Chiral Thiol-Stabilized Silver Nanoclusters with Well-Resolved Optical

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Chiral Thiol-Stabilized Silver Nanoclusters with Well-Resolved Optical Transitions Synthesized by a Facile Etching Procedure in Aqueous Solutions Nicole Cathcart,† Pretesh Mistry,† Christy Makra,† Brendan Pietrobon,† Neil Coombs,‡ Masoud Jelokhani-Niaraki,† and Vladimir Kitaev*,† †

Chemistry Department, Wilfrid Laurier University, 75 University Ave. W, Waterloo, Ontario, Canada N2L 3C5, and ‡Centre for Nanostructure Imaging, Chemistry Department, University of Toronto, 80 St George Street, Toronto, Ontario, Canada M5S 3H6 Received September 26, 2008. Revised Manuscript Received March 17, 2009

A novel approach of cyclic reduction in oxidative conditions has been developed to prepare a single dominant species of chiral thiol-stabilized silver nanoclusters (AgNCs). Such AgNCs, which are stable in solution for up to a few days, have been obtained for the first time. The generality of the established procedure is proven by using several enantiomeric water-soluble thiols, including glutathione, as protective ligands. The prepared AgNCs featured prominent optical properties including a single pattern of UV-vis absorption with well-resolved peaks. The chirality of the clusters has been investigated by circular dichroism (CD) spectroscopy. CD spectra displayed strong characteristic signatures in the visible range. Tentative identification of the cluster composition is discussed.

Introduction Noble metal clusters (NMCs) have received increasing attention in recent years. The scientific interest in these clusters originates from their unique properties1 that bridge the gap between those of molecular and nanoparticle systems.2,3 The main distinctive features of NMCs compared to relatively more investigated nanoparticles are their smaller sizes (typically less than 2 nm) and lack of characteristic plasmonic properties due to a limited number of delocalized electrons.2 NMCs typically display atomic-like electronic transitions and are often referred to as “superatoms”.4,5 One of the established routes to prepare NMCs in solution is to use ligand stabilization with strongly binding thiols and phosphines.6 A recent breakthrough in the total structure determination of Au102(SR)44 significantly advanced understanding of the ligand-stabilized clusters and invigorated further research.7 It has been demonstrated in several studies that thiol-protected NMCs consist of a central metal core and tightly coordinated metal thiolates surrounding the core.5,8 Experimentally, gold clusters are the most extensively studied NMCs due to their stability and relative ease of preparation.9 Mixed gold-silver clusters have been explored in several studies.10 Small silver clusters11 *Corresponding author. E-mail: [email protected]. (1) Castleman, A. W.Jr.; Keesee, R. G. Annu. Rev. Phys. Chem. 1986, 37, 520. (2) Wilcoxon, J. P.; Abrams, B. L. Chem. Soc. Rev. 2006, 35, 1162. (3) Risse, T.; Shaikhutdinov, S.; Nilius, N.; Sterrer, M.; Freund, H.-J. Acc. Chem. Res. 2008, 41, 949. (4) Akola, J.; Walter, M.; Whetten, R. L.; Hakkinen, H.; Gronbeck, H. J. Am. Chem. Soc. 2008, 130, 3757. (5) Walter, M.; Akola, J.; Acevedo, O. L.; Jadzinsky, P. D.; Calero, G.; Ackerson, C. J.; Whetten, R. L.; Gronbeck, H.; Hakkinen, H. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9157. (6) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000 33, 27. (7) Jadzinsky, P. D.; Calero, G.; Ackerson, C. J.; Bushnell, D. A.; Kornberg R. D. Science 2007, 318, 430. (8) Negishi, Y.; Chaki, N. K.; Shichibu, Y.; Whetten, R. L.; Tsukuda, T. J. Am. Chem. Soc. 2007, 129, 11322. (9) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293. (10) (a) Teo, B. K.; Keating, K. J. Am. Chem. Soc. 1984, 106, 2224. (b) Chaki, N. K.; Tsunoyama, H.; Negishi, Y.; Sakurai, H.; Tsukuda, T. J. Phys. Chem. C 2007, 111, 4885. (11) (a) Peyser, L. A.; Vinson, A. E.; Bartko, A. P.; Dickson, R. M. Science 2001, 291, 103. (b) Richards, C. I.; Choi, S.; Hsiang, J.-C.; Antoku, Y.; Vosch, T.; Bongiorno, A.; Tzeng, Y.-Li.; Dickson, R. M. J. Am. Chem. Soc. 2008, 130, 5038.

