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Assigned Reading
1) Nie, S. and S.R. Emory, Probing single molecules and
single nanoparticles by surface-enhanced Raman scattering.
Science, 1997. 275: p. 1102-1106.
2) Zheng, J. and R.M. Dickson, Individual water-soluble
dendrimer-encapsulated silver nanodot fluorescence.
J Am Chem Soc, 2002. 124(47) p. 13982-3.
3) Peyser, L.A., et al., Photoactivated fluorescence from
individual silver nanoclusters. Science, 2001. 291: p. 103-106.
4) Alvarez, M.M., et al., Optical Absorption Spectra of Nanocrystal
Gold Molecules. J. Phys. Chem. B, 1997. 101: p. 3706-3712.
Critique: Biophys. J, vol 89, 572-580 (2005) Makareva et al
Outline
1) Metal Enhancement of Raman, SHG
2) Dendrimer Encapsulated nanodots
Extinction coefficient ε:
Strong absorbers have ε between 20,000-100,000
Absorption cross section δ also used: 1x 10-16 cm2 = 23,000
Oscillator strength is integral of the absorption band
Sum rule: oscillator strength, f, for one electron is:
1x 10-16 cm2 eV
Or n* 1x 10-16 cm2 eV for n electrons
Limits absorption strength of dye molecules
How to overcome for better contrast?
Add or “borrow” more electrons
Implication of oscillator strength and absorption spectra
Oscillator strength must be conserved
Spectra with large maximum must be narrow
Broad spectra will have smaller extinction coefficient
SHG and Raman Enhancement by Metals
· Surface Enhancement of Second Harmonic Generation,
Raman Scattering of dyes on Bulk Surfaces (factor of 105 ) 1972
· More Recently Extended to Nanoparticles (factors of 1-1014 )
(Nie, Feld groups, ~1999)
Possible Mechanisms
· Surface Plasmon Resonance
· Metallic atoms have delocalized d orbitals
· Metal Colloids or Surfaces have sea of electrons
· Optical excitation is collective- huge absorptions
Induced dipole coupling to dye molecule
· Corona or Lightning Rod Effect
Metal acts like antenna, concentrates electromagnetic energy
Charge Transfer Process : Between metal electrons and dye
Colloidal Gold Absorption Spectra
Surface Plasmon Resonance
Small particles blue shift, broaden
1240/eV =nm
Whetten, J. Phys. Chem, 1997
NLO Imaging of NIE-115 Neuroblastoma Cells
SHG
TPEF
SHG much weaker than TPEF:
Very hard imaging
Improve by SPR with gold?
Not well-defined experiment:
Processes highly distant dependent:r-6
100 nm Gold Nanoparticle-Dye Conjugates
Polymer Coated (styrene, methacrylic acid mixture)
Gold Colloids linked to Styryl ANEPPS via Succinimydyl
Ester
•Well-defined distance
between dye and metal
•Hope to be less toxic
Dye-Nanoparticle Conjugates are unique:
Both Components can SHG under the
Right conditions
TEM images of 100 nm Particles
Uncoated
Polymer Coated:
3 nm thick uniform
Thickness controlled by relative polymer concentrations
Depend on dye and gold?
For dye concentration
Fluorescence QY
Lifetime shorter
If quenching
Enhancement
Factor was 20
Will use for CARS (two weeks)
Surface enhancement of spontaneous Raman also
Provides large enhancements.
Just like resonance enhanced SHG of dyes
Laser overlaps
With absorption
Band:
Enhances but
Will now bleach
May be
Necessary for
Adequate S/N
Surface Enhanced Raman Scattering
•6 orders of magnitude larger than spontaneous Raman
cross sections (10-30 cm2)
•More chemical/structural information than fluorescence
(vibrational spectra, like CARS)
•May be more bleach resistant (off resonance)
•Arises from surface plasmon resonance, lightning rod effect
Nie, Feld groups showed some particles have enhancements
of 1010-14: comparable to absorption cross sections (10-16
cm2) of fluorescent dyes
But most do nothing
SPR Enhancement:
overlap fluorescence/SHG/Raman
excitation with SPR of metal surface
Silver is bluer, more narrow than gold:
Silver usually better enhancement than gold
SERS of Single Rhodamine Molecules on Ag Nanoparticles
Light scattering
No dye
10-11 M
10-9 M
10-9 M less than 1 dye per nanoparticle
Nie, Science 1997
Size and Shape of “hot particles”?
