Energy Transfer between Conjugated-Oligoelectrolyte-Substituted

Jun 2, 2011 - Xiang Li , Ehsan Eftekhari , Ivan S. Cole , Nam-Trung Nguyen , Qin Li ... nanoclusters as a new fluorescence probe for Sudan I–IV sens...
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Energy Transfer between Conjugated-Oligoelectrolyte-Substituted POSS and Gold Nanocluster for Multicolor Intracellular Detection of Mercury Ion Kan-Yi Pu, Zhentao Luo, Kai Li, Jianping Xie, and Bin Liu* Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore

bS Supporting Information ABSTRACT: Although metal nanoclusters (NCs) with sizes close to the Fermi wavelength of electrons are well-known for their molecule-like luminescent behaviors, their energy-transfer properties remain unrevealed. Herein, fluorescence resonance energy transfer (FRET) between blue-fluorescent conjugatedoligomer-substituted polyhedral oligomeric silsesquioxane (POSSFF) and red-fluorescent gold NCs (R-AuNCs) is investigated and subsequently utilized for mercury-ion sensing both in solution and in cell. By virtue of their opposite charges and good spectral overlap, efficient FRET from POSSFF to R-AuNC occurs upon electrostatic complex formation, leading to dual-emissive pink fluorescence upon donor excitation. The pink fluorescence specifically turns blue in the presence of mercury ions rather than other metal ions because of the strong metallophilic Hg2þ/Auþ interaction that quenches the red fluorescence from R-AuNCs. This consequently allows for visual detection and precise quantification of mercury ions with a limit of detection of ∼0.1 nM in aqueous solution. Moreover, the whole-cell permeability of the complexes and the preserved ion-selective FRET in cells make these complexes effective for multicolor intracellular sensing of mercury ion. This study thus not only develops a promising mercury-ion nanoprobe for diagnostic and clinical applications but also provides fundamental guidelines for the design of metal-NC-based intracellular nanosensors.

’ INTRODUCTION Noble-metal nanoclusters (NCs) comprising several to tens of atoms are now under intensive investigation because of their distinct optical, electrical, and chemical properties compared to those of their larger counterparts, plasmonic nanocrystals.1,2 With sizes smaller than 2 nm, which is comparable to the Fermi wavelength of electrons, metal NCs exhibit discrete and sizetunable electronic transitions, dramatically featuring moleculelike luminescent properties.35 In contrast to fluorescent semiconductor quantum dots (QDs),6,7 metal NCs are much smaller and do not contain toxic elements as the ingredients; therefore, they are biocompatible and more attractive for biological sensing and imaging.8 Investigations of metal NCs for fluorescence applications are rare, however, and generally limited to the solution state.9,10 Moreover, existing metal-NC-based solution sensors simply capitalize on fluorescence quenching as the signal readout,11,12 which makes them less convenient for direct visual detection by the naked eye. For intracellular detection, fluorescence turn-on or emission profile variation signatures are preferred over turn-off signals so as to maximize optical spatial resolution.13 As a consequence, current metal-NC systems are less suitable for intracellular molecular imaging. Fluorescence resonance energy transfer (FRET), a photophysical process involving the nonradiative transfer of energy r 2011 American Chemical Society

between two nearby fluorophores through long-range dipole dipole coupling, is widely used as a powerful spectroscopic tool to generate emission profile variations or multicolor ratiometric signals for biological sensing and imaging.14,15 Although organic fluorophores, fluorescent proteins, and QDs have been widely utilized to construct FRET assays,16,17 metal NCs have not been involved in such applications to date. Fluorescence principles reveal that fluorescent molecules with spherical three-dimensional structures have orientation advantages over linear fluorophores to interact with optical partners in a more favorable manner for efficient FRET.18 In addition, unlike soft small molecules, which are liable to be barred by the cellular membrane, rigid spherical nano-objects are more likely to enter cells through passive diffusion, consequently meeting the prerequisite of detection in cells.19 These studies clearly imply that it is promising for metal NCs to constitute FRET-based probes with visual detection and intracellular sensing functionalities. A primary challenge in the development of metal-NC-involving FRET-based probes lies in the selection of suitable optical partners to form efficient FRET pairs. In this regards, conjugated Received: April 5, 2011 Revised: June 1, 2011 Published: June 02, 2011 13069

