Assembly of Nanoions via Electrostatic Interactions: Ion-Like Behavior of Charged Noble Metal Nanoclusters

The assembly of ultrasmall metal nanoclusters (NCs) is of interest to both basic and applied research as it facilitates the determination of cluster structures and the customization of cluster physicochemical properties. Here we present a facile and general approach to assemble noble metal NCs by selectively inducing electrostatic interactions between negatively-charged metal NCs and divalent cations. The charged metal NCs, which have well-defined sizes, charges and structures; and behave similarly to multivalent anions, can be considered as nanoions. These nanoions exhibit step-like assembly behavior when interacting with the counter cations – assembly only occurs when the solubility product (Ksp) between the carboxylate ions on the NC surface and the divalent cations is exceeded. The assembly here is distinctively different from the random aggregation of colloidal particles by counter ions. The nanoions would assemble into fractal-like monodisperse spherical particles with a high order of regularity that mimic the assembly of ionic crystals.

Herein, we report a facile method to assemble negatively-charged metal NCs in water via the electrostatic interactions between the dissociated carboxyl groups on the NC surface and divalent counter cations (e.g., Zn 21 and Cd 21 ) which are introduced to the solution. The divalent metal cations are used to electrostatically cross-link the negatively-charged NCs into large assemblies. An interesting ion-like behavior of the charged NCs was observed, where the assembly of NCs occurred in a step fashion and only when the metal-ionto-thiolate-ligand ratio (denoted as R [M]/[-SR] ) exceeded specific threshold values. The NC assemblies formed as such were spherical, fractal-like; and monodisperse in shape and size. The difference from the random aggregation of large colloidal NPs by counter ions is distinct and also suggests a high order of regularity in the assembly structure which is more akin to the formation of ionic crystals from real ions in solution. More interestingly, photoluminescence (PL) of the NCs was significantly enhanced after assembly, implying the existence of strong synergistic effects between the close-packed NC building blocks.

Results
Zn 21 -induced assembly of Au NCs. As a proof-of-concept, highly luminescent Au NCs protected by the tripeptide glutathione (GSH or GS-H) were chosen as the model nanoion. The as-synthesized Au NCs showed strong orange emission which peaked at a wavelength of ,610 nm. As shown in Figure 1a, the orange-emitting Au NCs were small spheres with a core size below 2 nm. The Au NCs synthesized as such had a high GSH content on the NC surface (the GSH-to-Auatom ratio was ,0.84 as reported in a previous study) 8 . The GSH ligands are likely to be present as oligomeric GS-[Au(I)-SG] n motifs 8 on the NC surface and determine the charge and surface properties of the Au NCs. Each GSH moiety carries two carboxyl groups with pK a below 4 51 . Hence at the near neutral condition of pH 6.5 in our experiments, all of the carboxyl groups on the NC surface were deprotonated to give rise to a negative charge on the Au NCs. The f-potential plot in Supplementary Fig. S1 (see Supplementary  Information) illustrates the pH dependence of the charge on the Au NCs.
Zn 21 was the counter cation used in this study. Zn 21 carries two positive charges and can electrostatically bind to two monovalent -COO 2 anions. Therefore, Zn 21 can be used as a bifunctional crosslinker to electrostatically bridge between two negatively-charged Au NCs to build large NC assemblies. In a typical Zn 21 -induced NC assembly, 1 mL of Au NCs at an optimized concentration of 0.46 mM [on Au atom basis as determined by inductively coupled plasma mass spectrometry (ICP-MS)] was mixed with 0.25 mL of ZnCl 2 solution at a given concentration. The pH of the mixture was brought to ,6.5 by the addition of 0.1 M NaOH. The solution was well-mixed in a vortex mixer (1500 rpm, 30 s) and kept at room temperature for ,15 min to allow the reaction to complete. The procedure was repeated by using different concentrations of ZnCl 2 while keeping the concentration of the Au NCs constant.
The size of the assembled NCs formed with different concentrations of Zn 21 was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The degree of assembly was determined by the ratio of divalent cations (Zn 21 ) to negativelycharged NCs (poly-anions). Since the latter depends on the GSH ligands on the NC surface, a controllable experimental variable would be the ratio of divalent cation (Zn 21 ) to GSH on the Au NC surface (taking into account that there are two carboxylate anions in one GSH moiety), termed as Zn 21 (Figure 1a). The discrete nature of the Au NCs was also supported by DLS measurements, where a hydrodynamic diameter (HD) of ,2.7 nm was calculated (Figure 1e, black line). When R [Zn21]/[-SG] was increased to 0.3, some small and irre-gularly-shaped NC assemblies began to appear in the TEM image ( Figure 1b). The slight increase in HD in the DLS measurements ( Figure 1e, red line) is consistent with the formation of small NC assemblies. The small amount of counter cations introduced to the NC solution at a low R [Zn21]/[-SG] value (0.3) could only induce controlled aggregation of the negatively-charged NCs to a limited extent; and only NC assemblies smaller than 10 nm were formed ( Figure 1b). The small size of the NC assemblies was also corroborated by UV-vis spectroscopy ( Supplementary Fig. S2) where scattering intensity increase (indication of a significant particle size increase) 8 was not found relative to the discrete Au NCs.