Examine by AFM
Hot
cylinder
Brightness
Is Raman
intensity
Hot faceted
sphere
Hot
aggregate
Panel A: 1,2 hot; 3, 4 were not: 100 nm vs 35 nm
C,D also hot different shapes:
No obvious correlation
Probably edges: lightning rod enhancement
Nie, Science 1997
Strong SERS Polarization Excitation dependence
SERS, ordinary Raman similar spectra (with cm-1)
Consistent with electric dipole,
Surface plasmon interaction
Nie, Science 1997
Strong SERS Polarization signal dependence
Excitation was scrambled polarization
Signal polarization selected (dichroic)
Signal polarized along long axis of dye
By contrast,
Bulk SERS largely depolarized
Unique aspect of nanoparticle SERS
Nie, Science, 1997
Time dependent SERS Spectrum of one particle
Different bands for same particle
come and go and change intensity
Probably Changes in orientation
Dye finally bleaches
(resonance Raman 514 nm)
Nie, Science, 1997
Relative Single Molecule SERS and fluorescence Intensities
B= dye bound to nanoparticle
A= dye bound to surface (non-metallic)
Integrated single molecule SERS
4 fold larger than single molecule
fluorescence
Nie, Science, 1997
Metal Particle Size effects leading to SPR:
•Sizes>~2-10 nm required for true surface plasmon
•Resulting absorption spectrum is broad
•Continuous distribution of excited states: conductor
(unlike dyes which have discrete states, although
Broadened in solution)
Small clusters (few atom aggregates) have discrete
energy levels
Quantum confined like Semiconductor Quantum Dots
Quantum Dot Overview
•Semiconductor Nanocrystals: CdSe, ZnSe 1-5 nm
(invented in mid 1980’s at Bell Labs, Brus,
Alivisatos, Bawhendi)
•Broad Absorption spectrum (UV)-electron hole pair
narrow emission (visible)
Quantum confinement: particle size smaller than
electron-hole Bohr radius
•Spectrum Red Shifts for larger particles: like dyes
•Blue shifts for small particles
Select desired wavelength by size of particles
• Spin forbidden emission~longer lifetimes 40 ns
(NOT fluorescence)
Bioimaging
First Applied to bioimaging in 1998
•10-50 fold brighter than organic dyes
•High quantum efficiency ~ “70%”
Highly photostable: “bleach free”: no bonds to
break
Labeling not specific without functionalization
Replace organic dyes?
Common Problems with Quantum Dots
•Normal synthesis have hydrophobic ligands for
Stability against aggregation; not water soluble
•Exchange with polar species for solubility:
Lose stability against aggregation
Reduced luminescence for hydrophilic QDs
•Multi-layer coatings are somewhat more stable:
Arduous fabrication
•Can coat with proteins, conjugates
Still can aggregate and bind non-specifically
when intracellular (even if ok in solution)
•Small silver and gold nanoclusters or nanodots (few atoms)
have strong absorption (SPR like):
Much stronger than organic dyes
•Absorption coefficient Comparable to Semiconductor
Quantum dots (CdSe)
•Strong emission when surface bound (none in solution)
•Not true SPR (too small) but energy of bands has same
spectral size dependence:
As SPR and (and quantum dots): smaller particles blue shift
But: free metal nanodots do not emit in solution
Water quenches emission completely
Only when usrface bound: protected and
fewer nonradiative decay pathways
Particles on Films limited in use as probes or biosensors
How to exploit optical properties of gold and
silver nanoparticles for biology?
