www.sciencemag.org/content/353/6299/571/suppl/DC1

Supplementary Materials for

Permanent excimer superstructures by supramolecular networking of metal quantum clusters

Beatriz Santiago-Gonzalez,* Angelo Monguzzi,*† Jon Mikel Azpiroz, Mirko Prato, Silvia Erratico, Marcello Campione, Roberto Lorenzi, Jacopo Pedrini, Carlo

Santambrogio, Yvan Torrente, Filippo De Angelis, Francesco Meinardi,† Sergio Brovelli†

*These authors contributed equally to this work.

†Corresponding author. Email: angelo.monguzzi@mater.unimib.it (A.M.); meinardi@mater.unimib.it (F.M.); sergio.brovelli@unimib.it (S.B.)

Published 5 August 2016, Science 353, 571 (2016)

DOI: 10.1126/science.aaf4924

This PDF file includes:

Materials and Methods Figs. S1 to S11 Tables S1 and S2 Full Reference List

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1. Materials and Methods

Synthesis of Gold Clusters. Au8 clusters were synthesized following the etching approach. (18) First, small gold nanoparticles (AuNPs) were obtained by adding 500 µL of 1M NaOH (Sigma Aldrich, ≥98% pellets, anhydrous) to 45 mL of ultrapure water (Chromasolv plus, for HPLC) followed by 1 mL of Tetrakis(hydroxymethyl)phosphonium chloride (THCP) solution (Sigma Aldrich, 80% in water), which has been prepared by mixing 12 µL THCP to 1 mL of water. The mixture was stirred for 5 min, followed by the addition of 1.5 mL of 0.03 M HAuCl4·3H2O (Sigma Aldrich, 99.999% trace metal bases). The brown color of the obtained solution indicates the reduction of Au+3 to Au0 and the formation of 2-3 nm gold particles (30). Synthesis of encapsulated Au clusters. By combining the etching approach and the Brust-Schiffrin method, (31) the monodispersed Au clusters were prepared by adding 10 mL of tetraoctylammonium bromide ([CH3(CH2)7]4NBr, Sigma Aldrich 98%) 0.1 M in toluene (Sigma Aldrich, 99.8%, anhydrous) to 20 mL of the as-synthesized AuNPs. The biphasic mixture was vigorously stirred. After 2 hours, aqueous phase became colorless indicating that phase transfer was completed and then the aqueous phase was removed. Next, 218 mg of 11-mercaptoundecanoic acid (HS(CH2)10CO2H, MUA, Sigma Aldrich, 95%) were added into the solution and stirred for 24 hours. The resulting solution was centrifuged at 6000 rpm for 20 minutes in order to collect and remove the residual Au nanoparticles. Then, the toluene phase was washed by adding 20 mL of ultrapure water and shaking the two immiscible solutions in order to remove any water-soluble impurities. This step was repeated three times. Synthesis of Au clusters superstructures (Au-pXs). The synthesis of thiol protected Au clusters was adapted from Huang’s protocol, by etching the previously obtained AuNPs.( 18)5 mL of HPCE buffer (100 mM sodium phosphate, pH 7, Sigma Aldrich) were injected to a 25 mL of the as-synthesized AuNPs (previously stored at 4°C), followed by the addition of 5 mL of 0.1 M MUA containing an equivalent amount of NaOH. The mixture was protected from the light and left to react for 72 h at room temperature. The resulting pale yellow solutions were purified through centrifugation at 3500 g fo r 30 min to remove excess thiols, and filtrated through a syringe membrane filter Whatman (0.22 μm pore size) to remove any remaining particle aggregates from solution. The obtained solution was concentrated and finely collected by a 10 kDa membrane MWCO Centrifuge Filter (Amicon) through ultracentrifugation at 25000 g for 40 min.

Mass Spectrometry Electrospray ionization mass spectrometry (ESI-MS) experiments in positive-ion mode were performed on a hybrid quadrupole/time-of-flight (qTOF) instrument equipped with a nanoelectrospray ion source (AB Sciex, ForsterCity, CA, USA). The samples were infused by borosilicate-coated capillaries of 1 μm internal diameter (Thermo Fisher Scientific, Waltham, MA USA). The main instrumental parameters were: ion-spray voltage 1.1 kV; curtain gas 20 PSI; declustering potential 80 V. The recorded spectra were averaged over 1 minute acquisition time. The simulation of the isotopic distribution was performed by the software IsoPro 3.1 (Windows).

