The long-term goal of this research is to create small, highly fluorescent nanomaterials to enable a whole new array of single molecule optical microscopy experiments in biological systems. Beautifully adept at probing mechanistic heterogeneity, single molecule experiments hold great potential to unravel the all of the complex steps leading to biological activity. Unfortunately, all single molecule experiments are at some level limited by the properties of available fluorescent labels. Thus, any significant advances in single molecule methods will only be possible through the development of new, extremely photostable, highly fluorescent labels with the potential for both facile in vitro and in vivo labeling. Through the three proposed Specific Aims, we will adapt our recently discovered single Ag nanodot fluorescence to create extremely bright, general use fluorescent labels. As these materials are very strongly absorbing, they can be easily excited with weak Hg lamps, thereby greatly reducing both the autofluorescent background associated with in vivo imaging and the experimental complexity associated with laser-based methods.
In Specific Aim I, we will fully chemically and photophysically characterize the different biocompatibie dendrimer-encapsulated Ag nanodots on both the single molecule and bulk levels. With a high probability of success, we will greatly expand our preliminary results to create and fully characterize highly fluorescent, water-soluble Ag nanodots, thereby providing the basis for the subsequent Aims.
In Specific Aim II, we will combine biological diversity with materials science to identify specific peptide sequences that bind and stabilize Ag nanodots. Using multiple peptide libraries, identified Ag nanodot-binding peptides will yield fluorescence complementary to that of the dendrimers currently stabilizing the Ag nanodot fluorescence. Thus, we will study the properties and specificity of peptide-Ag nanodot interaction through mass spectrometry and single molecule / bulk fluorescence experiments. The dendrimer - encapsulated nanodots characterized in Aim I will be investigated for Ag nanocluster transfer to the optimized Ag-binding peptides identified by library screening.
Specific Aim III probes attaching these identified peptides to two independently quantifiable proteins to assay labeling efficiency and retention of protein function. These experiments are crucial to the potential uses of genetically attaching the Ag nanodot binding peptide to the protein of interest and transferring Ag to it for use as a highly fluorescent, robust in vivo single molecule label. ? ?
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|Richards, Chris I; Hsiang, Jung-Cheng; Dickson, Robert M (2010) Synchronously amplified fluorescence image recovery (SAFIRe). J Phys Chem B 114:660-5|
|Richards, Chris I; Hsiang, Jung-Cheng; Khalil, Andrew M et al. (2010) FRET-enabled optical modulation for high sensitivity fluorescence imaging. J Am Chem Soc 132:6318-23|
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|Yu, Junhua; Choi, Sungmoon; Dickson, Robert M (2009) Shuttle-based fluorogenic silver-cluster biolabels. Angew Chem Int Ed Engl 48:318-20|
|Jung, Soonkyo; Dickson, Robert M (2009) Hidden markov analysis of short single molecule intensity trajectories. J Phys Chem B 113:13886-90|
|Patel, Sandeep A; Richards, Chris I; Hsiang, Jung-Cheng et al. (2008) Water-soluble Ag nanoclusters exhibit strong two-photon-induced fluorescence. J Am Chem Soc 130:11602-3|
|Richards, Chris I; Choi, Sungmoon; Hsiang, Jung-Cheng et al. (2008) Oligonucleotide-stabilized Ag nanocluster fluorophores. J Am Chem Soc 130:5038-9|
|Yu, Junhua; Choi, Sungmoon; Richards, Chris I et al. (2008) Live cell surface labeling with fluorescent Ag nanocluster conjugates. Photochem Photobiol 84:1435-9|
|Zheng, Jie; Nicovich, Philip R; Dickson, Robert M (2007) Highly fluorescent noble-metal quantum dots. Annu Rev Phys Chem 58:409-31|
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