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Scheme 1. (a) Schematic Illustration of Visual Detection of Mercury Ions Based on FRET between POSSFF and R-AuNC and (b) Chemical Structure of POSSFF

polyelectrolytes (CPEs) with π-electron-delocalized backbones and water-soluble ionic side chains have proven to be efficient light-harvesting antennas for a variety of fluorescent substances, including organic fluorophores,2022 fluorescent proteins,23 dyelabeled bioprobes,2426 and QDs.27,28 Recently, we developed a new generation of CPE derivatives consisting of a polyhedral oligomeric silsesquioxane (POSS) cage surrounded by conjugated oligoelectrolytes (COEs) as the arm on its globular periphery.29,30 These COE-substituted POSS molecules can be recognized as fluorescent unimolecular nanodots featuring unique whole-cell permeability for FRET throughout cells. In view of these desirable features, we envision that COE-based nanodots could be used to pair with metal NCs to constitute FRET-based probes for visual detection and intracellular sensing. In this work, we investigate FRET between COE-substituted POSS and gold NCs and subsequently apply the hybrid nanocomplex as an efficient multicolor probe for visual detection and intracellular sensing of mercury(II) ions. As mercury pollution is a global environmental problem that seriously threatens the health of human beings, development of fluorescent mercuryion probes is of vital importance.31 Among various existing probes for mercury-ion sensing, very few rely on the FRET mechanism and can also be operated in cells.32,33 In particular, gold NCs themselves have recently been used for mercury-ion sensing,9,10,34 but they were applied only in aqueous media. Moreover, the simple adoption of fluorescence quenching as the signal makes them less suitable for intracellular sensing because of their limited spatial resolution upon light excitation.13 As a consequence, this study opens up new opportunities for bringing metal NCs into intracellular molecular imaging. The sensing mechanism is proposed in Scheme 1a. Bluefluorescent cationic-oligofluorene-substituted POSS (POSSFF, Scheme 1b) and red fluorescent gold NCs (R-AuNCs) were

chosen as the energy donor and acceptor, respectively. In the absence of mercury ions, electrostatic attractions between the oppositely charged POSSFF (þ) and R-AuNC () induce the formation of hybrid complexes within which the donoracceptor distance is short enough to allow efficient FRET. In this case, the hybrid complex emits pink fluorescence. In contrast, in the presence of sufficient amounts of mercury ions, the R-AuNC () emission is quenched by a strong metallophilic interaction between Hg2þ and Auþ on the surface of the R-AuNCs,34 whereas the donor emission remains. Under these conditions, the hybrid complex emits blue fluorescence. According to such different fluorescence responses, mercury ions can be visually detected using POSSFF/R-AuNC complexes.

’ RESULTS AND DISCUSSION The donor POSSFF was synthesized by the Heck coupling reaction between cationic oligofluorene and octavinyl POSS as described in our previous report.29 This unimolecular nanodot has eight fluorescent cationic arms in eight different directions, making it a perfect three-dimensional light-harvesting molecule for FRET. The acceptor R-AuNC was prepared by a green biomineralization method using bovine serum albumin (BSA) to sequester and reduce ionic Au(III) precursor in situ according to the previous report.35 Each R-AuNC contains 25 gold atoms encapsulated by a BSA molecule, exhibiting extremely good aqueous stability. Transmission electron microscopy (TEM) reveals that POSSFF and R-AuNC have the average diameters of 3.6 and 0.8 nm, respectively (Figure S1 in the Supporting Information). The optical properties of POSSFF and R-AuNCs were studied in water. As shown in Figure 1a, POSSFF has absorption and emission maxima at 390 and 435 nm, respectively, whereas 13070

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Figure 1. (a) Normalized UVvis absorption and emission spectra of POSSFF and normalized PL excitation and emission spectra of R-AuNC in water. (b) PL spectra of POSSFF in PBS (15 mM, pH 7.4) in the absence and presence of R-AuNC upon excitation at 390 nm. [POSSFF] = 0.5 μM, and [R-AuNC] changes from 0 to 4 μM in intervals of 0.5 μM.