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(e10 atoms) have recently received significant attention due to their remarkable fluorescence.12 With regard to size-selected cluster preparation, significant advances have been made for gold through selective resistance to etching.13,14 At the same time, current progress in size-selected silver clusters is constrained due to the laborious size separation15 compounded by limited cluster stability. Recent notable studies of larger (than ca. 10 atoms) silver clusters have focused predominantly on circular dichroism (CD) properties.16,17 Indeed, nanoscale chirality is attracting an increasing interest due to important emerging applications of chiral clusters and nanoparticles in optics, catalysis, and sensing.18 Therefore, development of simple and reliable methods of chiral NMC preparation is essential to advance the fields of NMCs and nanoscale chirality. Herein we report on the synthesis of size-selected chiral silver clusters (AgNCs) that feature well-defined characteristic optical absorption and CD spectra in the visible range. The synthetic preparation is facile and employs cyclic reduction in oxidative conditions (CROC) in aqueous solutions or water-alcohol mixtures.

Methods Reagents. Silver nitrate (99%), (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl]pyrrolidine-2-carboxylic acid (captopril), glutathione reduced (98%), L-cysteine (97%), hydrogen peroxide (99.999%), citric acid (Aldrich 99%), sodium borohydride (99%), and potassium hydroxide pellets (99.99%), all supplied by Aldrich, were used as received. Anhydrous ethyl (12) Zheng, J.; Nicovich, P. R.; Dickson, R. M. Annu. Rev. Phys. Chem. 2007, 58, 409. (13) Boyen, H.-G.; Kastle, G.; Weigl, F.; Koslowski, B.; Dietrich, C.; Ziemann, P.; Spatz, J. P.; Riethmuller, S.; Hartmann, C.; Moller, M.; Schmid, G.; Garnier, M. G.; Oelhafen, P. Science 2002, 297, 1533. (14) Shichibu, Y.; Negishi, Y.; Tsunoyama, H.; Kanehara, M.; Teranishi, T.; Tsukuda, T. Small 2007, 3, 835. (15) Negishi, Y.; Takasugi, Y.; Sato, S.; Yao, H.; Kimura, K.; Tsukuda, T. J. Am. Chem. Soc. 2004, 126, 6518. (16) (a) Nishida, N.; Yao, H.; Kimura, K. Langmuir 2008, 24, 2759. (b) Nishida, N.; Yao, H.; Ueda, T.; Sasaki, A.; Kimura, K. Chem. Mater. 2007, 19, 2831. (17) Shemer, G.; Krichevski, O.; Markovich, G.; Molotsky, T.; Lubovitz, I.; Kotlyar, A. B. J. Am. Chem. Soc. 2006, 128, 11006. (18) Kitaev, V. J. Mater. Chem. 2008, 18, 4745.