Make dendrimers (branched polymers) to
encapsulate (and shield) nanoclusters (silver
and gold)
New class of probes
General Scheme for Dendrimer Formation
Generation (e.g. G2 or G4) is number of branched layers
Ions reduced to neutrals by white light activation
Also being investigated as drug delivery devices
Balogh et al
Absorption of Dendrimer Encapsulated Silver Clusters
NaBH4 reduction makes
Larger clusters: SPR nonemitting (1) Fluorescent dendrimers
are photoactivated:
No NaBH4 reduction for
photoreduced
Emitting species
From ions to neutrals (3)
Emitting species have
Dickson, JACS 2002
a few silver atoms, <8
Emission of Encapsulated Silver Nanodots in Solution
•Brightness increases as photoactivation occurs
•Blinking is observed, single particles (like single dye molecules)
•Anisotropic Emission, like surface bound
•Very photostable over 30 minutes with Hg cw radiation
•Emission is like dye fluorescence
Dickson, JACS 2002
Emission Spectra of Silver nanodot Dendrimers in solution:
400 nm excitation
Distinct spectral types: average to bulk
AgO surface bound nanodots
Only 5 sizes substantially contribute
Dickson, JACS 2002
Gold nanodot/dendrimers
n=8 is “magic number” geometric shell closing
Energetically favorable
Max is 360 nm-Not SPR band at 500 nm
Dickson, JACS 2003
Absorption Emission of Gold Nanodots/G4 Dendrimers n=8
No surface plasmon peak
particles <2 nm
High Quantum Yield: 45-50% ( at least 100 fold over free particles)
Dendrimer shields nanoparticle from water,
Greatly reduces quenching
Smaller dendrimers (G2) do not adequately protect the nanodot:
no emission
Dickson, JACS 2003
Fluorescent Lifetimes of Gold Nanodot/Dendrimers
Au8
Short (nanosecond): singlet-singlet (dsp) 93%
Long (microsecond):triplet-singlet emission
Analogous to fluorescent dyes
Dickson, JACS 2003
Size tunable Au: dendrimers –small particles blue shift
Analogous to semiconductor quantum dots
Dashed=Absorption
Solid=Emission
Larger Sizes prepared by increasing Au concentration
Dickson, Phys. Rev. Lett 2004
Size dependence of photophysical properties of Au/ dendrimers
330 nm
765 nm
Smaller particles shift towards the blue (like QDs and larger Gold
colloids): Have larger quantum yields
Larger sizes have more non-radiative decay pathways (librations)
Lower emission quantum yields (like red fluorescent dyes)
Consistent with “energy gap” law: nonradiative rate increases
At lower energy separation (probability)
Dickson, Phys. Rev. lett 2004
Classify emission: fluorescence or luminescence?
Like dyes or quantum dots?
Natural lifetime:
τ/QY
4.9
22
Longer lifetime at longer wavelengths consistent with
Spontaneous emission: just like fluorescent dyes,
τ~λ3
Unlike quantum dots
consistent with dye type fluorescence emission
Size scaling of emission for nanodots and Quantum Dots
Small Au nanodot spectra fit well to “Jellium” model:
continuous sea of d electrons scale as n-1/3 (number of atoms)
Quantum confinement in metals and semiconductors
Have different mechanisms: QD are pseudo-one electron atoms:
n-2/3 scaling for electron-hole formation
Dickson, Phys. Rev. lett 2004
Advantages of Au, Ag dendrimers over
semiconductor quantum dots
1) Water soluble without coatings
2) Simple synthesis, no high temperatures, pressures,
Molecular beam epitaxy, multiple layers
3) Maintain polarization (QD’s do not): better sensors of
Environment?
4) Comparable brightness to quantum dots
5) Can do FRET with nanodots: QD absorption too broad
But will not bleach likes dyes
TEM imaging of Cells labeled with Silver Nanodot Dendrimers
3T3s
On surface
In cytoplasm
U937
In vesicles
On surface
Balogh, Nanoletters
Live Cell Imaging with Silver Nanodot Dendrimers
Aqueous
With silver
Aqueous
Without silver
labeled cells
fluorescence
DIC
Control cells
fluorescence
DIC