Figure 2. (a) PL spectra of POSSFF/R-AuNC complex upon excitation at 390 and 480 nm and of R-AuNC upon excitation at 390 nm in PBS (15 mM, pH 7.4). (b) Time-resolved PL decay at 435 nm for POSSFF and POSSFF/R-AuNC complex upon excitation at 390 nm. [POSSFF] = 0.5 μM, and [R-AuNC] = 4 μM.

R-AuNCs show photoluminescence (PL) excitation and emission maxima at 480 and 665 nm, respectively. The good overlap between the emission spectrum of POSSFF and the PL excitation spectrum of R-AuNCs indicates the feasibility of FRET between them. The PL quantum yields of POSSFF and R-AuNC in water and are ∼85% and 7%, respectively, measured using quinine sulfate in 0.1 M H2SO4 (quantum yield = 0.55) as the standard. The fluorescence of POSSFF and R-AuNCs is only slightly affected by ionic strength and pH, featuring quantum yields of 80% and 6.5%, respectively, in 150 mM phosphate-buffered saline (PBS, pH 7.4). The stable optical properties suggest the suitability of these materials for biological sensing and imaging in complicated physiological systems. To optimize the donoracceptor molar ratio for metal-ion sensing, changes in the PL spectrum of POSSFF upon addition of R-AuNCs in PBS (15 mM, pH 7.4) were monitored by excitation of the donor at 390 nm. In these experiments, the concentration of POSSFF was fixed at 0.5 μM while that of R-AuNC was varied from 0 to 4 μM in intervals of 0.5 μM. As shown in Figure 1b, the blue emission centered at 435 nm gradually decreased with increasing concentration of R-AuNC, which is concomitant with a progressive increase in the red emission with the maximum at 665 nm. The spectral transformation clearly indicates the occurrence of FRET from POSSFF to R-AuNC, which originates from the complex formation due to electrostatic attraction between the oppositely charged POSSFF and R-AuNCs at pH 7.4. At an R-AuNC concentration of 4 μM, the blue emission no longer decreases, reflecting the saturation of donoracceptor interaction.

At this point, the emission intensities at 435 and 665 nm are nearly the same, making the solution fluorescence pink. As such, the optimum molar ratio of R-AuNC to POSSFF for mercury-ion sensing was chosen to be 4:0.5. To gain insight into FRET within the hybrid complex at the optimized molar ratio ([POSSFF] = 0.5 μM, [R-AuNC] = 4 μM), selective-excitation PL experiments and time-resolved fluorescence measurements were conducted. Figure 2a shows the PL spectra of POSSFF/R-AuNC complex in PBS (15 mM, pH = 7.4) upon excitation at the absorption maxima of donor (390 nm) and acceptor (480 nm), respectively. Furthermore, the intrinsic PL spectrum of R-AuNC (4 μM) itself in PBS upon excitation at 390 nm is also depicted in Figure 2a. It is obvious that the emission intensity of POSSFF/R-AuNC complex at 665 nm upon excitation at 390 nm is nearly twice of that upon excitation at 480 nm, which is also much higher than that for R-AuNC itself upon excitation at 390 nm. Accordingly, FRET from POSSFF to R-AuNC occurs within the complex, leading to amplified fluorescence of R-AuNC that facilitates visual detection. Time-resolved PL decays of POSSFF and POSSFF/R-AuNC complex at 435 nm upon excitation at 390 nm are shown in Figure 2b. These decays are well fitted by double-exponential, revealing the average lifetimes of 0.82 and 0.60 ns for POSSFF and POSSFF/R-AuNC complex, respectively. As R-AuNC is encapsulated by BSA, the PL spectrum and time-resolved PL decay of POSSFF in the presence of BSA (4 μM) are also studied (Figure S2 in the Supporting Information). The PL spectra and decay of POSSFF in the presence of BSA are similar to those in 13071