Spectroscopy Photoluminescence studies. Photoluminescence (PL) and photoluminescence excitation (PLE) measurements were performed at room temperature using a Varian Eclipse spectrometer with a Xenon lamp as a continuous wave light source. Absorption spectra were collected with a Cary Varian 50 spectrophotometer at normal incidence in1 mm quartz cuvettes (bandpass 1 nm). Time-resolved photoluminescence profiles were recorded with an Edinburg Instruments FLS 980 spectrometer using a 3.65 eV EP-LED as excitation source (pulse width 900 ps). To measure the PL quantum yield (QY), a solution of perylene in THF was used as fluorescence standard (32, 33). Photoluminescence signals were recorded by a nitrogen cooled CCD camera coupled to a

3

spectrograph with spectral resolution of 0.5 nm. For single particle PL measurements, a 10-4M solution of Au-pXs in carboxymethyl cellulose was deposited on a glass slide and mounted on the Ti-U inverted microscope described below. All spectra were corrected for the instrumental response. FT-IR Spectroscopy. Absorption spectra in the range 400−4000 cm−1 were collected in reflection configuration with a micro-FTIR Nicolet iN10 under nitrogen flux, with spectral resolution of 4 cm−1. X-ray Photoelectron Spectroscopy. XPS measurements were performed using a Kratos Axis UltraDLD spectrometer with monochromatic Al K source operated at 15 kV and 10 mA. The specimen for XPS was prepared by drop casting 200 l of a clean and concentrated solution onto a silicon wafer. All the analyses were performed over an area of 300 x 700 microns. High-resolution analyses were carried out with a pass energy of 10 eV. In order to minimize the damaging of the organic ligands, fast spectra were collected on several areas of the specimen (acquisition time no longer than 8 minutes on each area) and successively averaged. The Kratos charge neutralizer system was used during data acquisition. Spectra have been charge corrected to the main line of the carbon 1s spectrum set to 284.8 eV (C-C bond). Spectra were analysed using Casa XPS software (version 2.3.16).

Microscopy TEM measurements were carried out using a Jeol JEM 1220 operating at 120 kV. Samples were prepared by dropping the Au-pX solution on formvar/carbon-coated copper-grids. Fluorescence micrographs were collected using a Canon EOS 400D camera coupled to a Nikon Ti-U inverted microscope. Samples were excited with a Xenon lamp whose emission was spectrally filtered with a DAPI excitation filter (320-400 nm). Blue, green and red channels were collected separately using standard DAPI, FITC and TRITC emission filters, respectively. For confocal imaging, a Nikon C1 confocal head has been mounted on the same Nikon Ti-U inverted microscope. The excitation wavelength was 390 nm, obtained via duplication of a 780 nm Coherent Mira 900 Ti:Sapphire laser.

DFT and TD-DFT Calculations All ground state geometries were optimized using the B3LYP(34) functional, in conjunction with the LANL2DZ (Au) and 6-31G* (H, C, O, S) basis sets, as implemented in the Gaussian09 program package. To perform excited state TDDFT calculations, we switched to the CAM-B3LYP(35) functional, which was shown to better describe the optical features of bare and ligand-protected gold clusters.(36) In particular, this kind of long-range corrected functionals are able to properly describe the charge transfer excitations, which might contribute to the absorption spectrum of the title compound. In all cases, the effect of water solvation was included by means of the implicit CPCM scheme.(37) To estimate the effect of entropic contributions to the calculated energetics we resort to a simple statistical mechanics model in which the translational and rotational entropy of each species is calculated assuming a particle in a box and rigid rotator behavior, respectively. Vibrational contributions to the entropy are neglected, since these are expected to represent only a minor contribution to the entropy variation for a bimolecular reaction. To simulate the spectrally resolved oscillator strength we employed a Gaussian convolution of calculated transition energies and oscillator strengths with a broadening (FWHM) of 0.3 eV. Cell Culture, Staining Protocols and Viability Tests. NIH/3T3 cells (ATCC® CRL-1658™) were thawed and plated on cell culture dish in DMEM high glucose (Gibco) supplemented with 10% FBS (Euroclone) for 48 hours before use. For immunofluorescence experiments, 3T3 cells were plated in a 12 multi-well plate at a density of 5x104 cells/well; in each well a round cover glass slide was added in order to growth the cells on its surface and then visualise them by confocal microscopy. Before seeding, cells were stained with PKH Red Kit (PKH26GL, Sigma), following the producer protocol. Briefly, cells were centrifuged

