Surface Enhanced Raman Nanotags for Ultrasensitive and Multiplexed Detection of Caner Ximei Qian and Shuming Nie Emory Univ. Dept. of Biomedical Engineering Atlanta, GA For SNM, Feb 1st, 2010

Overview  Brief background of SERS spectroscopy  Comparing three optical tagging particles: Organic chromophores Organic chromophores Semiconductor quantum dots Semiconductor quantum dots SERS nanotags (Au nanoparticles) SERS nanotags (Au nanoparticles)  Evolution of SERS nanotags First generation (Porter group; Anal. Chem. 1999, 71(21), ) First generation (Porter group; Anal. Chem. 1999, 71(21), ) Second generation (Glass-coated tags; Anal. Chem. 2003, 75(22), ) Second generation (Glass-coated tags; Anal. Chem. 2003, 75(22), ) Third generation (Polymer-coated tags; Nature Biotech. 2008, 26(1), 83-90) Third generation (Polymer-coated tags; Nature Biotech. 2008, 26(1), 83-90)  Biomedical application of SERS nanotags In vitro cellular tagging — cancer biomarker detection In vivo non-invasive transcutaneous tumor detection

Surface plasmon resonance Noble Metal Nanoparticles Electrons undergo collective oscillation.

Sensitivity Raman Scattering Surface Enhanced Raman Scattering While most photons are elastically scattered, 1 in 10 7 incident photons undergo the Raman effect. Rayleigh Scattering SERS enhanced sensitivity fold (single molecule detection)

Key components of SERS nanotags Core: Au nanoparticle provides EM enhancement for SERS Reporter molecule: provides fingerprint signature; chemical enhancement for SERS Thin-layer protection: reduces non- specific binding/aggregation Function group: for specific targeting

Molecular weight(g/mol) ~10 9 ~ Molecular weight(g/mol) ~10 9 ~ SERS tag (60nm) QD705Atto610 dye N = f  V/V a N: number of gold atom in one nanoparticle f: FCC packing density 0.74 V: volume of one nanoparticle V a : volume of one gold atom a: radius of gold atom 1.44Å Comparison of SERS tags, QDs and dye molecules

Size comparison 15nm 10nm QD Core ~57nm Calculated ~1nm C-C bond length = 1.44Å Volume ratio Au QD705 SERS tagsQDs Dye molecules 50 nm

Molecular weight ~10 9 ~ Molecular weight ~10 9 ~ Core size 57nm 10-15nm 1nm Core size 57nm 10-15nm 1nm Hydrodynamic size 78±11nm 18±6nm Hydrodynamic size 78±11nm 18±6nm SERS tag QD705 Atto610 dye

Absorption, fluorescence, and SERS spectra

Molecular weight ~10 9 ~ Molecular weight ~10 9 ~ Core size 57nm 10-15nm 1nm Core size 57nm 10-15nm 1nm Hydrodynamic size 78±11nm 18±6nm Hydrodynamic size 78±11nm 18±6nm Bandwidth 2nm 63nm 37nm Bandwidth 2nm 63nm 37nm Structural information fingerprint broad structureless Structural information fingerprint broad structureless Multiplex detection >5 with NIR excitation limited limited Multiplex detection >5 with NIR excitation limited limited SERS tag QD705Atto610 dye

Photo-stability After 1 min.

Photo-stability QD705

Photo-stability  SERS nano-tag

Molecular weight ~10 9 ~ Molecular weight ~10 9 ~ Core size 57nm 10-15nm 1nm Core size 57nm 10-15nm 1nm Hydrodynamic size 78±11nm 18±6nm Hydrodynamic size 78±11nm 18±6nm Bandwidth 2nm 63nm 37nm Bandwidth 2nm 63nm 37nm Structural information fingerprint broad structureless Structural information fingerprint broad structureless Multiplex detection >5 with NIR excitation limited limited Multiplex detection >5 with NIR excitation limited limited Photo-stability insensitive to photobleach Photo-stability insensitive to photobleach decay under laser spot decay under laser spot decay under weak excitation decay under weak excitation SERS tag QD705Atto610 dye

Nature Biotech. 2008, 26(1), Brightness comparison of SERS nanotags and Quantum dots (QD705) (single particle based) SERS nanotagsQD705 Excitation:633±5nm; Emission:LP665nm; Exposure time:750ms; Average 50 images

Molecular weight ~10 9 ~ Molecular weight ~10 9 ~ Core size 57nm 10-15nm 1nm Core size 57nm 10-15nm 1nm Hydrodynamic size 78±11nm 18±6nm Hydrodynamic size 78±11nm 18±6nm Bandwidth 2nm 63nm 37nm Bandwidth 2nm 63nm 37nm Structural information fingerprint broad structureless Structural information fingerprint broad structureless Multiplex detection >5 with NIR excitation limited limited Multiplex detection >5 with NIR excitation limited limited Photo-stability insensitive to photobleach Photo-stability insensitive to photobleach decay under laser spot decay under laser spot decay under weak excitation decay under weak excitation Single particle brightness Single particle brightness Bulk brightness/particle Bulk brightness/particle Bulk brightness/volume  10 5 Bulk brightness/volume  10 5 Quantum yield(633nm) Quantum yield(633nm) Toxicity not toxic toxic toxic Toxicity not toxic toxic toxic SERS tagQD705Atto610 dye

SERS nanotags  1st generation Co-adsorption of reporter molecules and targeting ligands to metal nanoparticles Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, (21), Reporter molecules  spectral signature Reporter molecules  spectral signature Anti-body labeling  selectivity, Anti-body labeling  selectivity, specific targeting specific targeting A major limitation  these tags are prone to uncontrolled spectral changes and aggregation because they are not physically sequestered from targeting molecules, solvent, or analytes.

