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.

16

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

17

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