Quantum Dots in Frogs David J. Norris Chemical Engineering & Materials Science University of Minnesota Fluorescent imaging with semiconductor nanocrystals 1
Quantum Dots via Gas-Phase Deposition: Semiconductor nano-particles Grown on a substrate e.g. InAs on GaAs Stranskii-Krastanow growth Pyramidal shape Extensive research... e.g. electrically pumped lasers Not the focus of this talk A. Brown, Georgia Tech 2
Colloidal Semiconductor Quantum Dots (Nanocrystals): Me 2 Cd Se CdSe NCs 320 C diameters between 2nm to 12nm size distributions <3-5% Crystallites of semiconductor Chemically synthesized ~1000 atoms (3nm diameter) Coated with surfactants: trioctylphosphine (TOP) trioctylphosphine oxide (TOPO) Murray, Norris & Bawendi; JACS 115, 8706 (1993). In addition to CdSe... Many materials are now available 3
Size Control: Burst of nucleation Form seeds concentration grow seeds nucleation threshold growth threshold time Burst of nucleation controls size distribution Growth time controls mean size LaMer & Dinegar; JACS 72, 4847 (1950). Murray, Norris & Bawendi; JACS 115, 8706 (1993). 4
Strong fluorescence CdSe Quantum Yield ~10% at 300K Surfactant CdSe Core Quantum Yield ~80% at 300K ZnS Shell Hines & Guyot-Sionnest, J. Phys. Chem. 100, 468 (1996). 5
Size-dependent optical properties: CdSe Nanocrystals under uv light increasing size photo by Felice Frankel; samples by Bawendi Group (MIT) 6
Size-dependent optical properties of CdSe: Molecules Nanocrystals Semiconductor Energy Cd 5s LUMO Conduction Band -e hv Se 4p sp 3 HOMO +h Valence Band Size Brus; J. Chem. Phys. 79, 5566 (1983). 7
Size-dependent optical properties: Dot r Bulk Semiconductor Conduction Band -e V e (r) Energy hv +h hv Valence Band V h (r) Efros & Efros; Sov. Phys. Semi. 16, 772 (1982). Brus; J. Chem. Phys. 79, 5566 (1983). 8
Why Study Nanocrystals? Original Motivation: Fundamental science: What happens when a semiconductor becomes small? Also of practical importance: Semiconductor devices are becoming small Use optical properties in optoelectronic devices Lasers Photovoltaics (solar cells) Light-emitting diodes (LEDs) Progress: Tremendous progress over 20 years of research: Not only synthesis, but fundamental understanding... 9
Optical transitions understood in detail: Wavelength (nm) 600 500 400 300 CdSe ~1.5nm radius Absorption Intensity (arbitrary units) ~4.3nm radius 10K 2.0 2.4 2.8 3.2 3.6 4.0 4.4 Energy (ev) Norris & Bawendi; PRB 53, 16338 (1996).
Semiconductor Nanocrystals: Current status: Physics for many properties is understood Can be manipulated to make novel materials artificial solids, etc.. Applications? Traditional focus has been on opto-electronics lasers photovoltaics (solar cells) light-emitting diodes (LEDs) Biology? Can quantum dots be useful in biology? 11
Nature Biotechnology, January 2003 Dubertret, Skourides, Norris, Noireaux, Brivanlou & Libchaber; Science 298, 1759 (2002). Wu, Liu, Liu, Haley, Treadway, Larson, Ge, Peale & Bruchez, Nat. Biotech. 21, 41 (2003). Jaiswal, Mattoussi, Mauro & Simon, Nat. Biotech. 21, 41 (2003). 12
Fluorescent imaging in biology Microscopy: Many biological experiments utilize fluorescent tags Organic dyes (fluoroscein, rhodamine, etc.) Fluorescent proteins (green fluorescent protein, GFP) Problem: even the best fluorescent tags have poor photostability Fluorescence fades quickly over time Severely limits experiments Bio-object Organic dye 13
Replace organic dyes with nanocrystals? Bruchez, Moronne, Gin, Weiss & Alivisatos; Science 281, 2013 (1998). Chan & Nie; Science 281, 2016 (1998). Advantages of Nanocrystals: Semiconductor nanocrystals exhibit high photostability Rock that can be pounded with light Fluorescence can last days Change size; get different colors in fluorescence Excite different size nanocrystals with a single laser Bio-object Nanocrystal 14
Replace organic dyes with nanocrystals? Bruchez, Moronne, Gin, Weiss & Alivisatos; Science 281, 2013 (1998). 15
Problem Cd Se CdSe NCs 320 C As synthesized nanocrystals are hydrophobic Murray, Norris & Bawendi; JACS 115, 8706 (1993). Need to make the nanocrystals hydrophilic! Simultaneously maintain fluorescence and colloidal stability! Initial strategy: exchange surfactants on surface... 16
Different Strategies siloxane shell Bruchez, Moronne, Gin, Weiss & Alivisatos; Science 281, 2013 (1998). monolayer of carboxylic acids Chan & Nie; Science 281, 2016 (1998). CdSe Core ZnS Shell carboxylic acid + fusion protein Matoussi, Mauro, Goldman, Anderson, Sundar, Mikulec, & Bawendi; JACS 122, 12142 (2000). amine-modified polyacrylic acid Wu, Liu, Liu, Haley, Treadway, Larson, Ge Peale & Bruchez; Nature Biotech. 21, 41 (2003). oligomeric phosphines Kim & Bawendi; JACS 125, 14652 (2003). 17