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The Journal of Physical Chemistry C the absence of BSA, which indicates that BSA does not contribute to the change in the lifetime of POSSFF in the presence of R-AuNCs. As such, the faster PL decay at the donor emission maximum for the POSSFF/R-AuNC complex relative to that for POSSFF is due to FRET from POSSFF to R-AuNCs. Based on the changes in donor lifetime, the FRET efficiency within the POSSFF/R-AuNC complex is calculated to be 28% To examine the sensing ability of the POSSFF/R-AuNC complex, the fluorescence response of the complex ([POSSFF] = 0.5 μM, [R-AuNC] = 4 μM) toward mercury ion was first studied in aqueous solution. Titration experiments were conducted in PBS (15 mM, pH 7.4) with Hg2þ concentrations ranging from 0 to 2.7 μM in intervals of 0.3 μM. As shown in Figure 3a, with increased Hg2þ concentration, the red emission at 665 nm gradually decreases, whereas the blue emission at 435 nm changes slightly only. The saturation occurs at [Hg2þ] = 2.4 μM, where the red emission is totally quenched and the blue emission is recovered to 73% of its original intensity. In contrast to Hg2þ, other metal ions including Mn2þ, Pb2þ, Cd2þ, Zn2þ, Ni2þ, Pd2þ, Co2þ, Ca2þ, Ce3þ, and Cu2þ cannot induce significant fluorescence variations for the POSSFF/R-AuNC complex. For instance, the PL spectra of POSSFF/R-AuNC complex in the absence and presence of Cd2þ at [Cd2þ] = 2.4 μM are nearly the same (Figure S3 in the Supporting Information). The fluorescence change of the hybrid complex is mainly associated with the efficient fluorescence quenching of R-AuNCs toward mercury ions as a result of the strong metallophilic interaction between Hg2þ and Auþ on the NC surface. Moreover, the ion-resistant fluorescence of POSSFF is partially responsible, which preserves the blue emission in the presence of other metal ions at elevated concentrations. To determine the detection selectivity for mercury ion, ΔI was defined as the decrease in the emission intensity of the POSSFF/ R-AuNC complex at 665 nm after addition of different metal ions. Figure 3b summarizes ΔI for the POSSFF/R-AuNC complex as a function of metal ion at a metal-ion concentration of 2.4 μM. Hg2þ gave a ΔI value of 545, which is significantly larger than those for other metal ions (∼10). Because of the obvious difference in ΔI, the solution fluorescent color of the POSSFF/R-AuNC complex turns blue in the presence of Hg2þ, whereas it remains pink in the presence of other metal ions (Figure 3c). In addition, the POSSFF/R-AuNC complex shows a blue fluorescence signature in the mixed sample containing Hg2þ and other metal ions (Figure 3c). As a result, the POSSFF/RAuNC complex can be used as a highly selective multicolor probe for the visual detection of mercury ions. To quantify mercury ions, changes in the PL spectra of the POSSFF/R-AuNC complex in Figure 3a were correlated with the concentration of Hg2þ. Figure 4a shows ΔI as a function of Hg2þ concentration coupled with its fitting line. The good overlap between the original data and the linear fitting line confirms the effectiveness of the POSSFF/AuNC complex in mercury-ion quantification. The deviation from the linear fitting line above 1.8 μM is attributed to analyte saturation. On the other hand, the linearly decreased red emission and well-maintained blue emission of the POSSFF/AuNC complex obviously cause the solution fluorescent color to vary corresponding to Hg2þ concentration. As shown in the inset of Figure 4a, the fluorescent color of the hybrid complex solution is pink, heliotrope, steel blue, and blue at [Hg2þ] = 0, 0.6, 1.2, and 1.8 μM, respectively, allowing for visual quantification of Hg2þ. To determine the limit of detection (LOD) of the complex, PL experiments were