Published on Web 4/9/2009

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Cathcart et al. alcohol was acquired from Commercial Alcohol Inc. Highpurity deionized water (>18.3 MΩ 3 cm) was produced by Millipore A10 Milli-Q. Formation of Clusters. The silver clusters were prepared using multistage cyclic reduction in oxidative conditions (CROC) in aqueous solutions. Mixtures of water with ethanol (up to 80 vol %) have been found to be suitable media as well. Reactions were performed in small glass vials (20 mL, VWR) with total synthesis volumes ranging from 2 to 18 mL. A typical first stage of cluster formation started with silver concentration of 0.83 mM, and captopril concentrations varied from 0.3 to 2 mM (0.60 to 0.65 mM was found to yield the best clusters). A typical citric acid concentration was 3.3 mM, and the reaction was brought to a desired pH using KOH before reduction with an excess of sodium borohydride (10 mM total concentration in solution). The onset of AgNC formation was accompanied by a color change from clear to vibrant deep yellow and then darkening to deep red over a time period of an hour. Hydrogen peroxide was subsequently added to the colored solution (total concentration of 20 mM or 24:1 molar ratio to silver), which was usually left overnight to nearly completely oxidize crude AgNCs that have been produced during the first reduction. In the subsequent stages of CROC, additional amounts of thiols and silver nitrate were added: 20-40% of the original amounts present in faded solution afforded the best results. The addition of captopril to the oxidized solution was typically followed with a slight cloudiness, indicating the formation of less soluble thiolates. The subsequent addition of silver nitrate cleared the cloudiness, resulting in a clear solution. Small amounts of sodium borohydride (0.8 mM total concentration in solution) were then used for the subsequent reduction. The reduction of the second stage solution produced a dark orange to brown solution with high absorbance and well-defined UV-vis spectra. The cycles were repeated until the maximum absorbance (normalized per silver concentration) and peak resolution were achieved. Typically, four or five cycles were sufficient for refining. Characterization. Electron microscopy was performed using Hitachi HD-2000 ultrahigh-resolution dedicated scanning transmission electron microscope (STEM), operating at 200 kV. AgNP dispersions (diluted to below 0.3 mM in silver concentration) were deposited on ultrathin carbon-coated (∼3 nm) grids (SPI). Samples were decontaminated at 60000 magnification by exposing them to the defocused electron beam (beam current of 15 μA). Direct imaging was performed at magnifications from 3000000 to 5000000. UV-vis spectra were acquired with either an Ocean Optics QE-65000 fiber-optic UV-vis spectrometer or Cary 50Bio UV-vis spectrophotometer. CD spectra were obtained using an AVIV model 215 circular dichroism spectrometer. Fluorescence measurements were carried out with a Cary Eclipse fluorescence spectrometer. Raman spectra were recorded using an R-3000QE fiber-optic Raman spectrometer equipped with a 290 mW laser at 785 nm (RSI). Waters Micromass ZQ 4000 was used for ESI mass spectroscopy with a cone voltage varied between 30 and 70 V, a capillary voltage between 3 and 4 kV, cone temperatures of 80-150 °C, and desolvation temperatures between 180 and 300 °C. Native polyacrylamide gel electrophoresis (PAGE) was carried out in a gel with concentrations of the acrylamide monomer varying from 20 to 32%, with 3% bis(acrylamide) for cross-linking, buffered at pH = 8.8 with tris(hydroxymethylamine) and HCl. The eluting buffer contained 25 mM tris (hydroxymethylamine) and 192 mM glycine. Samples were mixed with 5% glycerol to increase their density and then loaded into a 10-lane, 0.75 mm gel and eluted for 2.5 h at 100 V. Bromophenol blue was used as a loading marker as well a polypeptide SDS and Native PAGE markers. The Beckman Coulter Allegra 64R centrifuge was used for ultracentrifugation tests. Langmuir 2009, 25(10), 5840–5846

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Figure 1. UV-vis spectra of (a) representative chiral AgNCs prepared by CROC in aqueous solution using three different thiol ligands: 1, captopril; 2, glutathione; 3, cysteine. (b) Different stages of CROC preparation of AgNCs using captopril; stages 1-4 are labeled with the corresponding numbers, and oxidized stage 1 is labeled with 0. The insets show the photographs of the vials with AgNC solutions labeled with the numbers corresponding to the spectra.