Fractal-like spheres displaying limited self-similarity 52 (spherical nanoion to spherical colloidal assembly at the scale of ,1 and 100 nm, respectively) were present in the TEM image when R [Zn21]/[-SG] was increased to 0.6 ( Figure 1c). The spherical assemblies had a uniform core size of 136.2 nm (100 colloidal spheres counted) and a HD of ,137.6 nm (Figure 1e, blue line). The assembled structure at a higher R [Zn21]/[-SG] value of 0.9 was visually similar (Figure 1d). The diameter of the NC assemblies was however smaller, at ,92.8 nm (100 colloidal spheres counted) and likewise the measured HD decreased to ,93.5 nm (Figure 1e, green line). An increase in the scattering intensity of the NC assemblies was noted when R [Zn21]/[-SG] was increased from 0.6 to 0.9 ( Supplementary Fig.  S2). This is an indication of the increase in the number of NC assemblies at R [Zn21]/[-SG] of 0.9. Hence at the higher R [Zn21]/[-SG] value of 0.9, there were more NC assemblies formed but the size was smaller (vs R [Zn21]/[-SG] 5 0.6). This finding can be understood based on the common nucleation-growth mechanism 53 . At a higher R [Zn21]/[-SG] (0.9), more Zn 21 ions were available as electrostatic cross-linkers, leading to a faster formation of ''assembly nuclei''. More ''nucleation sites'' were available for growth through continuous assembly of NCs. Consequently smaller NC assemblies were formed when the same NC concentration was used. The dependence of the NC assembly size on the concentration of the counter cations (Zn 21 ) is summarized in the inset of Figure 1e, where the maximum in the size of the NC assemblies was found to be around ,137.6 nm.
The structure and composition of fractal-like NC assemblies assimilated by Zn 21 were also examined by high-resolution TEM (HR-TEM). The HR-TEM images of assemblies formed at R [Zn21]/[-SG] of 0.6 and 0.9 are shown as insets in Figure 1c and 1d respectively, where NCs smaller than 2 nm are clearly visible within the assembly. UV-vis spectroscopy also provided supporting evidence for the assimilation of primary NCs ( Supplementary Fig. S2). The characteristic absorption peak of isolated (primary) Au NCs at ,400 nm was mostly retained in the UV-vis spectra of the NC assemblies. The surface plasmon resonance (SPR) of large spherical Au NPs, which absorbs at ,520 nm (for ,50 nm Au NPs) 54 was conspicuously absent, suggesting that the size and structure of the Au NC building blocks were preserved in the assemblies. No increase in the size of the NC building blocks was detected in the assemblies, confirming that the ion-induced NC assembly is a soft approach. The relatively weak electrostatic interaction between the NCs and the counter ions was unable to alter the size and structure of the NC building blocks. The chemical composition of the NC assemblies was analyzed by energy dispersion X-ray spectroscopy (EDX). Line scan analysis across the diameter of a representative assembly ( Figure 2a   properties (step-like assembly). The possession of ion-like structural features in the Au NCs not only made it possible for the step-like assembly, but also produced an ordered structure mimicking the formation of ionic crystalline compounds. In nature oppositely charged ions are often packaged into crystalline ionic compounds with long-range order. Similarly, the NC assemblies here adopted a fractal-like spherical geometry (Figure 1c and 1d). It should be reiterated that such fractal-like assembly was different from the counter-ion-induced assembly of large NPs (.3 nm) where the aggregates display only a low symmetry 55,56 . However, it should be noted that the spherical NC assemblies did not exhibit long-range crystalline order. This could be attributed to the relatively large difference in size between the negatively-charged NCs and the counter cations (Zn 21 ).
