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C. Jehn, B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker Institute of Physical Chemistry University of Würzburg Germany

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Presentation on theme: "C. Jehn, B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker Institute of Physical Chemistry University of Würzburg Germany"— Presentation transcript:

1 C. Jehn, B. Küstner, F. Schöppler, F. Baum, M. Schütz, M. Gellner and S. Schlücker Institute of Physical Chemistry University of Würzburg Germany SERS Microscopy (µSERS) Selective and Sensitive Localization of Proteins in Tissue Specimens 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 Tissue Section + 1. Label 2. Remove Unbound Antibodies 2 Protein Target Protein/Antigen Tissue Section Label

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

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

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

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

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

9 Wavenumber / cm -1 Raman Intensity 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, Schlücker, Küstner, Punge, Bonfig, Marx, Ströbel, J. Raman Spectrosc. (rapid comm.) 2006, 37,

10 SERS Microscopy (µSERS): Proof of Principle A B C E D 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, S(-) L(-) E(+) 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

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

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

14 Route 1: Novel SERS labels Absorption SERS 12

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

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

17 Route 2: Novel SERS labels Absorption SERS 15

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

19 SERS Imaging of PSA in Prostate Biopsies 20 µm Brightfield Image SERS Image 1340 cm -1 17

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

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

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


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