Results and Discussion Synthesis. In the developed AgNC synthesis, a silver salt was reduced by a common reducing agent, sodium borohydride, in the presence of water-soluble thiols as strongly binding stabilizing ligands and hydrogen peroxide that served as an oxidizing agent. Citric acid was employed as a charge-stabilizing ligand. In the presence of stronger binding thiol ligands, the citrate may not play a primary role but still serves for charge costabilization19 and is important for fine-tuning pH of the synthesis. To attest to the general applicability and universality of CROC, we were able to prepare comparable AgNCs with well-resolved UV-vis (Figure 1a) and CD (Figure 2) signatures using several optically active thiols: captopril ((2S)-1-[(2S)-2-methyl3-sulfanylpropanoyl]pyrrolidine-2-carboxylic acid), glutathione (GSH = γ-Glu-Cys-Gly), and cysteine. Captopril and glutathione yielded the highest quality AgNCs. Most of the results discussed below are presented for captopril-protected AgNCs, while all experiments have been performed using all three thiol ligands. It is noteworthy that well-defined AgNCs could not be prepared by this method using nonchiral thiols, such as sodium 2-mercaptoethyl sulfonate, 3-mercaptopropionic acid, and 11-mercaptoundecanoic acid (see Figure S1 for UV-vis and Figure S2 for CD spectra of silver clusters prepared with these ligands in similar conditions of the synthesis). Experimental findings clearly suggest that successful cluster preparation requires either chirality or, perhaps, the presence of an amino group adjacent to thiol and carboxyl groups in the ligand molecule. (19) Henglein, A.; Giersig, M. J. Phys. Chem. B 1999, 103, 9533.

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Figure 2. Circular dichroism spectra of chiral AgNCs protected with three different enantiomeric thiol ligands: 1, captopril; 2, glutathione; 3, cysteine.

Development of the CROC procedure was based on a general strategy of oxidative etching, which was successfully applied in the synthesis of shape-selected silver nanoparticles by several research groups.20 The oxidation of the freshly reduced clusters was performed using hydrogen peroxide. The reduced AgNCs formed at the first stage displayed relatively featureless absorption spectra (Figure 1b, curve 1). These AgNCs oxidized within several hours to form nearly transparent solutions with limited absorption in the visible range (Figure 1b, curve 0). At this stage, a fresh portion of sodium borohydride was added, together with extra amounts of silver nitrate and thiols (usually 40 mol % of those present in the original solution). After this reduction cycle, newly formed AgNCs started to display a broad peak near ca. 480 nm in their UV-vis spectra (Figure 1b, curve 2). Allowing these AgNCs to oxidize, followed by several repeated reductions with borohydride, led to further refinement of clusters, as evidenced by their optical signature (Figure 1b, curves 3 and 4), which typically reached its optimum after the fourth or fifth reduction cycle. An optimum pH for this synthesis has been found to be in the range of near-neutral to weakly basic at 8-9 (Figure 3a). The pH was adjusted using sodium or potassium hydroxide, while citric acid, employed in the synthesis, served as a natural acidifying agent. Because of limited AgNC stability, which makes it difficult to produce nonaggregated samples of clusters in a precipitated form required for the elemental analysis, and limitations of mass spectroscopy resolution because of the presence of two silver isotopes (in contrast with a single isotope of gold), we had to rely on solution-based techniques to determine cluster composition. Since in its late stages CROC equilibrium in the formation of the most stable cluster species is reached, we have performed several series of experiments with variation of the silver-to-thiol ratio, rAg/S, and monitoring stability of the clusters. We argue that, by direct analogy with well-established methods for the determination of the composition of metal complexes and supramolecular compounds, this approach yields informative evidence on cluster composition due to a small cluster size of few tens of metal atoms, strongly binding ligands, and resulting well-defined cluster composition. The UV-vis peaks only changed in intensities, and not in their position, during the variation of rAg/S, so the yield of clusters could be quantitatively monitored. Further supporting arguments for reaching the equilibrium, (20) (a) Metraux, G. S.; Mirkin, C. A. Adv. Mater. 2005, 17, 412. (b) Wiley, B. J.; Sun, Y.; Xia, Y. Acc. Chem. Res. 2007, 40, 1067.

