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Silver Nanoclusters: Single-Stage Scaleable Synthesis of Monodisperse Species and Their Chirooptical Properties† Nicole Cathcart and Vladimir Kitaev* Chemistry Department, Wilfrid Laurier UniVersity, 75 UniVersity AVenue W, Waterloo, Ontario, Canada, N2L 3C5 ReceiVed: February 26, 2010; ReVised Manuscript ReceiVed: June 2, 2010
Silver nanoclusters (AgNCs) with well-resolved electronic transitions in UV-vis spectra and chirooptical signatures have been synthesized via a versatile single-stage preparation utilizing stabilization with chiral thiols in aqueous solution. The most successful strategy proved to be the use of a combination of chiral ligands. No purification or size separation of synthesized AgNCs was required. AgNCs have been confirmed to be a single species and displayed significant stability in ambient conditions. Further improvement in AgNC stability has been achieved through their encapsulation in polymer films. Chemical properties of the prepared AgNCs were explored. Chirooptical properties of AgNCs have been studied in detail by circular dichroism (CD) spectroscopy, including nanocluster systems synthesized using different combinations of chiral ligands, where the tunability of chirality is demonstrated. Improved stability of AgNCs offers a good potential for future applications, for example, in optical sensors. Introduction In recent years, there has been growing interest and significant research developments in the synthesis and characterization of noble metal clusters (NMCs).1,2 Advantageous features of NMCs arise due to their dimensions, which fall between those of atoms and larger plasmonic nanoparticles. Within this size range, a plethora of unique electronic and optical properties can be observed that can be attributed to the electrons of metal atoms being effectively confined to molecular dimensions.3 The energy levels of confined electrons become more atomic-like rather than being similar to conduction bands.4 Consequently, NMCs with several to several tens of atoms display so-called superatom properties.5,6 The boundary at which NMCs start to display plasmonic properties is an area of ongoing research7 with theoretical studies contributing to better understanding in the field.8,9 Theoretical estimates using the free-electron model approximate the critical size at which electronic energy quantization is comparable to thermal energy at ca. 1.7 nm.10 Clusters smaller than this critical size display superatom properties rather than plasmonic behavior. Theoretical modeling has been proven instrumental in the interpretation of NMC properties11,12 and is paving the way for NMC use in a variety of applications.13,14 In fact, several applications of NMCs in fluorescent detection,15,16 labeling,17 and catalysis18,19 have been recently demonstrated. Among NMCs, gold clusters (AuNCs) are the most extensively studied experimentally, owing to their superior stability and relative ease of preparation.20,21 In an important breakthrough, the total structure determination of Au102(SR)44 has significantly advanced the field and opened new research directions.22 This work of Jadzinsky et al.,22 together with earlier predictions,23 has convincingly established that thiol-protected NMCs consist of an outer shell of tightly coordinated metal thiolates surrounding a central metal core. It has also reinforced † Part of the special issue “Protected Metallic Clusters, Quantum Wells and Metallic Nanocrystal Molecules”. * To whom correspondence should be addressed. E-mail: vkitaev@ wlu.ca.
