1. SERS Microscopy (µSERS) Selective and Sensitive Localization of Proteins in Tissue Specimens
C. Jehn, B. Küstner, F. Schöppler, F. Baum,
M. Schütz, M. Gellner and S. Schlücker
Institute of Physical Chemistry
University of Würzburg
Germany
SERS Round Table Discussion, Poltersdorf, October 3-5, 2007

2. Outline Immunohistochemistry: Basics Biofunctionalized Nanoparticles
SERS Microscopy: Proof of Principle
Novel SERS labels
SERS Imaging of PSA in Prostate Biopsies
Outlook
Label 1

3. Immunohistochemistry
Protein
Target Protein/Antigen
Tissue Section
Tissue Section
Label + 1.
Label
Remove Unbound Antibodies 2

4. Immunohistochemistry (IHC): Quo Vadis ?
Multiplexing Capacity of
Different Labelling Approaches
ca. 1-3
ca. 1-3
ca. 3-10 ca
Dyes
Fluorophores
Quantum Dots
SERS Labels
Absorption
Fluorescence Emission
Raman Scattering
small Raman line width
minimal spectral overlap 3

5. Functionalized Nanoparticles for Nanodiagnostics
Spacer
Immuno-SERS Label +
Spacer + +
Gold
Nanoparticle
Antibody
Raman Label 4

6. Self-Assembled Monolayer (SAM) on Gold Nanoparticles
Spacer 5

7. Hollow Gold Nanoparticles: Synthesis
Solid Ag spheres
Hollow Au shells
Ag colloid
max ~ 400 nm
Au-Ag alloy
max 550 – 900 nm
3 Ag + Au 3+
3 Ag +
+ Au
Nanoparticles with tunable surface plasmon resonance ! 6

8. Hollow Gold Nanoparticles: Characterization
50 nm
Plasmon resonance energy depends on shell thickness 7

9. Spectrum of Immuno-SERS lable
Wavenumber / cm-1
Raman Intensity
1750
1500
1000
750
1250
Selected Raman bands:
SERS distance dependence
SERS selection rules
Electronic resonance
D. S. Grubisha, R. J. Lipert, H.-Y. Park, J. Driskell, M. D. Porter, Anal. Chem. 2003, 75, 5936.
Schlücker, Küstner, Punge, Bonfig, Marx, Ströbel, J. Raman Spectrosc. (rapid comm.) 2006, 37, 8

10. S(-) L(-) E(+) SERS Microscopy (µSERS): Proof of Principle B A D E C
Localization of PSA in Prostate Biopsies A B C E D
S(-)
L(-)
E(+)
A - C: Epithel PSA (+)
D: Stroma PSA (–)
E: Lumen PSA (–)
Schlücker, Küstner, Punge, Bonfig, Marx, Ströbel, J. Raman Spectrosc. (rapid comm.) 2006, 37, 9

11. Outline Immunohistochemistry: Basics Biofunctionalized Nanoparticles
SERS Microscopy: Proof of Principle
Novel SERS labels
SERS Imaging of PSA in Prostate Biopsies
Outlook
Label 1 11

12. Bioconjugation: Sterical hindrance by SAM
Route 1
Route 2
Dual SAM
Silica encapsulated
Novel SERS labels
Sterical hindrance 10

13. Route 1: Novel SERS labelsAu Au
shorter spacer unit Au
longer spacer unit 11

14. Route 1: Novel SERS labels
Absorption 12

15. Silica encapsulated nanoparticles
Route 2: Novel SERS labels
gold
nanoparticle
spacer
Self Assembled Monolayer
silica
encapsulation Au
Sub Monolayer
vs.
P. Mulvaney, M. D. Musick, C. D. Keating, M. J. Natan, Langmuir 2003, 19, 4784. 13

16. Route 2: Novel SERS labels
50 nm
silica
encapsulation
100 nm
100 nm
50 nm 14

17. Route 2: Novel SERS labels
Absorption
SERS 15

18. Raman Microspectroscopic Imagingy
Spatial
Coordinates
Wavenumber x
Point Mapping
Line Mapping L
Spectrometer
Global Imaging
Spectrometer
LCTF
S. Schlücker, M. D. Schaeberle, S. W. Huffman, I. W. Levin, Anal. Chem. 2003, 75, 16

19. SERS Imaging of PSA in Prostate Biopsies
SERS Image
Brightfield Image
cm-1
20 µm 17

20. SERS Imaging of PSA in Prostate Biopsies
cm-1 18

21. Outlook: Multiplexing
µSERS vs. Fluorescence:
Multiplexing capability
Only single laser excitation
No photobleaching
Quantitation 1 1 2 2 3 3 4 4 19

22. Acknowledgement Pathology (Mannheim/Heidelberg) P. Ströbel A. Marx
Organic Chemistry (Würzburg)
C. Schmuck, M. Bauer, P. Wich