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Figure 3. (a) UV-vis spectra of captopril-protected AgNCs demonstrating effect of the synthesis pH: 1, 4.7; 2, 5.6; 3, 6.8; 4, 7.9; 5, 8.8; 6, 9.4. (b) Plot of the ratios of peak maxima at ca. 490 nm to the peak minima at ca. 430 nm (I480/I430 - 1) calculated from UV-vis spectra as a function of silver-to-thiol ratio in AgNC synthesis for several independent series of experiments.

necessary for such analysis, is that the later cycles of CROC yield the same species of AgNCs, as can be monitored by UV-vis and CD spectroscopy. The presence of a single prominently optically active cluster type is clear from only one unique optical signature during the last stages of CROC cycles with no other peaks emerging. Therefore, two different major scenarios can be considered for our cluster system: (i) In the case of the shortage of thiols (excess of silver), no free or partially protected silver core can survive since the sample is subjected to oxidative-reductive cycles to ensure the equilibrium of stable cluster formation is reached. Thus, the excess of silver is in an oxidized form, and the absorption intensity is reduced. (ii) In the case of the excess of thiols, some silver is readily converted to thiolates so the system consists of the stable cluster and thiolates (thiolates have featureless absorption with maxima in UV and only absorption tails in the visible), so the intensity of absorption peaks is lower as well. As a result, the maximum intensity of the cluster peaks is observed for the optimal thiol-to-silver ratio corresponding to its chemical composition. The broad optimal range of the silver-to-thiol ratio, rAg/S, for AgNC preparation varied from 1.2 to 1.45, depending on ligands and synthesis conditions. Figure 3b shows an assessment of the quality of captopril-stabilized AgNCs expressed as a ratio of maximum peak intensity at ca. 480-490 nm to the trough of this peak at ca. 430 nm. Having performed multiple series of experiments, we consistently observed that there was a significant change in the regime of peak intensity ratios at rAg/S of ca. 1.37-1.40, and some series had the best clusters prepared close to that range. (Typically, the maxima had a tendency to shift to a lower ratio since some thiol excess should be necessary for Langmuir 2009, 25(10), 5840–5846

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Figure 4. UV-vis spectra illustrating (a) AgNC stability. The sample was prepared using captopril as a ligand. No deaeration of solution was performed. Spectra were recorded every 2 h following the reduction. Half of the intensity of the peak at 490 nm was remaining after ca. 8 h. Peak vanished completely after ca. 24 h. (b) Slow formation of captopril-protected AgNCs in late cycles of CROC. From the lowest to the highest, the spectra were taken after 2, 10, 14, 22, 30, and 50 min, respectively, after addition of sodium borohydride to the system.

complete AgNC formation.) This critical value of rAg/S = 1.38 is close to 25/18 that, in combination with the maximum stability to etching of the cyclically oxidized clusters, suggests a close similarity with etching-resistant Au25(SR)18 as recently reported.8,14 AgNCs, produced using the method described above, demonstrated reasonable stability and remained intact for at least several hours in solution exposed to air. A characteristic aging pattern is shown in Figure 4a. Less than optimal synthesis conditions caused faster cluster deterioration; e.g., AgNCs could lose one-half of their optical activity in less than 30 min for rAg/S < 0.7. At the same time, several AgNC samples with the least excess of thiol (rAg/S in the range from 1.35 to 1.40) retained about half of the peak intensity at 480 nm for as long as 30 h. It should be pointed out that the most stable AgNCs in the late-stage cycles of CROC formed slowly upon reduction, reaching maximum peak intensity in ∼50 min after borohydride addition, as shown by UV-vis spectra (Figure 4b). Such slow forming clusters typically displayed the best stability and optical properties. Optical Properties. AgNCs prepared by the CROC procedure exhibited quite prominent optical properties: well-defined absorption peaks in UV-vis spectra (Figure 1a) and strong characteristic CD bands in the visible range (Figure 2). The UV-vis spectrum of captopril-protected AgNCs featured three prominent peaks: the strongest and best-resolved at 490 nm (2.53 eV) with fwhm less than 40 nm (∼0.1 eV), shallow peak at 660 nm (1.87 eV), and peak in UV at 335 nm (3.70 eV). These peaks were slightly different and less resolved for glutathioneprotected AgNCs (480, 635, and 325 nm, respectively), while Langmuir 2009, 25(10), 5840–5846