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Figure 3. (a) PL spectra of POSSFF/AuNC in PBS (15 mM, pH 7.4) in the absence and presence of Hg2þ at Hg2þ concentrations ranging from 0 to 2.7 μM in intervals of 0.3 μM. [POSSFF] = 0.5 μM, and [R-AuNC] = 4 μM. Excitation at 390 nm. (b) ΔI for POSSFF/R-AuNC complex (15 mM, pH 7.4) as a function of metal ion species. [POSSFF] = 0.5 μM, [R-AuNC] = 4 μM, and [metal ion] = 2.4 μM. The mixed sample contains Hg2þ (2.4 μM) and other metal ions (2.4 μM each). (c) Photographs of the corresponding fluorescent solutions of POSSFF/R-AuNC complex in the absence and presence of metal ions under UV radiation at 365 nm.

performed using a dilute solution with [POSSFF] = 5 nM and [R-AuNC] = 40 nM in PBS (15 mM, pH 7.4). As shown in Figure 4b, a progressive intensity decrease at 665 nm was clearly observed upon addition of Hg2þ in intervals of 0.1 nM. Further decreasing the complex concentration failed to induce detectable fluorescence responses. As such, the practical LOD of the POSSFF/R-AuNC complex for solution detection of mercury ion is ∼0.1 nM (0.02 ppb), which is much lower than the maximum level (2.0 ppb) of mercury in drinking water permitted by the United States Environmental Protection Agency (EPA). Having demonstrated the feasibility of the POSSFF/R-AuNC complex in the visual detection and quantification of mercury ion in aqueous solution, we started to test the sensing performance of the hybrid complex in cells. As POSSFF has been found to have whole-cell permeability,28 the cell permeability of R-AuNC was evaluated first upon incubation with breast cancer cells (MCF-7) for 3 h. The excitation wavelength was 488 nm, and the fluorescence signals were collected above 650 nm using confocal laser scanning microscopy (CLSM). As shown in Figure S4 (Supporting Information), red fluorescence was observed from the entire cell, revealing that R-AuNC also has a whole-cell permeability similar to that of POSSFF. Accordingly, the POSSFF/R-AuNC complex is expected to detect metal ions not only in the cytoplasm but also in the nuclelus. 13072

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Figure 5. CLSM fluorescence images of MCF-7 cells stained by POSSFF/ R-AuNC complex in the (ac) and (df) presence of Hg2þ. Excitation at 405 nm. Fluorescence collection (a,d) from 430 to 455 nm and (b,e) above 650 nm. (c,f) Overlapped images of a/b and d/e, respectively. The scale bar is the same for all images.

Figure 4. (a) ΔI as a function of Hg2þ concentration. Inset: Photographs of the corresponding fluorescent solutions of POSSFF/R-AuNC at [POSSFF] = 0.5 μM; [R-AuNC] = 4 μM; and [Hg2þ] = 0, 0.6, 1.2, or 1.8 μM under UV irradiation at 365 nm. (b) PL spectra of POSSFF/ AuNC in PBS (15 mM, pH 7.4) upon addition of Hg2þ in intervals of 0.1 nM. [POSSFF] = 5 nM, and [R-AuNC] = 40 nM. Excitation at 390 nm.

The utility of the POSSFF/R-AuNC complex in intracellular detection of mercury ion was subsequently investigated by conducting CLSM experiments on MCF-7 cells treated with the hybrid complex ([POSSFF] = 0.05 μM, [R-AuNC] = 0.4 μM) for 3 h. Parts ac and df of Figure 5 show the fluorescence images of the stained cells in the absence and presence of Hg2þ (0.24 μM), respectively. The excitation wavelength was 405 nm, and the fluorescence signals were collected in two channels: 430455 nm for Figure 5a,d and above 650 nm for Figure 5b,e. It should be noted that there are no observable CLSM fluorescence signals above 650 nm for POSSFF, as its emission intensity approaches zero above 620 nm (Figure 1a), which indicates that the fluorescence signals collected above 650 nm come from the emission of R-AuNCs. This is consistent with the images shown in Figure 5a,d, where bright blue fluorescence is observed for both Hg2þ-free cells and cells containing Hg2þ, whereas red fluorescence is detectable only from Hg2þ-free cells (Figure 5b). In addition, the two-channel overlapped images show pink and blue fluorescence in the absence and presence of Hg2þ, respectively. These cellular images clearly indicate that the fluorescence of R-AuNCs is quenched by Hg2þ. This phenomenon accords with the PL spectra and photographs of the POSSFF/RAuNC complex in the solution state, confirming its well-preserved fluorescence response toward Hg2þ in cells. Considering that the images in the absence and presence of other metal ions were nearly the same, these data clearly demonstrate that multicolor detection of mercury ions can be realized using the POSSFF/R-AuNC complex as an intracellular probe.

