DOI: 10.1002/adma.200800498 Bifunctional Fe 3 O 4 –Ag Heterodimer Nanoparticles for Two-Photon Fluorescence Imaging and Magnetic Manipulation** By Jiang Jiang, Hongwei Gu, Huilin Shao, Eamonn Devlin, Georgia C. Papaefthymiou, and Jackie Y. Ying* Superparamagnetic nanoparticles are of great interest for applications such as contrast-enhancing agents for magnetic resonance imaging (MRI), [1] and magnetically assisted cell sorting and separation. [2] Magnetic nanoparticles that are visible under an optical microscope would facilitate the cell separation process with real-time optical monitoring capa- bility. Previous studies have examined semiconductor nano- crystals or quantum dots (QDs) as the optical tag due to their non-bleaching and tunable fluorescent properties. [3] However, QDs are mostly cadmium-based, posing long-term environ- mental and health concerns. [4] Two-photon fluorescence (TPF) is an optical process whereby fluorophores are excited by simultaneous absorption of two infrared photons, followed by relaxation where a single photon of higher energy in the visible spectrum is emitted. Thus, the excitation probability is proportional to the square of the laser flux, with virtually no excitation outside the laser focal volume. Since its invention in 1990 by Webb et al., [5] two-photon fluorescence microscopy has become a powerful imaging tool for biologists because of its intrinsic optical sectioning capability, little near-infrared (NIR) absorption from endogenous species and water, [6] and large penetration depth. [7] Developing a cadmium-free inorganic two-photon fluorescence marker would be valuable for in vitro and in vivo imaging applications. Noble metals such as Au and Ag have been used as optical markers in scattering-based imaging modalities due to their tunable surface plasmon resonance in the visible to NIR spectral region and large scattering cross-sections. [8] Recently, gold nanoparticles [9] and nanorods [10] have been applied to cell imaging with two-photon fluorescence microscopy. They demonstrate an optical cross-section that is much larger than rhodamine dye molecules. Herein we report the development of noble metal nanocrystal-based two-photon fluorescence indicator com- bined with magnetic nanoparticles as a new class of bifunc- tional nanocomposites. An easy synthetic procedure has been developed for growing Ag nanocrystals with tunable sizes on magnetite (Fe 3 O 4 ) superparamagnetic nanoparticle seeds under mild conditions. [11] The resulting hydrophobic Fe 3 O 4 –Ag heterodimer nanocomposite was then rendered hydrophilic by functionalization of the iron oxide surface with hydroxyl groups, and the Ag surface with carboxyl and amine bearing thiol molecules. Live cells labeled with the hetero- dimer nanoparticles were successfully imaged with two-photon fluorescence microscopy, and manipulated using an NdFeB permanent magnet. Magnetite nanoparticles were first synthesized by thermal decomposition of iron-oleate complex following published procedures. [12,13] Heterodimer nanoparticles were then formed by reducing Ag in the presence of the pre-synthesized magnetite seed nanoparticles. As shown in Figure 1, Ag nanoparticles grew on spherical as well as cubic magnetite nanoparticles. The reaction proceeded under mild conditions, and Ag formation was visible at 60 8C within a few minutes. As a control (i.e., without adding the magnetite seed nanoparti- cles), Ag reduction was found to take at least 30 min under similar reaction conditions. This illustrated the importance of the magnetite nanoparticles in catalyzing Ag reduction on their surfaces. The size of the Ag nanoparticles could be tuned between 2 and 15 nm by changing reaction conditions, such as solvent type, temperature, and reducing reagents. X-ray diffraction (XRD) patterns of the heterodimer nanoparticles could be indexed to magnetite and Ag crystalline phases (Fig. S1). The Fe 3 O 4 –Ag heterodimer nanoparticles were superpara- magnetic with a blocking temperature of 225 K (Fig. S2), and a magnetization (M) value of 66 emu/g Fe 3 O 4 at an applied COMMUNICATION [*] Prof. J. Y. Ying, Dr. J. Jiang, Dr. H. Gu, H. Shao Institute of Bioengineering and Nanotechnology 31 Biopolis Way, The Nanos, Singapore 138669 (Singapore) E-mail: jyying@ibn.a-star.edu.sg Dr. E. Devlin, Prof. G. C. Papaefthymiou Institute of Materials Science NCSR Demokritos, Athens, 15310 (Greece) Prof. G. C. Papaefthymiou Department of Physics, Villanova University Villanova, Pennsylvania 19085 (USA) [**] The authors thank Dr. Yu Han and Dr. Lan Zhao, Dr. Shyh-Chyang Luo, and Chiang Huen Kang for their assistance with TEM, inductively coupled plasma-mass spectrometry (ICP-MS), and two-photon fluorescence microscopy, respectively. This work is funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). G. C. P. thanks the National Science Foundation and the European Union, Marie Curie Program, for support. Supporting information is available online from Wiley InterScience or from the authors. Adv. Mater. 2008, 20, 4403–4407 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4403