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only one peak could be clearly observed in the case of cysteine (488 nm). Out of all gold clusters reported in the literature,21 the UV-vis spectra of AgNCs most closely resemble UV-vis spectra of size-selected glutathione-stabilized Au25(SG)18 with three major peaks at ca. 1.8, 2.7, and 3.8 eV. The recent theoretical modeling of the Au25(SH)18, Ag25(SH)18, and their mixed clusters demonstrated reasonably close similarity of the spectra for gold and silver. The electronic transitions of the clusters can be understood and modeled in terms of their superatomic structure4 with the same number of electrons (eight) retained on an icosahedral core of 13 atoms for both silver and gold.22 Furthermore, it was clearly demonstrated that the thiolate shell has an important effect on the optical signature of the resulting clusters since the core spectra alone are noticeably different.22 The single most important characteristic of CROC synthesis of AgNCs is that only one type of optical signature has been observed for all the ligands used. Even significant variations in reagent concentrations and reaction conditions of the synthesis did not yield any noticeable variations from the characteristic pattern shown in Figures 1 and 3a. The observed peaks only decreased in intensity without appearance of any other peaks or peak shifts by more than 5 nm. These findings strongly suggest that a single characteristic species of AgNCs is formed. The ratio between the peak intensities in UV-vis spectra always remained the same, further supporting a single type of AgNCs. It can be noted that the peak at ca. 650-660 nm was most sensitive to reaction conditions (solvent, pH, etc.) in its wavelength shift, as it corresponds to the lowest in energy HOMO-LUMO transition in clusters.22 The circular dichroism (CD) spectra clearly demonstrated that the prepared AgNCs were chiral based on their characteristic CD signals, which closely corresponded to UV-vis absorption peaks at ca. 650, 490, and 430 nm (Figure 2). The CD spectra had several negative and positive maxima across the entire measurement range, which was limited by the signal-tonoise sensitivity of the instrument at wavelengths larger than 675 nm. Cysteine-protected AgNCs displayed the most complex signature, likely due to the fact that these clusters are not single species. A comparison of glutathione and captopril AgNC spectra in Figure 2 shows that the maximum ellipticity in the glutathione CD spectrum in the 500 nm range is red-shifted relatively to that of captopril system, while, similar to the UV-vis spectra, maximum ellipticity for glutathione in the 400 nm range is blue-shifted. Captopril and cysteine AgNCs displayed a well-defined split Cotton effect around the strongest transition peak at 490 nm, while glutathione AgNCs demonstrated a more complex behavior. A detailed investigation of chiral properties is currently in progress. All AgNCs showed only weak fluorescence due to the presence of the thiol ligands (Figure 5) with the quantum yield estimated at 5  10-4. All spectra featured the same excitation maxima at 440-445 nm (2.8 eV) and emission maxima at 625-630 nm (2.0 eV) for all clusters independent of ligands and AgNC concentrations as well as corresponding emission and excitation wavelength. The single characteristic pattern of the fluorescence spectra in combination with gel electrophoresis data (Figure 6) suggests that a single species of clusters with a similar electronic structure is prepared at least when glutathione and captopril serve as the ligands. The fluorescence pattern of (21) Wyrwas, R. B.; Alvarez, M. M.; Khoury, J. T.; Price, R. C.; Schaaff, T. G.; Whetten, R. L. Eur. Phys. J. D 2007, 43, 91. (22) Aikens, C. M. J. Phys. Chem. C 2008, 112, 19797.

