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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. http://www.phys-chemie.uni-wuerzburg.de.
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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 http://www.phys-chemie.uni-wuerzburg.de SERS Round Table Discussion, Poltersdorf, October 3-5, 2007
Label Outline • Immunohistochemistry: Basics • Biofunctionalized Nanoparticles • SERS Microscopy: Proof of Principle • Novel SERS labels • SERS Imaging of PSA in Prostate Biopsies • Outlook 1
Protein Protein Target Protein/Antigen Label + 1. Tissue Section Immunohistochemistry Label Label 2. Remove Unbound Antibodies Tissue Section 2
Immunohistochemistry (IHC): Quo Vadis ? Multiplexing Capacity of Different Labelling Approaches ca. 1-3 ca. 1-3 ca. 3-10 ca. 10-100 Dyes Fluorophores Quantum Dots SERS Labels Absorption Fluorescence Emission Raman Scattering • small Raman line width • minimal spectral overlap 3
Spacer Functionalized Nanoparticles for Nanodiagnostics Immuno-SERS Label + + + Spacer Gold Nanoparticle Antibody Raman Label 4
Spacer Self-Assembled Monolayer (SAM) on Gold Nanoparticles 5
Ag colloid max ~ 400 nm Au-Ag alloy max 550 – 900 nm 3 Ag + Au 3 Ag + Au 3+ + Nanoparticles with tunable surface plasmon resonance ! Hollow Gold Nanoparticles: Synthesis SolidAg spheres Hollow Au shells 6
50 nm Hollow Gold Nanoparticles: Characterization Plasmon resonance energy depends on shell thickness 7
Spectrum of Immuno-SERS lable • Selected Raman bands: • SERS distance dependence • SERS selection rules • Electronic resonance Raman Intensity 1750 1500 1250 1000 750 Wavenumber / cm-1 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. 8
SERS Microscopy (µSERS): Proof of Principle Localization of PSA in Prostate Biopsies B S(-) A D L(-) E(+) E C 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. 9
Label Outline • Immunohistochemistry: Basics • Biofunctionalized Nanoparticles • SERS Microscopy: Proof of Principle • Novel SERS labels • SERS Imaging of PSA in Prostate Biopsies • Outlook 1
Bioconjugation: Sterical hindrance by SAM Route 1 Dual SAM Novel SERS labels Route 2 Stericalhindrance Silica encapsulated 10
Route 1: Novel SERS labels Au Au shorter spacer unit Au longer spacer unit 11
Route 1: Novel SERS labels SERS Absorption 12
Silica encapsulated nanoparticles Route 2: Novel SERS labels spacer silica encapsulation Au gold nanoparticle vs. Self Assembled Monolayer Sub Monolayer P. Mulvaney, M. D. Musick, C. D. Keating, M. J. Natan, Langmuir2003, 19, 4784. 13
50 nm 50 nm 50 nm 100 nm 100 nm 100 nm 100 nm 100 nm Route 2: Novel SERS labels silica encapsulation 50 nm 14
Route 2: Novel SERS labels Absorption SERS 15
Point Mapping Line Mapping L Spectrometer Raman Microspectroscopic Imaging y Spatial Coordinates Wavenumber x Global Imaging Spectrometer LCTF S. Schlücker, M. D. Schaeberle, S. W. Huffman, I. W. Levin, Anal. Chem.2003, 75, 4312-4318. 16
20 µm SERS Imaging of PSA in Prostate Biopsies SERS Image Brightfield Image Intensity @ 1340 cm-1 17
SERS Imaging of PSA in Prostate Biopsies Intensity @ 1340 cm-1 18
Outlook: Multiplexing • µSERS vs. Fluorescence: • Multiplexing capability • Only single laser excitation • No photobleaching • Quantitation 1 1 2 2 3 3 4 4 19
Acknowledgement • Pathology (Mannheim/Heidelberg) • P. Ströbel • A. Marx • Organic Chemistry (Würzburg) • C. Schmuck, M. Bauer, P. Wich