Engineering Quantum Dots for Live-Cell Single-Molecule Imaging
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1 Engineering Quantum Dots for Live-Cell Single-Molecule Imaging Andrew M. Smith and Shuming Nie Georgia Tech and Emory University Department of Biomedical Engineering 2011 NSF Nanoscale Science and Engineering Grantees Conference December 5, 2011
2 Quantum Dots for Biological Imaging Quantum dots are nearly spherical nanocrystals composed of semiconductors with size-tunable optical and electronic properties. Versatile fluorescent probes In vitro bioassays Immunostaining In vivo animal imaging Single molecule imaging Electron microscopy contrast Smith AM & Nie SM, Acc. Chem. Res., 43: (2010).
3 Quantum Dots for Biological Imaging Advantages over conventional fluorophores Size-tunable fluorescence color Efficient near-infrared emission Multiplexing capabilities o Narrow emission bands o Wide excitation bands Large extinction coefficents: x brighter than dyes and proteins Photostability: x greater than dyes and proteins Disadvantages Large compared to dyes: one order of magnitude larger in hydrodynamic diameter Inefficient energy acceptors Smith AM et al., Biochem., 32: (2010).
4 Nanoparticle Design: Bandgap Engineering Smith AM & Nie SM, Acc. Chem. Res., 43: (2010).
5 Nanoparticle Design: Bandgap Engineering
6 Nanoparticle Design: Bandgap Engineering nm cores
7 Next Generation Quantum Dots 1. State-of-the-art quantum dots are hydrodynamically nm. Large size yields a lower diffusion rate (D ~ 1/r) Large size may sterically inhibit specific binding 2. Undefined valency 3. Rapidly blink on and off Smith AM & Nie SM, Nature Biotech., 27: 732 (2009).
8 Live Cell Imaging QD type Reference D (μm 2 /s) QD 655 strep Courty, Nano Letters QD800 with PEG2000 antibody conjugation kit to Herceptin Tada, Cancer Res QD655 PEG amine Yoo, Exp Cell Res QD-Strep 565 or 655 Nelson, Biophys J QD-655-PEG 5000 Nature Cell Biol QD 655 strep Yum, Nano Letters, QD 655 strep Yum, Nano Letters, QD 605 strep Won, Biophys J QDs demonstrate >90% Brownian motion and Multiple sub-populations of diffusion coefficients Diameter (nm) D cyto (μm 2 /s) D theor (μm 2 /s) D cyto / D theor
9 Next Generation Quantum Dots 1. Organic Coating Size-Minimization Size vs. stability tradeoff 2. Inorganic Core Size-Minimization Size & brightness vs. stability tradeoff Smith AM & Nie SM, Nature Biotech., 27: 732 (2009).
10 Quantum Dot Size-Minimization Two major strategies are implemented to stabilize QDs in biological media Polymer Encapsulation Thick bilayer shell Colloidally & photohemically stable Thin organic monolayer Colloidally & photochemically unstable Smith AM et al., Phys. Chem. Chem. Phys., 8: (2006).
11 Hydrophilic Multidentate Ligands Polymeric ligands yield stable and compact nanoparticles Thiolated, aminated polyacrylic acid ~1800 Da polyacrylic acid: ~25 carboxylic acids on a linear polymer 35% of carboxylic acids modified with cysteamine or protected ethylenediamine: 3.5 thiols, 3.0 amines, 16 carboxylates per polymer ~2300 Da product Smith AM & Nie SM, J. Am. Chem. Soc., 130: (2008).
12 Hydrophilic Multidentate Ligands Multidentate Polymer Amphiphilic Polymer Dynamic light scattering 2.5 nm CdTe nanocrystal cores Amphiphilic polymer: 12 nm Multidentate polymer: 5.5 nm Gel permeation chromatography 2-6 nm CdTe nanocrystal cores Overall size similar to proteins ( nm), nm hydrodynamic shell >2 year stability: multidentate binding Smith AM & Nie SM, J. Am. Chem. Soc., 130: (2008).
13 Limitations of Modern Quantum Dots 1. Organic Coating Size-Minimization Size vs. stability tradeoff 2. Inorganic Core Size-Minimization Size & brightness vs. stability tradeoff Smith AM & Nie SM, Nature Biotech., 27: 732 (2009).
14 Quantum Dot Size-Minimization Conventional QDs are composed of core/shell CdSe/ZnS nanocrystals Use of II-VI sulfides can enhance stability and brightness
15 CdS Oxidation Controlled oxidative etching with benzoyl peroxide in benzylamine/toluene Similar results with photooxidation CdSe CdTe CdS CdSe CdTe CdSe CdTe CdS
16 Absorption Red and Near-Infrared Sulfides: Mercury Exchange Mercury cation exchange red-shifts absorption and emission spectra of CdS nanocrystals CdS Hg x Cd 1-x S Wavelength (nm)
17 Mercury Cation Exchange in Nonpolar Solvents CdTe Hg x Cd 1-x Te Near lattice match between CdTe and HgTe a CdTe = 6.48 Å a HgTe = 6.46 Å Large difference in bandgap E g (CdTe) = 1.5 ev E g (HgTe) = ev Independent control over size and bandgap Smith AM & Nie SM, J. Am. Chem. Soc., 133: (2011).
18 Mercury Cation Exchange in Nonpolar Solvents Smith AM et al., J. Am. Chem. Soc., 133: (2011).
19 Mercury Cation Exchange in Nonpolar Solvents CdSe/CdZnS HgCdS/CdZnS 5x brighter CdSe/CdZnS QDs 2.8 nm core, 2 ML CdZnS shell ~45% quantum yield HgCdS/CdZnS QDs 2.7 nm core, 2 ML CdZnS shell ~70% quantum yield Excitation at 400 nm
20 Mercury Cation Exchange in Nonpolar Solvents Fluorescence (Normalized) Wavelength (nm) 3 minimally overlapping colors from blue to near-infrared Bandwith dependent on nanoparticle size Limited by inhomogeneous broadening from alloying Current work aims to further narrow the emission bandwidths
21 Current and Former Lab Members Amit Agrawal, Ph.D. Gang Ruan, Ph.D. Aaron Mohs, Ph.D. Matthew Rhyner, Ph.D. Hongwei Duan, Ph.D. Brad Kairdolf, Ph.D. Mary Wen, B.S. Collaborators Emory Adam Marcus Lily Yang Costas Hadjipanalis Yeshiva University: John Condeelis David Entenberg Georgia Tech: Gang Bao ZL Wang Funding NCI/NIH K99/R00 Pathway to Independence Award Emory University/Georgia Tech CCNE Acknowledgements NIH Nanomedicine Center for Nucleoprotein Machines National Institutes of Health R01 CA108468
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