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Figure 5. Characteristic excitation and emission spectra of AgNCs stabilized with both glutathione and captopril and measured at total silver concentration of 0.2-0.4 mM. Excitation spectra are measured for emission at 625 nm (upper and lower curve correspond to different captopril-stabilized clusters, rAg/S = 1.38 with silver concentration of 1 and 0.7 mM, respectively). Emission spectra were measured with excitation at 430 and 450 nm (lower curve corresponds to glutathione-stabilized clusters rAg/S = 1.38 with silver concentration of 0.5 mM and two upper curves correspond to captopril-stabilized clusters with rAg/S = 1.38 and rAg/S = 1.0 and silver concentration of 1 and 0.7 mM, respectively).

the reported AgNCs is close to that reported for Au23 stabilized by dendrimers12 with emission at ∼740 nm and glutathionestabilized14,15 Au25(SG)18. Raman spectra featured only strong background fluorescence (Figure S3), which is expected considering that excitation at 785 nm is in the close vicinity of electronic transitions in the clusters. This observation is consistent with the description of Raman spectra reported in the literature for benzenethiolate monolayer-protected AuNCs.23 Tentative AgNC Identification. Three main methods have been used for identification of AgNCs in conjunction with the optical characterization: native polyacrylamide gel electrophoresis (PAGE), high-resolution transmission electron microscopy (HR-TEM), and electrospray ionization (ESI) mass spectroscopy. At first, we tested all the samples of AgNCs by ultracentrifugation at 50000 RCF for 2 h. No sediment or color gradients were observed in contrast with many AgNCs prepared by us using different but related procedures, where larger silver nanoparticles were often present. The ultracentrifugation experiments strongly corroborated that the CROC procedure does not produce any silver nanoparticles larger than ca. 2 nm. Given the absence of larger AgNPs, the observed optical transitions are inherent to superatomic clusters22 and are clearly not related to plasmons, especially since plasmonic peaks at 480 nm would correspond to larger than 30 nm and/or anisotropic silver NPs.20,24 Furthermore, the wavelength of the absorption maxima in UV-vis varied by less than 5 nm in different water-ethanol mixtures, which is not characteristic of plasmonic transitions sensitive to the dielectric constant of the media.25 The fact that the absorption peaks of the AgNCs are insensitive to solvents indicates that the thiolate shell shields the inner metallic core well. Most crucially, several PAGE experiments performed at different gel concentrations (as high as 32%) clearly demonstrated a single band for the separation of the most stable glutathione and captopril clusters (Figure 6) in full accordance with well-resolved UV-vis and CD spectra, which strongly supports the single AgNC species present in these two systems. (23) Price, R. C.; Whetten, R. L. J. Phys. Chem. B 2006, 110, 22166. (24) Maillard, M.; Giorgio, S.; Pileni, M. P. J. Phys. Chem. B 2003, 107, 2466. (25) Templeton, A. C.; Pietron, J. J.; Murray, R. W.; Mulvaney, P. J. Phys. Chem. B 2000, 104, 564.

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Figure 6. PAGE separation results using the highest density of acrylamide gels (32 wt %) for AgNPs stabilized with (1) captopril, (2) glutathione, and (3) cysteine; blue spot (4) in (b) corresponds to bromophenol blue indicator.

Figure 7. HR-TEM images of captopril-protected AgNPs (a) before and (b) after decontamination by exposure to the beam at 200 kV for 10 min. The scale bar is 10 nm for (a) and 5 nm for (b).

At the same time, for cysteine-protected AgNCs, two major lines were observed for the batch shown in Figure 6, while for some batches several more minor bands could be observed, which correlates well with less defined UV-vis spectra (Figure 1a). In other words, we have not been able to prepare a single species in the case of the cysteine-protected AgNCs but clearly succeeded in doing so for clusters stabilized with glutathione and captopril. As can be expected due to large size of glutathione Langmuir 2009, 25(10), 5840–5846

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Figure 8. (a) Negative-ion ESI mass spectra of captopril-stabilized AgNCs with peak assignment. (b) Deconvoluted spectrum based on the data shown in (a). The displayed peak closely corresponds to the cluster composition of Ag24(Capt)16, one Ag(Capt)2 less than tentatively proposed Ag25(Capt)18. “Capt” stands for captopril.