the framework of stable electron configuration of NMCs postulated by a superatom concept.24 The research in silver clusters (AgNCs) has been significantly developed in the early 90s by Henglein and colleagues25,26 and has received increasing attention since the pioneering reports of Dickson and co-workers on small fluorescent AgNCs.27,28 There has been a growing number of studies on fluorescent silver clusters stabilized with DNA.29,30 Tunable fluorescent silver clusters have also been prepared using water-soluble polymers and reduction under light irradiation in gels31,32 and in solutions.33 In another photochemical approach, synthesis of stable strongly fluorescent AgNCs has been recently reported using photochemical reduction in the presence of amines.34 Significant efforts in studies of AgNCs (similar to AuNCs35) are focused on their chirooptical properties.36,37 In general, the chiral properties of NCs38 are an important aspect of understanding chirality on a nanoscale39 that attracts significant research interest. Importantly, enantiomeric structures have been reported by X-ray studies of crystallized AuNCs for Au102(SR)44 clusters in the absence of chiral ligands.22 Consequently, both ideas of an intrinsically chiral metal core and ligand chirality imprinting are finding their support in experimental data.40 We have previously reported on the preparation of chiral silver clusters with several enantiomeric ligands using a procedure of cyclic reduction and oxidation (CROC).41 CROC, as the name implies, is a cyclic process in which the clusters are continually refined. Initially, a crude cluster solution is prepared by reducing a silver salt in the presence of thiols (captopril, in our case). The crude clusters are then oxidized by hydrogen peroxide in order to retain only the most stable clusters and then subsequently reduced with excess silver and thiols. Cycles are repeated until no further improvement in optical spectra of the clusters can be observed, usually 5-6 cycles. While proving the formation of single species of AgNCs, we were not able to unequivocally assign their composition due to the presence of two silver isotopes that significantly broadened mass-spectroscopy peaks and prohibited conclusive identification. The stability of the previously prepared AgNCs was limited
10.1021/jp101764q 2010 American Chemical Society Published on Web 06/14/2010
Silver Nanoclusters and Their Chirooptical Properties to several hours due to the presence of peroxide traces, and AgNC formation required several cycles (a few days) to reach optimal properties. Clusters with a similar optical signature have been recently reported by Bakr et al,42 who described intensely and broadly absorbing nanoparticles (IBANs) that were stable for 18 months at subzero temperatures. Recently, Wu et al.43 described a large-scale synthesis of thiolate-protected optically active AgNCs with a UV-vis absorption peak at ∼500 nm similar to recently reported AgNCs41,42 and identified them as Ag7 by mass spectroscopy. Meso-2,3-dimercaptosuccinic acid (DMSA) was used to stabilize these AgNCs and to enable mass spectroscopy determination. The presence of Ag7(DMSA)4- species has been convincingly proven. At the same time, it is not clear why the postulated structure of Ag7(DMSA)4 with four core electrons and half of the sulfur atoms effectively not bound to the cluster (and proposed to be in a S-S form) is the most energetically stable compared to cyclic thiols with all sulfur atoms bound to silver cations. More studies are necessary to establish composition of this family of AgNC clusters. Herein we report a single-stage room-temperature aqueous synthesis of thiolate-protected chiral silver clusters using single and mixed ligands. These clusters develop within several hours of reduction and exhibit well-defined UV-vis spectra. They are present as a single species, as proven by optical properties and gel electrophoresis, and remain stable in ambient conditions for several days to weeks. Their detailed characterization including chemical stability, behavior of mixed ligand systems, and chirooptical properties is presented and discussed. Experimental Methods Reagents. Silver nitrate (99.9%), (2S)-1-[(2S)-2-methyl-3sulfanylpropanoyl] pyrrolidine-2-carboxylic acid (captopril), glutathione (GSH ) γ-Glu-Cys-Gly) reduced (98%), sodium borohydride (99%), tetrabutylammonium (TBA) borohydride (98%), borane-tetrahydrofuran complex (1.0 M solution in THF), potassium hydroxide pellets (99.99%), tetrachloroauric acid (99.9%), 2-dimethylaminoethane thiol (98%), thiobenzoic acid (90%), 11-mercaptoundecanoic acid (95%), mercaptoethane sulfonate (99%), meso-2,3-dimercaptosuccinic acid (98%), D-glucose (anhydrous), ascorbic acid (99+%), potassium bromide (analytical grade), ammonium hydroxide (28-30%), sodium hydroxide pellets (>99%), potassium chloride (analytical grade), polyvinylpyrrolidone (99%, Mw ) 40K), polystyrenesulfonate (18 wt % in water, Mw ) 75K), poly(acrylic acid) (Mw ) 18K), polyethylenimine (50 wt % in water, Mw ) 750K), and polydiallyldimethylammonium chloride (35 wt % in water, Mw < 100K), all supplied by Aldrich, were used as received. High-purity deionized water (>18.3 MΩ3 cm) was produced by Millipore A10 Milli-Q. Characterization. UV-vis spectra were acquired with either an Ocean Optics QE-65000 fiber-optic UV-vis spectrometer or a Cary 50Bio UV-vis spectrophotometer. Circular dichroism (CD) spectra were obtained with an AVIV model 215 Circular Dichroism Spectrometer. Fluorescence measurements were carried out using 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). For negative ion electrospray (ESI) experiments, a Waters/Micromass QTOF Ultima Global mass spectrometer was employed. Samples were injected at 1 µL/min in a mixture of 1:3 CH3CN/H2O + 0.5% NH4OH. Typical operating conditions were as follows: source temperature of 80 °C, capillary voltage of 3 kV, cone voltage of 35-80 V, and collision energy of
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Figure 1. (a) Circular dichroism spectra and (b) UV-vis spectra of representative AgNCs stabilized using different ligands 1 captopril, 2 glutathione, 3 captopril and glutathione (molar ratio 62:38). Inset in (b) shows the chemical structure of captopril.
