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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.
                                           Laser overlaps
                                           With absorption
                                           Band:
                                           Enhances but
                                           Will now bleach
                                           May be
Just like resonance enhanced SHG of dyes
                                           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
 Brightness
                                                   cylinder
 Is Raman
 intensity



 Hot                                               Hot faceted
 aggregate                                         sphere




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
No NaBH4 reduction for                are photoactivated:
Emitting species                      photoreduced
                                      From ions to neutrals (3)
               Emitting species have
               a few silver atoms, <8         Dickson, JACS 2002
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                             Aqueous
 With silver                         Without silver


labeled cells
fluorescence                          DIC


Control cells

fluorescence                          DIC