’ CONCLUSIONS We have developed a FRET-based hybrid complex nanoprobe using blue-fluorescent COE-substituted POSS and red-fluorescent metal NCs as the energy donor and acceptor, respectively. The opposite charges and good spectral overlap between POSSFF and R-AuNCs lead to complex formation and efficient intracomplex FRET, which results in dual-emissive pink fluorescence upon donor excitation. Because of the specific metallophilic Hg2þ/Auþ interaction on the NC surface, the acceptor fluorescence of the POSSFF/ R-AuNC complex is significantly quenched by mercury ions rather than other metal ions, consequently bringing about the distinct fluorescence color variation from pink to blue as the signature for the visual discrimination of mercury ions. Meanwhile, the linear optical response of the hybrid complex toward mercury ions allows for mercury-ion quantification with a practical LOD of ∼0.1 nM in aqueous solution. In addition, whole-cell permeability and ion-selective FRET were observed in cells for this hybrid complex, which together enable multicolor intracellular sensing of mercury ions. The high selectivity, trace sensibility, and unique intracellular detecting capability make the POSSFF/ R-AuNC complex a promising mercury-ion probe for diagnostic and clinical applications. To the best of our knowledge, this is the first time that metal NCs have successfully been utilized to construct FRET probes. Further improvement can be made to increase FRET efficiency by surface modification of metal NCs or structural variation of COEs. In addition to water-soluble conjugated polymers or oligmers, other fluorescent materials such as QDs and fluorescent proteins could also be used to form FRET pairs with metal NCs, potentially leading to the detection of other chemical and biological substances of interest. Considering their aqueous stability, cell permeability, sufficient brightness, and ingredient biocompatibility, metal-NC-based probes merit further investigation with respect to in vitro and in vivo sensing and imaging. As a result, this study provides fundamental guidelines and new opportunities for the development of hybrid nanomaterials with complicated optical and biological functionalities. ’ EXPERIMENTAL SECTION Instruments. UVvis spectra were recorded on a Shimadzu UV-1700 spectrometer. PL measurements were carried out on a 13073

