C. Jehn , B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker
description
Transcript of C. Jehn , B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker
C. Jehn, B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker
Institute of Physical ChemistryUniversity of Würzburg
Germany
http://www.phys-chemie.uni-wuerzburg.de
SERS Microscopy (µSERS)
Selective and Sensitive Localization
of Proteins in Tissue Specimens
SERS Round Table Discussion, Poltersdorf, October 3-5, 2007
Outline
Immunohistochemistry: Basics
Biofunctionalized Nanoparticles
SERS Microscopy: Proof of Principle
Novel SERS labels
SERS Imaging of PSA in Prostate Biopsies
Outlook
Label
1
Immunohistochemistry
Tissue Section
+1. Label
2. Remove Unbound Antibodies
2
Protein
Protein
Target Protein/Antigen
Tissue Section
Label
Label
Multiplexing Capacity of Different Labelling Approaches
Fluorescence Emission
Fluorophores Quantum Dots
ca. 1-3 ca. 3-10
Absorption
Dyes
ca. 1-3
Raman Scattering
SERS Labels
ca. 10-100
Immunohistochemistry (IHC): Quo Vadis ?
3
• small Raman line width• minimal spectral overlap
Functionalized Nanoparticles for Nanodiagnostics
+ +
Immuno-SERS Label
AntibodyGold
NanoparticleRaman Label
Spacer +
Spacer
4
Self-Assembled Monolayer (SAM) on Gold Nanoparticles
Spacer
5
Hollow Gold Nanoparticles: Synthesis
Ag colloid
max ~ 400 nm
Au-Ag alloy
max 550 – 900 nm
Nanoparticles with tunable surface plasmon resonance !
3 Ag + Au3+ 3 Ag+ + Au
Solid Ag spheres Hollow Au shells
6
Hollow Gold Nanoparticles: Characterization
400 500 600 700 800 900 1000 1100
Ab
sorb
an
ce
Wavelength/nm
78
5
72
5
63
0
83
0
Plasmon resonance energy depends on shell
thickness
50 nm
7
Wavenumber / cm-1
Ra
ma
n In
ten
sity
1750 1500 1000 750 1250
Spectrum of Immuno-SERS lable
Selected Raman bands:
• SERS distance dependence
• SERS selection rules
• Electronic resonance
8
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, 719-721.
SERS Microscopy (µSERS): Proof of Principle
A
B
C
E
D
1750 1500 1250 1000 750
E
D
C
BA
Ram
an In
tens
ity
Wavenumber / cm-1
Localization of PSA in Prostate Biopsies
A - C: Epithel PSA (+)D: Stroma PSA (–) E: Lumen PSA (–)
Schlücker, Küstner, Punge, Bonfig, Marx, Ströbel, J. Raman Spectrosc. (rapid comm.) 2006, 37, 719-721.
S(-)
L(-)E(+)
9
Outline
Immunohistochemistry: Basics
Biofunctionalized Nanoparticles
SERS Microscopy: Proof of Principle
Novel SERS labels
SERS Imaging of PSA in Prostate Biopsies
Outlook
Label
1
Bioconjugation: Sterical hindrance by SAM
NH2
vs.NH2
NH2
vs.NH2
NH2
vs.NH2
Route 1
Route 2
Dual SAM
Silica encapsulated
10
Sterical hindrance
Novel SERS labelsNovel SERS labels
S
NO2
NH
O O O COOH
O
2
S
NO2
O
OH
2
S
NO2
NH
O OH
O*
2
Route 1: Novel SERS labels
shorter spacer unit
longer spacer unit
11
Au
Au
Au
2000 1800 1600 1400 1200 1000 800 600 400
dual samRam
an i
nte
nsi
ty
Wavenumber/cm-1
psa biofunctionalized
nhs activated
Route 1: Novel SERS labels
400 500 600 700 800
0
1
no
rmal
ized
ab
sorb
ance
Wavelength/nm
hollow Au dual SAM
~20nm
NHS activated
PSA biofunctionalized
Absorption SERS
12
N
O
O
O
O
NH
O
Route 2: Novel SERS labels
goldnanoparticle
spacer
Self Assembled Monolayer
silicaencapsulation
13
P. Mulvaney, M. D. Musick, C. D. Keating, M. J. Natan, Langmuir 2003, 19, 4784.
Au
Sub Monolayer
Silica encapsulated nanoparticles
vs.
50 nm
50 nm50 nm50 nm
100 nm100 nm
100 nm100 nm100 nm
Route 2: Novel SERS labels
silicaencapsulation
14
500 600 700 800 900
hollow gold
no
rmal
ized
ab
sorb
ance
Wavelength/nm
silica encapsulated
SAM
Route 2: Novel SERS labels
500 1000 1500 2000
Ram
an in
ten
sity
Wavenumber/cm-1
silica encapsulated
SAM
Absorption SERS
15
Global Imaging
LCTF
Line Mapping
L
Spectrometer
Point Mapping
Spectrometer
Raman Microspectroscopic Imaging
Spatial
Coordinates xWavenumber
y
S. Schlücker, M. D. Schaeberle, S. W. Huffman, I. W. Levin, Anal. Chem. 2003, 75, 4312-4318.
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1000 1100 1200 1300 1400 1500 1600
Ram
an in
tens
ity
Wavenumber/cm-1
SERS Imaging of PSA in Prostate Biopsies
20 µm
Brightfield ImageSERS Image
Intensity @ 1340 cm-1
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32psa-3
32psa-3
1600 1500 1400 1300 1200 1100 1000
Ram
an in
ten
sity
Wavenumber/cm-1
5 µm
30 40 50 60 70 80 90
400
300
200
100
0
Intensity @ 1340 cm-1
SERS Imaging of PSA in Prostate Biopsies
5 µm
30 40 50 60 70 80 90
400
300
200
100
0
18
Outlook: Multiplexing
SH
HOOC
SH
O2N
COOH
OH
HS
SH
Br
COOH
2
3
4
1000 1200 1400 1600
Ra
ma
n In
ten
stity
Wavenumber/cm-1
1
2
3
4
µSERS vs. Fluorescence:
Multiplexing capability
Only single laser excitation
No photobleaching
Quantitation
1
19
Acknowledgement
Pathology (Mannheim/Heidelberg)P. Ströbel A. Marx
Organic Chemistry (Würzburg)C. Schmuck, M. Bauer, P. Wich