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twice in serum free medium (400g, 5 minutes) and then stained for 5 minutes with Dye Solution, as reported in datasheet. Cells were then rinsed twice in complete medium and seeded at the right density after checking fluorescence. After 24 hours the nanoparticles were added to the cells at the concentration indicated and, at 24, 48 and 72 hours, DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride, Sigma) staining was performed in order to visualize cell nuclei. Cover glass were then mounted with a 1:1 v/v glycerol-PBS solution. MTT Test.For proliferation experiments, cells were seeded in a 96 multiwell at a density of 3x103

cells/well in triplicate; after 24 hours nanoparticles were added to the cell medium and at 24, 48 and 72 hours an MTT test was performed (Methylthiazolyldiphenyl-tetrazolium bromide, Sigma). Briefly: a 50μg/ml MTT solution was added to the samples; after 4 hours of incubation at 37°C the medium was removed, the converted dye solubilised with DMSO (dimethylsulfoxide, Sigma) and the absorbance measured at 560nm (GloMax Discover, Promega). Cells in growth medium were used as control condition. ROS Test.For the evaluation of the Reactive Oxygen Species (ROS) possibly produced in culture, cells were seeded as for the MTT test; the analysis were performed 24, 48 and 72 hours after the addition on nanoparticles in culture medium; a ROS-Glo™ H2O2 Assay (Promega) was used, following the producer protocol. Samples treated with H2O2 5mM were considered as positive control, while samples of only growth medium were reported as negative control. The Non-Lytic assay was applied and the relative luminescence units were measured by a plate reader (GloMax Discover, Promega).

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2. Experimental confirmation of the absence of energy-transfer effects

To unambiguously exclude energy transfer between sub-populations of clusters in the ensemble as a possible origin of the Stokes-shifted emission, we conducted PL experiments exciting the Au-pX sample with intense laser light at 2.62 eV, which, based on the Stokes shift observed for encapsulated clusters, would approximately match to the absorption energy of isolated Au clusters emitting at 2.36 eV. No PL was detected despite the even accumulation times and laser power was increased up to 1000 times those used for UV-excitation.

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3. TEM image of Au nanoparticles used to synthesized the investigated Au clusters

Figure S1.TEM image of AuNPs (average diameters measured on 100 particles d=2.2±0.5nm).

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4. ESI-MS analysis of encapsulated clusters

Figure S2. ESI-MS spectrum of encapsulated clusters. (a)Positive mode ESI-MS spectra of encapsulated clusters in toluene. The peaks are ascribed to encapsulated Au8 clusters (2*, 3*, 5* 6*) and to capping ligand adducts (1, 4, 7, 8, 9, 10 and 11). The details on the peaks assignment are reported in Table S1. (b) Enlargement of peaks 2*, 3*, 5* and 6* ascribed to encapsulated Au8 clusters. The inset shows the perfect agreement between the experimental and simulated isotopic patterns ofpeak 2* corresponding to[Au8(MUA)4(TOA)5H]2+.

a

8

ESI-MS analysis of encapsulated clusters is reported in Fig.S2a, showing a series of peaks for m/z ranging between 500 and 4000, the enlargement of the 200-3100 region is shown in Fig.S2b. The signals highlighted in Fig.S2b, corresponding to doubly-charged ions as indicated by the isotopic splitting of 0.5, are ascribed to protonated clusters composed of eight Au atoms capped by 4-5 MUA ligands. Specifically, the m/z peaks are assigned to [Au8(MUA)4(TOA)5H]2+ (peak 2*, m/z = 2380.4), [Au8(MUA)5(TOA)6H]2+ (peak 3*, m/z = 2722.2), [Au8(MUA)4(TOA)7Br2H]2+ (peak 5*, m/z = 2927.3), and [Au8(MUA)5(TOA)7BrH]2+(peak 6*, m/z=2995.8). The remaining two peaks in Fig.S2 correspond to adducts of MUA and TOABr ligands, respectively [(TOA)4Br3]

+

(peak 1, m/z=2106.9) and [(MUA)(TOA)5Br3]+ (peak 4, m/z=2790.4). The inset of Fig.S2b reports

the enlargement of peak 2*, highlighting the isotopic pattern of the [Au8(MUA)4(TOA)5H]2+ mass distribution. Remarkably, the experimental data match perfectly the corresponding simulated pattern calculated considering the isotopic distribution of the constituent atomic species (theoretical average m/z= 2380.2), thus confirming the formation of Au8 clusters capped by four MUA units. We highlight that such agreement between experimental data and theoretical predictions is found for all ESI-MS peaks as reported in Table S1.