3.0-10 eV. Other series of mass spectrometry experiments were performed using a Waters Micromass ZQ 4000 mass spectrometer with a cone voltage varied between 10 and 30 V, a capillary voltage between 3 and 3.2 kV, cone temperatures of 80 °C, and desolvation temperatures between 120 and 180 °C. Native PAGE gel electrophoresis in acrylamide gels (30% monomer and 3% cross-linker) in a tris(hydroxymethyl)aminomethane base buffer of a pH between 8.3 and 8.6 was used for AgNC purity confirmation. Samples were run for 2 h at 100 V. The Beckman Coulter Allegra 64R centrifuge was used for AgNC ultracentrifugation. Thin polymer films encapsulating AgNCs were prepared using a VWR 1410 vacuum oven (at room temperature) connected to Vacuubrand PC2001 Vario chemical resistant vacuum pump to evaporate AgNC solutions with added polymers. Formation of Clusters. The silver clusters were prepared in glass vials (20 mL, VWR) with typical total synthesis volumes (for exploratory series) of ca. 3 mL. The synthesis of the clusters with the best optical properties and the best stability has been carried out as follows: in a glass vial, 1.9 mL high-purity deionized water was combined with 91 µL of 0.02 M captopril and 57 µL of 0.02 M glutathione. To the thiols, silver nitrate (500 µL of 0.005 M solution) and potassium hydroxide (360 µL of 0.1 M solution) were added. No precipitate or opaqueness was observed at this stage. The solution was then reduced with 10 µL of 0.1 M sodium borohydride. Total concentrations in the resulting cluster solution were as follows: 0.62 mM captopril, 0.39 mM glutathione, 0.83 mM silver, 12.5 mM potassium hydroxide, and 0.34 mM sodium borohydride (borates after decomposition). At the initial stages of the reduction, the solution became pale reddish brown with the color deepening as the AgNC formation progressed. Over the period of several hours (typically, 6-12 h), clusters continued to develop slowly and improve in quality that is manifested in their well-resolved peaks in UV-vis spectra (as can be seen in Figures 1 and 8). After reaching their maximum optical intensity, AgNCs remained stable for a period of 7-10 days. The robustness of this cluster synthesis permitted for a fairly wide range of concentrations where AgNCs were still able to
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form. To provide an example of more sensitive preparation using pure captopril as a ligand, clusters could be formed in a range of thiol concentration between 0.5 and 0.8 mM with the concentration of silver at 0.83 mM. In terms of KOH amounts added, expressed as a total concentration in solution, AgNC formation were possible in the range between 1.18 mM and 2 M. The cluster formation was fairly insensitive to the concentration of strong reducing agents. For instance, AgNCs formed with total borohydride concentration in a range varying from 0.08 to 14.9 mM. Concentrations of clusters could be increased up to 2 mM of total silver concentration by using less water with minimal effect on stability (we have not attempted to use higher concentrations because of their high optical densities (A > 3 for the strongest absorption peak at 490 nm in a 0.5 cm cell). At least two-thirds of the water content could be replaced by methanol, ethanol, or isopropanol. To test polymer stabilization in cluster solutions, the above procedure was followed with polymer addition either before silver nitrate, or after reduction. Typical concentrations of polymers were 0.1 mM, which corresponds to 10:1 molar ratio of monomer units to silver. To prepare thin polymer films, the cluster preparation started according to the typical synthesis and then allowed the cluster initial formation for 1-2 h. Polymers were then added to the solutions in a solid powder form and then were either stirred until the polymers were dissolved or sonicated to assist dissolution. Polymer amounts varied between 0.1 and 0.3 g per 3 mL of as prepared cluster solution. Optimal addition amount was ca. 0.2 g, which provided enough polymer for the formation of a continuous robust film. 2-3 mL of solutions were then placed in a 8 mL cylindrical flat-bottom vial (VWR) inside a vacuum chamber, and the water was evaporated at low pressure (typically 18 Torr) and room temperature for a period of time varied between 10 and 72 h. Clusters for use in mass spectroscopic experiments (free of sodium and potassium ions) were prepared as follows: 0.74 mM captopril and 0.95 mM silver nitrate were combined with 3.5 mM ammonia, and reduced using 0.5 mM tetrabutylammonium borohydride in 73% methanol/water solution (the molarities given are total reagent concentrations in the final solution). Finally, the AgNC synthesis is readily scalable by increasing the amounts of the reagents proportionally. We have successfully performed several scale-up preparations with