Determination of the Size, Concentration, and Refractive Index of Silica Nanoparticles from Turbidity Spectra Boris N. Khlebtsov, † Vitaly A. Khanadeev, ‡ and Nikolai G. Khlebtsov* ,†,‡ Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt EntuziastoV, SaratoV 410049, Russia, and SaratoV State UniVersity, 83 Ulitsa Astrakhanskaya, SaratoV 410026, Russia ReceiVed March 31, 2008. ReVised Manuscript ReceiVed May 11, 2008 The size and concentration of silica cores determine the size and concentration of silica/gold nanoshells in final preparations. Until now, the concentration of silica/gold nanoshells with Sto ¨ber’s silica core has been evaluated through the material balance assumption. Here, we describe a method for simultaneous determination of the average size and concentration of silica nanospheres from turbidity spectra measured within the 400-600 nm spectral band. As the refractive index of silica nanoparticles is the key input parameter for optical determination of their concentration, we propose an optical method and provide experimental data on a direct determination of the refractive index of silica particles n ) 1.475 ( 0.005. Finally, we exemplify our method by determining the particle size and concentration for 10 samples and compare the results with transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering data. Introduction In the past few years, gold nanoshells (NSs) have attracted interest as novel plasmon-resonant structures for various ap- plications to nanobiotechnology. 1,2 These particles consist of a spherical silica core covered with a thin (15-30 nm) gold shell, which ensures both convenient surface bioconjugation with molecular probes and remarkable plasmon-resonant optical properties. It has been recognized that NSs provide an efficient platform for analytical biosensing, 3–5 integrated applications to the photothermal therapy 6,7 and optical imaging of cancer cells, 8–10 optical coherent tomography, 11 and diffusion-wave spectros- copy. 12 In contrast to usual colloidal gold particles, the absorption and scattering plasmon resonance of NSs can easily be tuned from the visible (VIS) to the near-infrared (NIR) spectral band by varying the core/shell geometry. 13,14 The resonance absorp- tion and scattering efficiency of NSs exceeds that of colloidal gold particles by more than 1 order, thus having the advantage of colloidal gold as labels for optical imaging of biospecific interactions. An accurate determination of the size and concentration of NSs is essential for most biomedical applications of NSs. For instance, the size and concentration of nanoparticles are crucial parameters determining their uptake by living cells 15 and their circulation and biodistribution in living bodies. 16 Whereas transmission electron microscopy (TEM) is a reliable method for sizing of metal nanoparticles, there are no analogous time- tested and convenient tools for concentration measurements. As a few examples, one can point out the paper by Green at al. 17 on small-angle X-ray and dynamic light scattering studies performed to monitor the nucleation of Sto ¨ber’s silica nano- particles, and studies of concentrated samples by using back- scattering, 18 diffuse photon density, 19 and fluorescence 20 tech- niques. Here, we consider gold NSs, which are fabricated in many laboratories by a two-step protocol. First, silica nanospheres are prepared, and then gold nanoshells are formed by functionalization of the silica surface with fine (1-3 nm) gold seeds followed by * To whom correspondence should be addressed. E-mail: khlebtsov@ ibppm.sgu.ru. † Russian Academy of Sciences. ‡ Saratov State University. (1) Hirsch, L. R.; Gobin, A. M.; Lowery, A. R.; Tam, F.; Drezek, R.; Halas, N. J.; West, J. L. Ann. Biomed. Eng. 2006, 34, 15–22. (2) Kalele, S.; Gosavi, S. W.; Urban, J.; Kulkarni, S. K. Curr. Sci. 2006, 91, 1038–1052. (3) Hirsch, L.; Jackson, J. B.; Lee, M.; Halas, N.; West, J. Anal. Chem. 2003, 75, 2377–2381. (4) Wang, Y.; Qian, W.; Tan, Y.; Ding, S. Biosens. Bioelectron. 2008, 23, 1166–1170. (5) Khlebtsov, B. N.; Dykman, L. A.; Bogatyrev, V. A.; Zharov, V. P.; Khlebtsov, N. G. Nanoscale Res. Lett. 2007, 2, 6–11. (6) Hirsch, L.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N.; West, J. Proc. Natl. Acad. Sci. U.S.A. 2003, 23, 13549–13554. 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