Table S1. ESI-MS analysis data of encapsulated clusters. m/z-values and chemical formula of the Auclusters and the adduct species extracted from the mass spectrum of the encapsulated clusters reported in Fig. S2.

Peak Chemical species Theoretical (m/z) Experimental (m/z)

1 [(TOA)4Br3]+ 2107.3 2106.9

2* [Au8(MUA)4(TOA)5H]2+ 2380.2 2380.4 3* [Au8(MUA)5(TOA)6H]2+ 2721.8 2722.2 4 [(MUA)(TOA)5Br3]

+ 2790.5 2790.4 5* [Au8(MUA)4(TOA)7Br2H]2+ 2927.0 2927.3 6* [Au8(MUA)5(TOA)7BrH]2+ 2995.2 2995.8 7 [(TOA)2Br]+ 1013.7 1013.8 8 [(MUA)(TOA)2]

+ 1150.1 1150.1 9 [(TOA)3Br2]

+ 1560.5 1560.4 10 [(TOA)5Br4]

+ 2654.1 2654.1 11 [(TOA)6Br5]

+ 3200.8 3200.7

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5. XPS Spectroscopy of Au-pXs.

Fig.S3 shows the XPS results after averaging over the spectra collected on 7 different areas of the specimen and subtraction of a Shirley-type background. The Au-4f spectrum (Fig.S3a) resembles the typical doublet profile of metallic gold with main component at 84.4 eV instead, slightly shifted with respect to the binding energy of bulk gold (84.0 eV) (38).The S-2p experimental profile is symptomatic of multiple sulphur chemical states in the sample (Fig.S3b). From a quantitative point of view, it is possible to estimate the Au:S atomic ratio in the specimen by calculating the ratio between the areas under the Au-4f and S-2p profiles, after normalization to the relative sensitivity factors (RSF), which depend on the cross sections for photoemission from the Au 4f and S 2p levels. For reference RSF (Au-4f) = 6.25 while RSF(S-2p) = 0.668. Such procedure yields a gold-to-sulphur ratio Au:S=52:48. However, the S-2p profile could be decomposed in several components due to different chemical states of sulphur in the sample. Due to spin-orbit coupling, each state is represented by a doublet, in which the intensity ratio between the S-2p3/2 (the most intense) and the S-2p1/2 components is fixed to 2:1, the spin-orbit splitting is set to 1.2 eV, and the FWHM of the two peaks is set to be the same. From now on, each chemical state of S will be identified by the binding energy position of its S-2p3/2 component. To simplify the fitting procedure, the same FWHM was used for all the components. The result of the deconvolution procedure is reported in Fig. S3b. The best fit was obtained with 5 components: S1 centered at 161.3 ± 0.2 eV, S2 centered at 162.5 ± 0.2 eV, S3 centered at 163.7 ± 0.2 eV, S4 centered at 166.6 ± 0.2 eV and S5 centered at 168.5 ± 0.2 eV. The observed components could be assigned to specific chemical states of S by comparison with literature.(39) Indeed, we could assign our S2 component to the thiolate compound formed after the reaction of the -SH molecular termination with the Au atoms of the clusters. Our S3 component is at a position compatible with that already observed for unbound thiols. Regarding the S1 component, it could be assigned to molecules damaged by low-energy photoelectrons.(39) This hypothesis is supported by the fact that, running a longer acquisition (approx. 1 hour long vs. the 8 minutes used to obtain the data reported in figure) resulted in a S 2p profile with a really intense S1 component. The minor S4 and S5 components could be assigned to oxidized forms of S, since they are in the binding energy range typical for sulphones, sulphites and sulphates. (40) In Table 2, the results on the Sulphur are summarized, including also the quantitative analysis.

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Figure S3. XPS data analysis for Au Au-pXs. (a) Gold (Au 4f) XPS spectrum showing the typical doublet profile of metallic gold. (b) Sulphur (S 2p) experimental XPS profile. The spectrum is symptomatic of multiple sulphur chemical states in the sample, and it can be decomposed in five components that are reported in Table S2.