total volumes of 150-200 mL. Results and Discussions AgNC Synthesis. Robust reproducible synthesis of silver nanoclusters is crucial for advancing their studies. Starting with our previously reported preparation using CROC and involving multiple stages,41 we have systematically explored synthetic parameters to simplify the procedure. As a result, we were able to achieve formation of the same type AgNCs directly in one stage with inherent scalability and greatly increased stability. Similar to our previous work,41 captopril was used as a primary ligand for cluster stabilization and also as a chiral thiol to imprint its chirality onto the clusters. Captopril is structurally and functionally similar to amino acids and consists of a carboxylic acid group, attached to a pyrrolidine ring and a thiol group connected to this ring through an amide linkage (inset in Figure 1b). By further developing our original approach and testing different factors and parameters, it has been discovered that the use of a combination of thiol ligands (in our case, captopril and glutathione) was advantageous for slowing the formation of clusters and consequently improving stability. Another crucial
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Figure 2. Plot of the definition of AgNC main absorption peak at 490 nm (defined by the ratio of maximum and minimum peak intensities) vs silver to ligand (captopril) molar ratio.
factor for improving AgNCs formation and stability was pH. Importantly, in the optimum pH range for the synthesis of AgNCs, formation of insoluble thiolates was completely avoided. Thus, no heterogeneous reactions were involved in the synthesis, and produced clusters did not require any purification or size separation. The representative data of the optical properties of AgNCs formed at optimal conditions with the mixed ligands, characterized by circular dichroism and UV-vis spectroscopy, are presented in Figure 1. AgNCs exhibited prominent sharp electronic transitions manifested in well-defined absorption peaks in UV-vis spectra (Figure 1b) and well-resolved strong characteristic CD bands in the visible range (Figure 1a) similar to the same type of clusters prepared by CROC.41 The UV-vis spectrum of AgNCs displayed three peaks, the strongest and best-resolved at 490 nm (2.53 eV) with fwhm of less than 40 nm (∼0.1 eV), a shallow peak at 660 nm (1.87 eV), and a peak at 335 nm (3.70 eV). CD spectra of these AgNCs show welldefined patterns with strong intensities and display essentially mirrorlike features for captopril and glutathione (discussed in more detail in the last section). The UV-vis data display the best resolved spectra of AgNCs formed with mixed ligands (62 mol % of captopril and 38 mol % of glutathione) with a more pronounced peak at ca. 665 nm together with two other welldefined peaks at 490 and 335 nm. This set of the peaks belongs to the same species; all the peaks decreased in intensity simultaneously and proportionally and exhibited minimal spectral shifts. The presence of a single AgNC species was also confirmed by gel electrophoresis (as described in Characterization of AgNCs). To establish an optimal ligand-to-silver ratio, first the molar ratio of silver to captopril (rAg/S) was varied in a range from 1.14 to 1.45. Figure 2 shows the quality of the AgNC (expressed as a ratio of the maximum intensity of the AgNC main absorption peak at ca. 480-490 nm to the trough of this peak at ca. 430 nm, ((Aλmax/Aλmin) - 1) plotted as a function of rAg/S. As demonstrated in Figure 2, the optimal ratio is quite welldefined at 1.28 ( 0.02, which can be considered either as the ratio of silver to captopril corresponding to the chemical composition of this type of AgNCs, or at least as the ratio where a possible excess of captopril is beneficial for cluster formation and stability. It is instructive to point out that for the same type of AgNCs formed by CROC,41 optimal rAg/S was higher likely due to the fact that under oxidative conditions not all silver atoms were incorporated into clusters. One of the crucial parameters for single-stage AgNC synthesis was pH during the reduction, which in our case was controlled by the addition of KOH (or other bases). AgNCs could be successfully formed in a pH range from 6.2 to higher than 12.5. The optimal pH was above 11, as shown in Figure 3. The relationship between pH of the cluster formation and quality of
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Figure 4. (a) Development and stability of large-scale preparation of AgNCs 1 immediately after reduction, 2 3 h, 3 9 h, 4 24 h, 5 48 h, 6 120 h, 7 336 h, and 8 1.5 months. (b) Optical photograph of large-scale synthesis of AgNCs (scaled 50 times). (c) Optical photograph of clusters encapsulated in thin polymer films: polystyrene sulfonate (left) and polyvinylpyrrolidone (right).