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The Journal of Physical Chemistry C Perkin-Elmer LS-55 instrument equipped with a xenon-lamp excitation source and a Hamamatsu (Hamamatsu City, Japan) 928 photomultiplier tube (PMT), using 90° angle detection for solution samples. The excitation energy at different wavelengths was automatically adjusted to the same level by an excitation correction file. PL quantum yields were measured using quinine sulfate as the standard, with a quantum yield of 55% in H2SO4 (0.1 M). High-resolution transmission electron microscopy (HR-TEM) images were obtained from a JEOL JEM-2010 transmission electron microscope with an accelerating voltage of 200 kV. Photographs of the polymer solutions were taken using a Canon EOS 500D digital camera under a handheld UV lamp with λmax = 365 nm. Time-resolved PL decays were obtained on a FluoTime 200 TCSPC fluorescence platform from Picoquant GmbH (Berlin, Germany). A titanium:sapphire 100-fs laser (Chameleon, Coherent) with second- and third-harmonic generation was used as the excitation source, and its excitation wavelength was 390 nm. In the time-correlated single-photon-counting (TCSPC) apparatus, the detector was based on a microchannel plate (MCP) PMT system (HAM-R3809U-50, Hamamatsu) and had a spectral sensitivity from 160 to 850 nm and an instrument response function of 30 ps. Fluorescence lifetimes were extracted from the decay curves using commercially available analysis software (FluoFit Pro, PicoQuant GmbH). Time-resolved PL decay curves were fitted using a two-exponential mode. All UV and PL spectra and time-resolved PL decay data were collected at 24 ( 1 °C. Milli-Q water (18.2 MΩ) was used for all experiments. Materials. Fetal bovine serum (FBS) was purchased from Gibco (Life Technologies AG, Basel, Switzerland). Dulbecco’s modified essential medium (DMEM) was a commercial product from the National University Medical Institutes (Singapore). Phosphate-buffered saline (PBS) buffer with pH 7.4 (ultrapure grade) was a commercial product from 1st BASE Singapore. Milli-Q water (18.2 MΩ) was used to prepare the buffer solutions from the 10 PBS stock buffer. The metal-ion solutions were prepared from Hg(CH3COO)2, ZnCl2, Ce(CH3COO)3, Pb(NO3)2, Ni(CH3COO)2, MnSO4 3 H2O, CdSO4 3 H2O, CaCl2 3 2 H2O, Pd(NO3)2, CuSO4 3 5H2O, and CoCl2 3 6H2O. POSSFF was synthesized according to our previous report.28 Other chemical reagents were purchased from Sigma-Aldrich Chemical Co. and were used as received. Synthesis of R-AuNC.34. All glassware was washed with aqua regia [HCl/HNO3 = 3:1 (v/v)] and rinsed with ethanol and ultrapure water. (Caution: Aqua regia is a highly corrosive oxidizing agent that should be handled with great care.) In a typical experiment, aqueous HAuCl4 solution (5 mL, 10 mM, 37 °C) was added to BSA solution (5 mL, 50 mg/mL, 37 °C) under vigorous stirring. Sodium hydroxide solution (0.5 mL, 1 M) was introduced into the mixture 2 min later, and the reaction was allowed to proceed under vigorous stirring at 37 °C for 12 h. The as-synthesized R-AuNCs (10.5 mL) were dialyzed in membrane tubing with a molecular-weight cutoff of 12 kDa against Milli-Q water at room temperature. After 24 h and three changes of water (at 8-h intervals), the tubing contents (R-AuNCs) were collected, and the solution was concentrated to 10 mL for further tests and experiments. Cell Cultures. MCF-7 tumor cells were cultured in DMEM containing 10% FBS and 1% penicillin streptomycin at 37 °C in a humidified environment containing 5% CO2. Before experiments, the cells were precultured until confluence was reached. Cellular Imaging. MCF cells were cultured in chambers (LAB-TEK, Chambered Coverglass System) at 37 °C for

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qualitative study. After 80% confluence, the medium was removed, and the adherent cells were washed twice with 1  PBS buffer. The solution of POSSFF/R-AuNC complex ([POSSFF] = 0.05 μM, [R-AuNC] = 0.4 μM) was then added to the chamber. After incubation for 3 h, cells were washed three times with 1  PBS buffer and then fixed with 75% ethanol for 20 min and washed twice more with 1  PBS buffer. Cells with and without exposure to Hg2þ (0.24 μM) were used as the control and target samples, respectively. Cellular imaging experiments were performed by CLSM (Zeiss LSM 410, Jena, Germany), with the imaging software Fluoview FV500. The cellular imaging experiments using R-AuNCs were conducted in a similar way.

’ ASSOCIATED CONTENT

bS

Supporting Information. TEM images of POSSFF and R-AuNC, PL spectrum and time-resolved PL decay of POSSFF in the presence of BSA, PL spectra of POSSFF/R-AuNC in the absence and presence of Cd2þ, and CLSM images of R-AuNCstained MCF-7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Fax: (þ65) 6779-1936. E-mail: [email protected].

