In Vivo Detection of Gold-Imidazole Self-Assembly Complexes: NIR-SERS Signal Reporters Glauco R. Souza, †,‡ Carly S. Levin, † Amin Hajitou, ‡ Renata Pasqualini, ‡ Wadih Arap, ‡ and J. Houston Miller* ,† Department of Chemistry, The George Washington University, Washington, D.C. 20052, and The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030 Here we report in vitro and in vivo detection of self- assembled Au-imidazole by using near-infrared surface- enhanced Raman scattering (NIR-SERS). In vivo, the Au- imidazole structures were administered into tumor- bearing mice and detected noninvasively. The self- assembled Au-imidazole complexes were generated by the adsorption of imidazole molecules onto Au nanopar- ticles (NP) and were then characterized as aqueous suspensions by using NIR-SERS, angle-dependent light scattering with fractal dimension analysis, and visible extinction spectroscopy. The structure and optical activity was sensitive to imidazole concentration and Au NP size. Specifically, the Au-imidazole assemblies formed at lower imidazole concentrations had the lowest fractal dimension (D f ) 1.2) and the largest Raman enhancement factors for the dominant NIR-SERS feature, a ring-breathing vibrational mode at 954 cm -1 . Changes in elastic scat- tering intensity, fractal dimension, and surface plasmon absorption were observed with increasing imidazole con- centration. The Raman enhancement factor was also found to range between 10 6 and 10 9 with different primary Au nanoparticle sizes. For the higher enhancement factor systems, NIR-SERS detection of Au-imidazole was per- formed with data acquisitions time of only 5 s. The largest enhancement was observed for the 954-cm -1 feature at an imidazole concentration of 1.9 μM when coupled to 54-nm-diameter Au NPs (the largest NP tested). Finally, we show the first demonstration of in vivo, noninvasive, and real-time SERS detection. Development of signal reporters based on near-infrared surface- enhanced Raman scattering (NIR-SERS) is required for application of this technology in biomolecular imaging. 1-3 Biological tissues show minimal NIR radiation (700-900 nm) absorption, thus allowing efficient light penetration for imaging and phototherapy applications in vivo. 4-6 We have coupled Au nanoparticles (NPs) with imidazole (Au-imidazole) into self-assembled NPs, which can be used for real-time, in vivo NIR-SERS detection. The strong affinity between imidazole and Au induces the aggregation of the Au NPs, which results in a shift in the surface plasmon resonance absorption into the NIR wavelengths along with changes in fractal structure and optical properties. All of these attributes are desirable for noninvasive, detection in tissue. Here we present proof of principle for the use of NIR-SERS in noninvasive, in vivo, and real-time detection of self-assembled nanoparticle complexes. We show the optical and structural characterization of these assemblies by using SERS, UV-visible extinction, and angle-dependent light scattering (ADLS) coupled with fractal dimension analysis. We also show detection in vivo by using a fiber-optic probe to deliver and collect light through the skin of tumor-bearing mice injected with Au-imidazole complexes. Imidazole has been the target of several prior SERS studies using Ag NPs and electrodes. 7-10 Holze 11 reported the only prior study of SERS for imidazole on gold, specifically on the surface of an Au electrode. The two nitrogens 12 of imidazole (Scheme 1a), the position 1 nitrogen (sp 3 as in pyrrole, pK a 6.5), and the position 3 nitrogen (sp 2 as in pyridine, pK a 14.0) 11 play a role in bridging metal particles 7 (Scheme 1b). Because the in vitro work was performed at pH 8.0, one would expect electrostatic interaction between the cationic form of imidazole (Scheme 1a, right) and the negatively charged Au NP (resulting from adsorbed citrate ions from Au synthesis). Imidazole and its derivatives are associ- ated with corrosion prevention and serve as precursors for the adsorption of other molecules onto metal surfaces. 7-10,13,14 The * Corresponding author. Phone: 202-994-7474. Fax: 202-994-5873. E-mail: Houston@gwu.edu. † The George Washington University. ‡ The University of Texas M. D. Anderson Cancer Center. (1) Souza, G. R.; Christianson, D. R.; Staquicini, F. I.; Ozawa, M. G.; Snyder, E. Y.; Sidman, R. L.; Miller, J. H.; Arap, W.; Pasqualini, R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1215-1220. (2) Kneipp, J.; Kneipp, H.; Rice, W. L.; Kneipp, K. Anal. Chem. 2005, 77, 2381- 2385. (3) Schwartzberg, A. M.; Grant, C. D.; Wolcott, A.; Talley, C. E.; Huser, T. R.; Bogomolni, R.; Zhang, J. Z. J. Phys. Chem. 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Chem. 2006, 78, 6232-6237 6232 Analytical Chemistry, Vol. 78, No. 17, September 1, 2006 10.1021/ac060483a CCC: $33.50 © 2006 American Chemical Society Published on Web 07/22/2006