Figure 3. (a) Plot of the definition of AgNC main absorption peak at 490 nm (defined by the ratio of maximum and minimum peak intensities) vs pH adjusted prior to reduction; (b) UV-vis spectra of representative samples at different pH values: 1 6.2, 2 9.4, 3 10, 4 12.5.
the clusters (expressed by ((Aλmax/Aλmin) - 1)) is shown in Figure 3a (Supporting Information Figure S1 presents cluster quality as a function of the base concentration added to the cluster system prior to the reduction). Other than potassium hydroxide, sodium hydroxide was often employed for pH control demonstrating that there is no cation specificity in the system. Ammonia also worked as a base despite its strong complexing ability with silver, although the stability of the clusters was appreciably reduced from days to several hours. To elucidate the role of reducing agents, several strong reducers have been tested, including sodium borohydride, tetrabutylammonium borohydride, and borane-tetrahydrofuran complex. Each of these reducing agents yielded the clusters as long as the pH of the solution was maintained in the optimal range described above. Only borane-tetrahydrofuran reduction did not produce appreciably stable AgNCs (the clusters formed initially, yet did not continue to develop as is typical in this synthesis) with ammonia as a base, thus some stabilizing cations such as of alkaline metals or tetraalkylammonium seem to be necessary. Having established the optimal parameters for the formation of AgNCs with captopril and glutathione, we have tested different thiols in identical synthesis procedures (Supporting Information Figure S2). In these experiments, mercaptoethane sulfonate (MES) produced plasmonic silver nanoparticles, dimethylaminoethanethiol (DMAE) yielded aggregated particles with a broad spectrum, while dimercaptosuccinic acid (DMSA) produced a mixture of silver clusters with a pale color and featureless spectrum. Then, capitalizing on a successful strategy of using a combination of thiols, we have tested several mixedligand systems. Glutathione, DMAE, DMSA, MES, thiobenzoic acid, and 11-mercaptoundecanoic acid were added both before reduction and right after reduction together with the optimal amount of captopril as a main ligand, while AgNC formation and stability were monitored by CD and UV-vis spectroscopy (Supporting Information Figure S3). Since AgNCs develop over the course of several hours, the results with thiol addition before and after reduction were nearly identical. All of the thiols coligands used improved AgNC stability compared to pure
captopril clusters with the exception of MES, which did not improve stability and also hindered AgNC development. In combination with captopril, glutathione was noticeably superior to all other thiols in terms of improvements in both quality and stability of the clusters. The main reason for these findings is that glutathione is the only thiol, other than captopril, that is capable of stabilizing high-quality AgNCs as a single ligand. The amount of a coligand thiol proved to be important as well; cluster improvement was observed in a range from 4.7[captopril]:1[coligand] to 2[captopril]:3[coligand] The higher ratio of added thiols resulted in slower AgNC formation, and UV-vis spectra became less defined with lower intensities. The UV-vis and CD graphs in Supporting Information Figure S3 are shown for the ratio of 38 mol % of an added thiol with 62 mol % of captopril, which is an optimal ratio for glutathione and an effective ratio for other thiols as well. The data of CD spectroscopy (Supporting Information Figure S3a) demonstrated that out of all coligands, DMAE actually improved the chirooptical response of the clusters compared to pure captopril and even the clusters formed using the mixtures of glutathione and captopril (Figures 1a and 8b). This observation indicated that DMAE (a nonchiral ligand) is advantageous for enhancing chirality imprinting, likely due to its amino group, which is capable of carrying a positive charge and associating electrostatically with the carboxylic groups of the thiols used. Thus, DMAE and similar aminothiols can be potentially useful for the preparation of chiral clusters and other nanostructures of silver and gold. A great merit of the developed one-stage preparation is that it is readily scaleable. We have prepared several large AgNC batches (scaled ca. fifty times relative to a typical vial synthesis) using common round-bottom flasks (Figure 4b) without deoxygenation, use of low temperature, or other special precautions. No limitations are expected for this AgNC synthesis to be performed at significantly larger scales. AgNC Stability. AgNCs prepared in a larger scale synthesis actually demonstrated improved stability, likely due to less cluster exposure to the environment in bulk. The formation of clusters is a gradual process that typically progresses over 5 to 8 h with the progress of the development of the absorption peaks conveniently monitored by using UV-vis spectroscopy. As a result, “an optimal point of development” can be defined as the time at which the cluster’s optical spectra are the most resolved. Peak resolution was quantified by using the ratio of the maximum intensity of the AgNC main absorption peak at ca. 480-490 nm to the trough of this peak at ca. 430 nm, ((Aλmax/