’ ACKNOWLEDGMENT The authors are grateful to the National University of Singapore (R-279-000-234-123, R279-000-301-646), Singapore Ministry of Education (R-279-000-255-112), and Ministry of Defense (R-279-000-301-232) for financial support. We also thank Prof. Gagik G. Gurzadyan for PL decay lifetime measurements. ’ REFERENCES (1) Wilcoxon, J. P.; Abrams, B. L. Chem. Soc. Rev. 2006, 35, 1162–1194. (2) Zheng, J.; Nicovich, P. R.; Dickson, R. M. Annu. Rev. Phys. Chem. 2007, 58, 409–431. (3) Wilcoxon, J. P.; Martin, J. E.; Parsapour, F.; Wiedenman, B.; Kelley, D. F. J. Chem. Phys. 1998, 108, 9137–9143. (4) Peyser, L. A.; Vinson, A. E.; Bartko, A. P.; Dickson, R. M. Science 2001, 291, 103–106. (5) Vosch, T.; Antoku, Y.; Hsiang, J. C.; Richards, C. I.; Gonzalez, J. I.; Dickson, R. M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12616–12621. (6) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (7) He, Y.; Su, Y. Y.; Yang, X. B.; Kang, Z. H.; Xu, T. T.; Zhang, R. Q.; Fan, C. H.; Lee, S. T. J. Am. Chem. Soc. 2009, 131, 4434–4438. (8) Yu, J. H.; Choi, S.; Dickson, R. M. Angew. Chem., Int. Ed. 2009, 48, 318–320. (9) Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Angew. Chem., Int. Ed. 2007, 46, 6824–6828. (10) Wei, H.; Wang, Z. D.; Yang, L. M.; Tian, S. L.; Hou, C. J.; Lu, Y. Analyst 2010, 135, 1406–1410. (11) Huang, C. C.; Chiang, C. K.; Lin, Z. H.; Lee, K. H.; Chang, H. T. Anal. Chem. 2008, 80, 1497–1504. (12) Guo, W. W.; Yuan, J. P.; Wang, E. K. Chem. Commun. 2009, 45, 3395–3397. (13) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008, 4, 168–175. (14) F€orster, T. In Modern Quantum Chemistry; Sinanoglou, O., Ed.; Academic Press: New York, 1965. 13074

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(15) Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2002, 124, 15208–15216. (16) Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2003, 21, 1387–1395. (17) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (18) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer, LLC: New York, 2006. (19) Pouton, C. W.; Wagstaff, K. M.; Roth, D. M.; Moseley, G. W.; Jans, D. A. Adv. Drug Deliv. Rev. 2007, 59, 698–717. (20) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339–1386. (21) Duarte, A.; Pu, K. Y.; Liu, B.; Bazan, G. C. Chem. Mater. 2011, 23, 501–515. (22) Pu, K. Y.; Liu, B. Adv. Funct. Mater. 2009, 19, 1371–1378. (23) Duan, X. R.; Liu, L. B.; Feng, X. L.; Wang, S. Adv. Mater. 2010, 22, 1602–1606. (24) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954–10957. (25) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168–178. (26) Pu, K. Y.; Liu, B. Biosens. Bioelectron. 2009, 24, 1067–1073. (27) Jiang, G. X.; Susha, A. S.; Lutich, A. A.; Stefani, F. D.; Feldmann, J.; Rogach, A. L. ACS Nano 2009, 3, 4127–4131. (28) Lutich, A. A.; Jiang, G. X.; Susha, A. S.; Rogach, A. L.; Stefani, F. D.; Feldmann, J. Nano Lett. 2009, 9, 2636–2640. (29) Pu, K. Y.; Li, K.; Liu, B. Adv. Mater. 2010, 22, 643–646. (30) Pu, K. Y.; Li, K.; Zhang, X. H.; Liu, B. Adv. Mater. 2010, 22, 4186–4189. (31) Wang, Q.; Kim, D.; Dionysiou, D. D.; Sorial, G. A.; Timberlake, D. Environ. Pollut. 2004, 131, 323–336. (32) Nolan, E.M .; Lippard, S. J. Chem. Rev. 2008, 108, 3443–3480. (33) Fang, Z.; Pu, K. Y.; Liu, B. Macromolecules 2008, 41, 8380–8387. (34) Xie, J. P.; Zheng, Y. G.; Ying, J. Y. Chem. Commun. 2010, 46, 961–963. (35) Xie, J. P.; Zheng, Y. G.; Ying, J. Y. J. Am. Chem. Soc. 2009, 131, 888–889.

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