays used to identify novel transporter antagonists.
In this paper we report two different methodologies for labeling ligand-gated receptors. The first of
these builds upon our earlier work with serotonin conjugated quantum dots and our studies with pegilated quantum dots
to reduce non specific binding. In this approach a pegilated derivative of muscimol was synthesized and attached via an
amide linkage to quantum dots coated in an amphiphillic polymer derivative of poly acrylamide. These conjugates were
used to image the GABAC receptor in oocytes. An alternative approach was used to image tissue sections to study
nicotinic acetylcholine receptors in the neuro muscular junction with biotinylated Bungerotoxin and streptavidin coated
The surface coating of quantum dots has been characterised using Z-stem. Quantum dots have been pegylated to increase stability in aqueous solution. The fluorescence intensity of the quantum dots was modulated pegylation. PEG was coupled using different ratios of EDC, PEG and NHS. Optimum coupling conditions were found to occur when 2000 equivalents of PEG were reacted with 1 equivalent of dot in the presence of 1500 equivalents of NHS and EDC. Angiotensin II was also conjugated to quantum dots and these conjugates were shown to be biologically active. Quantum dots have also been surface functionalised with other peptides such as NGR with subsequent demonstration of cell surface binding and can be characterized by flow cytometry.
Fluorescence is a tool widely employed in biological assays. Fluorescent semiconducting nanocrystals, quantum dots (QDs), are beginning to find their way into the tool box of many biologist, chemist and biochemist. These quantum dots are an attractive alternative to the traditional organic dyes due to their broad excitation spectra, narrow emission spectra and photostability. Non-specific binding is a frequently encountered problem with fluorescent labeling in biological assays. In these studies various cell lines were examined for non-specific binding to quantum dots. Evidence suggests that non-specific binding is related to cell type and, may be significantly reduced by functionalizing quantum dots with polyethyleneglycol ligands (PEG). In addition quantum dots were used to detect and monitor the progession of the viral glycoproteins ,F (fusion) and G (attachment), from Respiratory Syncytial Virus (RSV) in HEp-2 cells. RSV is the most common cause of lower respiratory tract infection in children worldwide and the most common cause of hospitalization of infants in the US. Antiviral therapy is available for treatment of RSV but is only effective if given within the first 48 hours of infection. Existing test methods require a virus level of at least 1000-fold of the amount needed for infection of most children and require several days to weeks to obtain results. The use of quantum dots may provide an early, rapid method for detection and provide insight into the trafficking of viral proteins during the course of infection.