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Aλmin) - 1). Monitoring this ratio allowed us to conclude that the clusters remained unchanged for more than 24 h, and only started to display minimal noticeable decay in 48 h, retaining more than a third of their original peak intensity after 1.5 months (Figure 4a). No special precautions were used other than closing the flask with the clusters and avoiding direct light exposure, which has a noticeable deteriorating effect on AgNCs. Another stability test involved the use of heat for cluster aging. AgNCs were stable at moderate temperatures of 50 °C for approximately 24 h, at which point they lost optical definition of the peaks monitored by UV-vis spectroscopy. At 100 °C, clusters degraded quickly, as evidenced by the rapidly diminished absorption peaks in UV-vis spectra as well as solution color, with complete disintegration into clear solutions within 30 min. It can be noted that these clear solutions of degraded clusters (most likely soluble thiolates) could be revived by the addition of a strong reducing agent (the same amount as for their initial formation). Experimenting with solvents in the AgNC synthesis, we found that water-methanol mixtures were noticeably advantageous for long-term stability compared to pure water and mixtures of water with other alcohols (ethanol, isopropanol, and t-butanol), as shown in Supporting Information Figure S4. Note that these synthetic series were only performed on a small scale, thus the combination of large scale and water-methanol mixtures should be able to offer the best route for the most stable solutions of AgNCs. In this regard, we have tested the influence of common mild reducing agents and antioxidants on cluster stability in a hypothesis that oxidation is the main degradation pathway. No positive influence could be observed (Supporting Information Figure S5b). Previously, we also could not achieve a positive effect using argon atmosphere,41 suggesting that cluster decomposition may not involve oxidation as a main factor. In a further advance to improve stability and practical handling of AgNCs we were able to encapsulate the clusters in solid films formed by water-soluble polymers (Figure 4c). Films were prepared by dissolving polymers in the cluster solution after their formation (see Experimental Methods). The solutions were then evaporated under low pressure. We have used several common polymers: polyethylene oxide, polystyrenesulfonate (PSS) and polyvinylpyrrolidone. It is instructive to note that we often did not dry the films completely using evaporation with the controlled low pressure at room temperature, thus leaving some water present so that the films remained elastic, while being transparent and mechanically robust (most of completely dried films became brittle with the polymers we have used, though polymer optimization is feasible). The best optical quality of the films has been achieved with PSS and PVP, with PVP providing superior cluster stability. Some films remained unchanged, as evident by their UV-vis spectra, for a period of two months. As a result, we have demonstrated a practical convenient approach to preserve optically active silver clusters for potential applications in optical devices and sensors. We have also investigated the effectiveness of several common water-soluble polymers to stabilize clusters in solution by introducing the polymers both during and after the formation of AgNCs. It was found that the presence of polymers did not significantly enhance stability, while the AgNC development became noticeably hindered (Supporting Information Figure S5d). A likely reason for the lack of stabilization with the polymers in solution is that the clusters are capped with chemically stable thiolate shell and therefore are lacking strong binding sites for polymers compared to metal nanoparticles. Also
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Figure 5. Optical photograph of native PAGE gel runs comparing AgNCs prepared using different ligands: 1 captopril, 2 captopril and glutathione (molar ratio 62:38), 3 glutathione.