Issue that we wanted to address: NON-SPECIFIC ADSORPTION Thus, lots of in vitro experiments No experiments in vivo! 18
Approach: Use Micelles hydrophilic head group hydrophobic tail amphiphilic molecule (surfactant) hydrophilic shell hydrophobic core H 2 O H 2 O micelle 19
Our Route: Encapsulate hydrophobic quantum dot: Keep initial hydrophobic ligand (TOP/TOPO) Thus, maintaining fluorescence Place inside a bubble to protect the dot And obtain water stability Use Micelles: Form spontaneously Surfactant molecules used in nature for membranes Should be bio-compatible 20
The surfactant we chose: hydrophilic head group hydrophobic tail N-poly(ethylene glycol) phosphatidylethanolamine [PEG-PE] poly(ethylene glycol) (PEG) double hydrophobic chain Block copolymer phospholipid: PEG block added to natural phospholipid PEG known as an extremely bio-compatible surface PEG will be on the outside of our micelle Can control the length of the PEG block 21
New Route: Evaporate Solvent Add water Micelles self-assemble: simple 10 minute synthesis 22
Why did we choose this surfactant? Previous studies on empty micelles Johnsson M. et al. J. Phys. Chem. B 105, 8420 (2001) [PEG-PE] PEG-X R t (Å) R c (Å) L (Å) 750 51 34 17 2000 67 32 35 5000 107 32 75 23
TEM of encapsulated quantum dots Negative Staining 24
Basic Properties: Size = ~13nm (TEM) Colloidal Stability: can boil in water stable over a broad range of quantum dot concentrations stable over a broad range of salt concentrations (up to 2M) stable over a broad range of ph 25
Stability: ph ph = 10 ph = 7 ph = 4 Days 26 Absolute Fluorescence Intensity 0 1 2 3 4 5 6 7
Basic Properties: Size = ~13nm (TEM) Colloidal Stability: can boil in water stable over a broad range of quantum dot concentrations stable over a broad range of salt concentrations (up to 2M) stable over a broad range of ph General Method: CdSe, PbSe, FePt... quantum dots and quantum rods 27
Are QD-micelles bio-compatible? In vitro I: DNA Attachment Coupler biotin-modified ssdna attached to streptavidin-modified 4% agarose beads 28
Are QD-micelles bio-compatible? In vitro II: directed self-assembly 29
In Vivo Imaging? Explore: Toxicity? Stability in a living organism? Non-specific adsorption? Aggregation? 30
Why Frogs? Xenopus: Embryos are large Very sensitive to perturbations (good for toxicity tests) Quick: 5 days from fertilization to feeding tadpole Extremely well studied system Large number of embryos can be obtained 31
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Injecting QD-micelles into frogs In vivo imaging with quantum dots Dubertret, Skourides, Norris, Noireaux, Brivanlou, & Libchaber; Science 298, 1759 (2002). 33
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Photostability Dubertret, Skourides, Norris, Noireaux, Brivanlou, & Libchaber; Science 298, 1759 (2002). 36
Toxicity? Dubertret, Skourides, Norris, Noireaux, Brivanlou, & Libchaber; Science 298, 1759 (2002). 37
Different colors... 38
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Why bright green spots? 42
Also learned something new... Quantum Dots become localized in nucleus: Occurs at specific stage of development Mid-blastula transition Embryo starts to make its own genetic material Pores in nuclear membrane open; dots go into nucleus Surprise: Green dots go in, red dots get stuck! First evidence for size of the nuclear pores 43
What leads to stability? H 2 O H 2 O H 2 O H 2 O micelle micelle + quantum dot ~ 1 week indefinitely 44
Conclusions Quantum Dot Micelles: New method to make nanocrystals bio-compatible Good stability in vitro and in vivo Low toxicity in vivo Low non-specific adsorption Easy coupling (bio-conjugation) In vivo imaging 45
Future Challenges: Loss of Stability: bovine serum at 37C: aggregation after 1 week ph 12: aggregation after days 46
Future Challenge: Whole Animal Imaging: Injected mice with quantum dots Image blood vessels through foot on confocal microscope After a few days the quantum dot fluorescence could not be detected anymore Organs harvested: quantum dots in kidney, not in lung or liver. 47
Detection of single quantum-dot-micelles: 48
Acknowledgements Dr. Benoit Dubertret* Vincent Noireaux Prof. Albert Libchaber* Prof. Ali Brivanlou Paris Skourides The Rockefeller University Center for Studies in Physics and Biology The Rockefeller University Lab. of Molecular Embryology Saurabh Agarwal Chulwoo Son Youngjong Kang Prof. Andrew Taton Prof. Marna Ericson * Research Institute, Princeton 49