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Table S2. Deconvolution of the S 2p XPS signal in Au-pXs.The experimental spectrum can be deconvolved in five components, each one corresponding to a specific chemical state.

BE position (eV) Atomic % Chemical species S1 161.3 ± 0.2 22.9 Molecule cleavage by low energy photoelectrons S2 162.5 ± 0.2 43.2 -S-Au S3 163.7 ± 0.2 22.1 -SH S4 166.6 ± 0.2 5 -SO3 S5 168.5 ± 0.2 6.8 -SO4

Now, considering the Au:S ratio, we could calculate the actual ratio between Au atoms and bound thiols. It is approx. 52:21, meaning that for each Au cluster of 8 Au atoms, we have 3.2 bound thiols. If we assume that the whole S1 component was bound to the Au clusters before irradiation, the Au: bound ratio would change to 52: 32, i.e. 4.9 bound thiols for each Au cluster. On average we could say we have 4.05 (±0.85) bound S per cluster.

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6. Synthesis of dendrimer-templated Au8 clusters.

Gold clusters in dendrimers were obtained by reducing HAuCl4.5H2O (1.5 µmol) with NaBH4 (1 equivalent 0.005M, slowly added) in the presence of PAMAM-OH dendrimer generation 4th (0.5 µmol) in 2 ml of ultrapure water. This mixture was stirred for two days at room temperature. Solutions were then centrifuged to remove large gold nanoparticles. Fig. S4 show the PL and PL excitation (PLE) spectra of the synthesized material (Fig. S4a), together with the PL decay traces collected at the emission maximum (Fig. S4b). The PL quantum yield is 24%, with an observed PL decay time of 3.5ns. Therefore, the radiative decay time for the fluorescence of dendrimer-templated Au8 clusters is /Φ 14.6 ns.

Figure S4. Optical properties of dendrimer templated Au8 quantum clusters. (a) Steady-state PL (solid line) spectrum under 3.25 eV excitation and PLE spectrum (dotted line) recorded by monitoring the PL intensity at 2.75 eV. The inset is digital picture of the sample irradiated with a UV lamp. (b) The PL decay has been recorded at 2.35 eV under pulsed excitation at 3.65 eV.

7.

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13

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14

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15

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16

10. Stability of different bare Au8 clusters in water

Figure S8. Relative stabilities of different Au8 isomers in water solution (kcal/mol) calculated at the PBE, TPSS, and B3LYP levels, in conjunction with the LANL2DZ basis set.

PBE TPSS

B3LYP

D4h D2d C2v Td

0.00 0.00 0.00

16.90 13.07 25.18

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11.

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18

12. Modeling the C2- and the real C11-alkyl chain systems.

To investigate the possible formation of excimers, we consider two interacting [Au8(MUA)4] monomers, both including C11 alkyl chains, corresponding to the real length of MUA moieties, and considering shorter C2 chain. The C11-dimer optimized geometry (A in Fig.S10) delivers a distance of ca. 30 Å between the Au8 cores, which precludes electronic communication between connected clusters and can thus not account for the large Stokes shift observed experimentally. We thus considered a second configuration in which the C11-MUA ligands are twisted, maintaining the formation of carboxylic acid dimers, to allow the Au8 cores to be close enough to interact. This new configuration (B in Fig.S10), however, lies 2.2 eV higher in energy than the previous one, due to the strain of the carbon chains, thereby ruling out its formation as responsible for the observed photophysical behavior.

Figure S10. Interacting geometries of the Au8 dimers, linked by 11 C long ligands, at their extended (A) and twisted (B) geometries. Yellow (orange) = Au, white = H, brown = C, red = O, and green = S atoms.

A" B"

19

13. ROS Control Test.

As a control experiment to verify the ROS scavenging effect of Au-pXs, the ROS test has been performed on an aqueous solution saturated with H2O2. Despite the high concentration of the hydrogen peroxide, the ROS level in presence of the 1:50 diluted Au-pXs solution increases of only ~50% after 72h, while in the control sample without Au-pXs the level is almost doubled, with an increment of ~100%, as showed in Fig.S11.

Figure S11. Radical oxygen scavenging using Au-pXs. In aqueous solution of H2O2 the ROS level is progressively buffered by the presence of an increasing amount of Au-pXs (from 1:100 to 1:50 dilution of the mother solution 740 M).

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