due to the smaller cluster size, a cooperative effect of polymer binding is largely lost leading to less effective steric stabilization. Chemical Properties of AgNCs. We have tested chemical properties of AgNCs in several reactions. First, it was instructive to check interactions with silver ions with a potential goal to form different types of clusters or to grow larger nanoparticles starting with the monodisperse clusters. The AgNC main absorption peak was shifted noticeably to the blue upon silver addition (Supporting Information Figure S5a). At the same time, the spectrum became more featureless, so it is unlikely that some well-defined species were formed. Exposure of AgNCs to a gold salt was also performed in order to test whether the galvanic replacement and formation of mixed clusters is possible. Again, judging by the minor shift in the AgNC peak position and peak broadening (Supporting Information Figure S5a), it is not possible to conclude positively that mixed clusters were formed. We are currently further exploring this direction. In another series of experiments, exposure to a strong reducing agent, such as borohydride led to deterioration of AgNCs, as shown by curve 3 in Supporting Information Figure S5a. The latter observation was effectively utilized in adding minimal amounts of borohydride to form the most stable AgNCs (see Experimental Methods). Finally, the effect of chemical agents known to etch silver nanoparticles has also been tested. Supporting Information Figure S5c presents concentrations of bromide, ammonia, and chloride, at which AgNCs were able to retain more than half of the intensity of the main UV-vis absorption peak after 18 h of exposure that ranged from 3.6 µM for bromide to 14.8 mM for ammonia. Characterization of AgNCs. Most importantly, gel electrophoresis experiments (Figure 5) prove unequivocally that synthesized AgNCs are single species both for captoprilstabilized clusters and a glutathione-captopril mixed ligand system. With the optimal high density of the polyacrylamide gel (which was in a range from 28 to 35% of the monomer), good AgNC separation is achieved. Only single lines were observed for captopril-stabilized AgNC and AgNCs stabilized with both glutathione and captopril (Figure 5). At the same time, some very minor traces of other fractions could be discerned for glutathione-stabilized AgNCs (Figure 5), which is consistent with the less-resolved UV-vis peaks for these clusters when compared to captopril-stabilized ones (Figure 1). Glutathionestabilized AgNCs were noticeably bulkier judging by their
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Figure 6. (a) Negative ion electrospray (ESI) mass spectra of AgNCs. (b) Normalized expansions of selected fragment peaks.
slower electrophoretic migration (Figure 5) due to appreciably larger glutathione molecules. At the same time, captoprilstabilized AgNCs and AgNCs stabilized with both glutathione and captopril were essentially the same type of clusters in their electronic transitions, based on optical properties (Figure 8), and their hydrodynamic mobility, judging by the migration length in electrophoresis runs (Figure 5). These results indicate that captopril plays the major role in AgNC stabilization, while glutathione serves a secondary stabilizing role and also assists slower cluster formation. Furthermore, the presence of one, and only one, optical signature of the clusters with three well-defined peaks, whose ratio of intensities always remained the same, in combination with electrophoresis evidence offers very strong support for a single cluster species. Furthermore, we have tested several samples of AgNCs by ultracentrifugation at 50 000 RCF for several hours. No sedimentation or appearance of separation gradients was noticeable, so the presence of silver nanoparticles with diameters larger than ca. 2 nm can be completely excluded. Several different experiments of mass-spectroscopy characterization have been performed with the prepared AgNCs. Synthetically, equivalent clusters have been prepared with due care to exclude smaller stable cations (K+ and Na+) and use labile NH4+ instead as a main counterion to assist MSdetermination. We have used tetrabutylammonium borohydride (TBAB) as a reducing agent, ammonium hydroxide as a base, and captopril as a single stabilizing ligand. (Unfortunately, a combination of borohydride-THF complex and ammonia produced very short living clusters that could not be used for MSidentification.) The clusters with ammonia and TBAB were formed slowly during 1-2 h and degraded within 3-5 h, which was sufficient for the measurements (see Experimental Methods). Monitoring these clusters by UV-vis spectroscopy ensured their optimal development and also confirmed that they were the same species as prepared by the standard procedure. It is important to reiterate that in contrast with AuNCs, the strong fundamental limitation of mass-spectroscopy for AgNCs is the natural presence of two silver isotopes that broadens the peaks significantly for clusters composed of several silver atoms. This limitation is clearly evident in the reports on silver nanoclusters, where only smaller (