SERS nanotags  2 nd generation Shield the nanoparticle- reporter complex from the external environment Shield the nanoparticle- reporter complex from the external environment Amenable to covalent bioconjugation Amenable to covalent bioconjugation Nanometer-size Nanometer-size Long coating time Coating process is competing with adsorption of reporter molecules on gold surface Glass particles tend to bind non-specifically to proteins and cell surfaces Core-shell structure silica-coated SERS tag Doering, W. E.; Nie, S. M. Anal. Chem. 2003, 75, (22),

SERS nanotags  2 nd generation First to achieve specific biomolecule targeting in a native cellular environment using SERS sensors First to achieve specific biomolecule targeting in a native cellular environment using SERS sensors Phage network does not protect the tags from spectral changes and aggregation High background of SERS signal of phage limits the assay signal-to-noise ratio Bacteriophage network Souza, G. R. et. al., Proc. Natl. Acad. Sci. U. S. A. 2006, 103, (5), ; Ana. Chem. 2006, 78, 6232.

SERS nanotags  3rd generation 57±10nm 62±9nm 78±11nm 533nm 534nm Coating 5nm Nature Biotech. 2008, 26(1),

PEG-SH “Lock-out” phenomena 30,000 PEG-SH per Au 300,000 PEG-SH per Au Without PEG-SH coating Reverse order, PEG-SH first, dye locked out

PEG-SH coating prevents cross-talk Au-MGITC Au-RBITC RBITC locked out 2 dyes co-adsorb

Stability test in PBS Nature Biotech. 2008, 26(1),

Long term stability

Application: cancer biomarker detection on cell surfaces SERS active SERS in-active 12 12

Carcinoma and non-carcinoma cells Minimal non-specific binding Minimal non-specific binding Superior signal-to-noise ratio Superior signal-to-noise ratio BT474 EpCAM positive cancer cell3T3 EpCAM negative cancer cell 633nm excitation

Carcinoma and non-carcinoma cells Minimal non-specific binding Minimal non-specific binding Acceptable signal-to-noise ratio Acceptable signal-to-noise ratio BT474 EpCAM positive cancer cell3T3 EpCAM negative cancer cell 785nm excitation

Carcinoma and non-carcinoma cells Reproducible Reproducible Applied to both live cell and fixed cell Applied to both live cell and fixed cell Quantification for practical biomedical application Quantification for practical biomedical application 633nm Excitation785nm Excitation S/N30:1 9:1 Cell density: 2  10 6 cells / cm 3 Laser collection volume: ~2  cm 3 ~ 40 cells

Dark Field Reflective Images EGFR positive EGFR negative

Bright field Pseudo- Raman Images Bright-field images EGFR Positive EGFR Negative

Multiplex Detection

QSY 1.3pM 10:1 5:1 1:1 1:3 1:5 1:10 MG 1.3pM

QSY 10:1 5:1 1:1 1:3 1:5 1:10 MG

KB-8-5 cells overexpress both Folate receptor and EGF receptor FA EGF

Sensitivity of NIR SERS spectroscopy at different tissue depth X210 X30 X1 Pure tag Skin spectrum (Control) Subcutaneous injection Deep injection 1-2mm 6-7mm 50uL 1nM SERS tags were injected

In vivo tumor targeting Nature Biotech. 2008, 26(1),

In-vivo cancer targeting NIR-SERS spectroscopy Nature Biotech. 2008, 26(1),

Biodistribution of gold nanoparticles 5 hours post injection

Nu Tumor uptake Nature Biotech. 2008, 26(1),

Summary We have developed stable SERS nano-tags by grafting PEG-SH onto Au nanoparticle-reporter molecule complexes. We have developed stable SERS nano-tags by grafting PEG-SH onto Au nanoparticle-reporter molecule complexes. Complete PEG-SH monolayer protection exhibits excellent stability under extreme conditions and long storage time. Complete PEG-SH monolayer protection exhibits excellent stability under extreme conditions and long storage time. Negligible non-specific binding and superior S/N in cell assay indicates PEG-SH coated SERS biosensors can be used as sensitive optical probes for biomedical application. Negligible non-specific binding and superior S/N in cell assay indicates PEG-SH coated SERS biosensors can be used as sensitive optical probes for biomedical application. We have demonstrated SERS nanoparticles for active targeting of both cancer cells and xenograft tumors in animal models We have demonstrated SERS nanoparticles for active targeting of both cancer cells and xenograft tumors in animal models Multi-modality compatible with fluorescence and TEM imaging system Multi-modality compatible with fluorescence and TEM imaging system

ACKNOWLEDGMENT Prof. Shuming Nie Prof. Shuming Nie Dr. Xu Wang and Dr. X. Winship Cancer Inst. Dr. Xu Wang and Dr. X. Winship Cancer Inst. Dr. Greg Fox Chase Cancer Center Dr. Greg Fox Chase Cancer Center MURI Program MURI Program NCI funding through CCNE Award NCI funding through CCNE Award