Development of Novel Probes for Intra-Cellular Studies
- Electronic Properties of Molecules Used for Opto-electronic Devices
- Emission Properties of Single Molecules and Aggregates
- RNA Splicing at the Single Molecule Level
- Optical Properties of Dye-Labeled DNA Gold Nanoparticle Constructs
- Development of Novel Probes for Intra-Cellular Studies
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Instrumentation:
- Single Molecule Fluorescence Microscopy
Recently, there has been explosive growth in the areas of sensors and cellular probes based on semiconductor quantum dots (QDs). Likewise, a number of probes have been developed that incorporate the capacity of metallic nanoparticles (AuNPs) or metallic surfaces to enhance or quench the emission of fluorophores depending on factors including distance and orientation. Typical probe design motifs are molecular beacons, nucleic acid hybridization sensors, and those in which the recognition element consists of a binding or a cleavage site between a fluorophore and an AuNP. The latter type of construct forms the design basis for our first generation probe. It consists of a streptavidin coated fluorescent QD linked to a 1.4 nm Nanogold™ particle via a biotin moiety. This ligand incorporates a disulphide linker that is susceptible to cleavage by a reducing agent. Nanogold is a commercially available gold cluster that contains a single functional group which, for this construct, was chosen to be an amine. This prototype is designed to be either partially or entirely quenched when outside of a cell (depending on the Nanogold to QD ratio and to ‘light up’ when internalized due to cleavage of the dithiol bond and consequent separation of the QD from its quencher. An added feature of the design is the incorporation of Nanogold™ which, following fixation of the cell and silver staining, can be localized by electron microscopy. Future modifications of the design will incorporate delivery peptides and/or use of multiply labeled fluorescent tags to facilitate cellular entry. Recognition sites for binding within the cell can be added as well.
