Quantum Dots in Frogs
David J. Norris
Chemical Engineering & Materials Science
University of Minnesota
Fluorescent imaging with semiconductor nanocrystals
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Quantum Dots via Gas-Phase Deposition:
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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
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Colloidal Semiconductor Quantum Dots (Nanocrystals):
Me2Cd
Se
CdSe NCs
320 °C
• diameters between 2nm to 12nm
• size distributions <3-5%
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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
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Size Control:
concentration
Burst of nucleation
Form “seeds”
nucleation
threshold
grow seeds
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).
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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).
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Size-dependent optical properties:
CdSe Nanocrystals under uv light
increasing
size
photo by Felice Frankel; samples by Bawendi Group (MIT)
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Size-dependent optical properties of CdSe:
Molecules
Nanocrystals
Cd 5s
Conduction
Band
LUMO
Energy
Semiconductor
-e
hv
HOMO
Se 4p
+h
Valence
Band
sp3
Size
Brus; J. Chem. Phys. 79, 5566 (1983).
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Size-dependent optical properties:
r
Dot
Bulk Semiconductor
Conduction
Band
Ve(r)
Energy
-e
hv
hv
+h
Valence
Band
Efros & Efros; Sov. Phys. Semi. 16, 772 (1982).
Brus; J. Chem. Phys. 79, 5566 (1983).
Vh(r)
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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 . . .
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Optical transitions understood in detail:
Wavelength (nm)
600
500
400
300
~1.5nm radius
“Absorption”
Intensity (arbitrary units)
CdSe
~4.3nm radius
2.0
2.4
2.8
3.2
Energy (eV)
3.6
10K
4.0
4.4
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?
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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).
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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
Organic dye
“Bio-object”
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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
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Replace organic dyes with nanocrystals?
Bruchez, Moronne, Gin, Weiss & Alivisatos; Science 281, 2013 (1998).
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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 . . .
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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
carboxylic acid + fusion protein
ZnS Shell
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).
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Issue that we wanted to address:
NON-SPECIFIC ADSORPTION
Thus, lots of in vitro experiments
No experiments in vivo!
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Approach: Use Micelles
hydrophobic tail
hydrophilic head group
amphiphilic molecule
(surfactant)
hydrophobic core
hydrophilic shell
H2O
H2O
micelle
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Our Route:
Encapsulate hydrophobic quantum dot:
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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
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The surfactant we chose:
hydrophilic head group
hydrophobic tail
N-poly(ethylene glycol) phosphatidylethanolamine
poly(ethylene glycol) (PEG)
[PEG-PE]
double hydrophobic chain
Block copolymer phospholipid:
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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
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New Route:
Evaporate Solvent
Add water
Micelles self-assemble: simple 10 minute “synthesis”
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Why did we choose this surfactant?
Johnsson M. et al.
J. Phys. Chem. B 105, 8420 (2001)
Previous studies on empty micelles
[PEG-PE]
PEG-X
Rt (Å)
Rc (Å)
L (Å)
750
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2000
67
32
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5000
107
32
75
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TEM of encapsulated quantum dots
Negative Staining
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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
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Absolute Fluorescence Intensity
Stability: pH
pH = 10
pH = 7
pH = 4
0
1
2
3
4
Days
5
6
7
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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
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Are QD-micelles bio-compatible?
In vitro I: DNA Attachment
Coupler
biotin-modified ssDNA attached to streptavidin-modified 4% agarose beads
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Are QD-micelles bio-compatible?
In vitro II: directed self-assembly
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In Vivo Imaging?
Explore:
• Toxicity?
• Stability in a living organism?
• Non-specific adsorption?
• Aggregation?
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Why Frogs?
Xenopus:
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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
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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).
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Photostability
Dubertret, Skourides, Norris, Noireaux, Brivanlou, & Libchaber; Science 298, 1759 (2002).
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Toxicity?
Dubertret, Skourides, Norris, Noireaux, Brivanlou, & Libchaber; Science 298, 1759 (2002).
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Different colors . . .
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Why bright green spots?
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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
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What leads to stability?
H2O
H2O
H2O
H2O
micelle
micelle + quantum dot
~ 1 week
indefinitely
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Conclusions
Quantum Dot Micelles:
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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
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Future Challenges:
Loss of Stability:
•bovine serum at 37C: aggregation after 1 week
•pH 12: aggregation after days
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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.
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Detection of single quantum-dot-micelles:
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Acknowledgements
Dr. Benoit Dubertret*
Vincent Noireaux
Prof. Albert Libchaber*
The Rockefeller University
Prof. Ali Brivanlou
Paris Skourides
The Rockefeller University
Center for Studies in Physics and Biology
Lab. of Molecular Embryology
Saurabh Agarwal
Chulwoo Son
Youngjong Kang
Prof. Andrew Taton
Prof. Marna Ericson
*
Research Institute, Princeton
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