molecules, the glutathione-protected clusters were the slowest migrating (Figure 6). We should also point out that in all PAGE experiments AgNCs migrated noticeably faster than the lowest molecular weight negatively charged standard available (1.5 kDa). Furthermore, smaller cysteine- and captoprilprotected AgNCs migrated even faster than dye molecules, e.g., bromophenol blue, as shown in Figure 6b. Very fast migration, even at the highest gel loadings of 32 wt %, confirms small sizes of AgNCs, and their highly charged state at pH = 8.8 that was used in PAGE experiments. Direct HR-TEM imaging of AgNCs corroborated that the clusters are less than or around 1 nm (Figure 7a). It should be mentioned that it was necessary to use dilute solutions to avoid aggregation of clusters upon drying. Only a small amount of aggregates was observed under carefully chosen preparation conditions. A noteworthy finding was that after exposure of the samples to the beam at 200 kV for 10 min, used for decontamination, an increased fraction of smaller ca. 0.7 nm clusters could be detected (Figure 7b) compared to ca. 1 nm AgNCs largely observed for less-decontaminated areas of the sample (Figure 7a). It is possible that we have witnessed partial removal Langmuir 2009, 25(10), 5840–5846

of the thiolate shells and largely imaged the central core of the clusters, which is also consistent with the thiolate defragmentation observed by mass spectroscopy. It is important to emphasize that, in contrast with AuNCs where ESI mass spectroscopy is a powerful tool to identify the composition, there is a clear fundamental limitation of mass spectroscopy in the case of silver. The reason for this limitation is the natural presence of two silver isotopes that broadens the peaks significantly for clusters composed of more than a few silver atoms. This limitation is clearly evident in the body of literature reporting upon small (a few atoms) silver clusters.12,26 As a result, we were not able to observe characteristic patterns of intact AgNCs (as routinely observed for AuNCs). At the same time, for most of the AgNC samples, we could detect MS spectra corresponding to diverse silver thiolate fragments, especially at higher desolvation and source temperatures (Figure 8a). Deconvolution of the fragmentation patterns15 (Figure 8b) yielded the assignment of the cluster composition as with 24 silver atoms (26) Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M. J. Am. Chem. Soc. 2004, 126, 5207.

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and 16 captopril ligands, which may not be overly relied upon, given the clear limitations in detecting larger fragments of clusters due to the isotope broadening of silver. Several other deconvolution spectra for different experiments, shown in Figure S4, are at the very least consistent with the clusters composed of 22-28 silver atoms. Taking into account all the experimental evidence, (i) single AgNC species resistant to etching, (ii) single species of clusters evident from PAGE, (iii) optimal silver-to-thiol ratio of the equilibrium cluster formation close to 1.38, (iv) fluorescence emission pattern and characteristic Stokes shift, (v) mass spectroscopy evidence, and (vi) cluster size of 0.9-1 nm directly observed by HR-TEM, we propose that the size-selected AgNCs produced are comprised of 22-28 silver atoms (most likely 25 silver atoms, based on strong parallels in size selection and stability with the Au25(SR)18).22 Given the limitations of the MS analysis for AgNCs (in contrast with AuNCs, where it serves as a primary identification method), it is quite challenging to produce more certain assignment for this system.

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Future work will explore more precise identification of AgNC composition and, ultimately, its structure based on high-resolution electron microscopy. At the moment, research efforts are directed toward further refinement of the AgNC preparation and systematization of the findings on the chiral behavior of the system. Acknowledgment. The authors gratefully acknowledge financial support from Natural Science and Engineering Research Council of Canada, Canada Foundation for Innovation, Ontario ORF-RI, Research Corporation (Cottrell Award), Wilfrid Laurier University, and STEP. The authors thank Gazelle Crasto, Payal Patel, and Dr. Lillian DeBruin for assistance with gel electrophoresis and Dr. Ian Hamilton for insightful comments on the manuscript. Supporting Information Available: Additional UV-vis and CD spectra, mass spectroscopy deconvolution patterns, and Raman scattering data. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(10), 5840–5846