The counter cations Zn 21 exerted their influence on the assembly of negatively-charged Au NCs through principally two effects. The first was the screening effect where Zn 21 reduced the negative charge on the Au NC surface through the formation of intra-cluster -COO 2 ???Zn 21 ??? 2 OOC-electrostatic linkage (referred to henceforth as intra-EL). The second was the bridging effect forming inter-cluster -COO 2 ???Zn 21 ??? 2 OOC-electrostatic linkages (inter-ELs). The bridging effect brought two separate NCs together while intra-EL did not bring about NC assembly per se. These two effects are illustrated schematically in   Several independent measurements also suggest the bridging effect of divalent Zn 21 ions as the primary factor in the assembly of Au NCs. First, common monovalent cations such as Na 1 ions (from dissolved NaCl), which could also screen the surface charge of Au NCs, were unable to cause a similar assembly of the Au NCs. Au NCs remained separate in 0.6 mM NaCl solution (same ionic strength as the 0.2 mM ZnCl 2 used to provide R [Zn21]/[-SG] 5 0.6, please refer to the Methods section for the details of the ionic strength calculation). The HD measurement in Figure 4 (second column) indicated likewise. A further increase in the ionic strength of the NaCl solution (e.g., by increasing the NaCl concentration to 0.5 M) also did not help to bring about the assembly of Au NCs (Figure 4, third column). Hence the screening effect of cations on the surface charge of the NCs is not critical to the assembly of NCs. The increase in the solution ionic strength could however disrupt the electrostatic interaction between Zn 21 and carboxylate anions. The presence of NaCl in large excess in the Au NC solution (,0.5 M in the final reaction mixture) prior to ZnCl 2 addition could suppress the assembly to a great extent. The HD of the NCs in the presence of both Na 1 and Zn 21 was also the same as that of discrete NCs, thereby suggesting no significant assembly of the Au NCs (Figure 4, fifth column).
Excess H 1 had the same effect as Na 1 in disrupting the assembly of NCs by Zn 21 . For example, by adjusting the pH of the NC solution to ,2.7 (a value lower than the isoelectric point of Au NCs, Supplementary Fig. S1), the assembly of Au NCs was significantly inhibited, as shown by the nearly invariant HD of the NCs in the presence and absence of Zn 21 at pH ,2.7 ( Figure 4, the last two columns). More interestingly, the assembly and disassembly of the Au NCs could be reversed by cycling the pH of the solution between 6.5 and 2.7. The zigzag changes in the HD of the Au NCs under pH cycling (Figure 5a) indicate that the Au NCs could undergo repeated assembly and disassembly at pH 6.5 and 2.7 respectively.
There was irreversibility shown, however, as the HD of the Au NCs at pH 6.5 gradually decreased with the increase in the cycle number. This trend could be attributed to the slow accumulation of ionic strength due to the way pH cycling was implemented: The pH of the solution was adjusted by the addition of a given amount of 0.1 M HCl or 0.1 M NaOH, leading to the gradual buildup of ionic strength with the increase in the cycle number. A higher ionic strength of the solution increased the screening effect on the electrostatic interactions between Zn 21 and negatively-charged NCs, thus leading to a  The Au NCs were initially assembled at pH ,6.5 (#1), and then pH was varied in the sequence of ,2.7 (#5) --6.5 (#2) --2.7 (#6) --6.5 (#3) --2.7 (#7) --6.5 (#4). In a separate experiment, solution #7 was dialyzed overnight followed by reassembly with Zn 21 at pH 6.5. The hydrodynamic diameter of the reassembled ''refreshed'' Au NCs was recorded as the red dot in (a). The inset in (b) shows the expanded view of the circled area. All assemblies were carried out at R smaller degree of assembly (decreasing size of the NC assemblies). The ionic-strength-induced irreversibility could be eliminated by removing the excess small ions. We ''refreshed'' the Au NC solution at the lower pH of cycle 3 (corresponding to point 7 in Figure 5a) by dialysis, where a semipermeable membrane with a molecular weight cut off (MWCO) of 3500 Da was used to filter away the small ions (e.g., H 1 , Na 1 , Zn 21 and Cl 2 ). After pH adjustment to 6.5 and Zn 21 addition, a HD of 124.4 nm (the red dot in Figure 5a) could be obtained from the ''refreshed'' Au NC solution, similar to the HD assembled at pH 5 6.5 in cycle 1 from a fresh Au NC solution (137.6 nm, point 1 in Figure 5a). This observation led us to conclude that the reversibility of assembly and disassembly could be greatly improved through the control of the ionic strength.
Assembly induced photoluminescent enhancement. The physical and chemical properties of the Au NCs could be altered by proximity effects in an ionically cross-linked close-packed structure. One most interesting property of the Au NCs is their strong optical luminescence, as shown in Figure 6 (black line). The assynthesized Au NCs had a unique Au(0)@Au(I)-thiolate core-shell structure, where the intra-cluster aurophilic interaction [Au(I)-Au(I)] contributed to the PL of the NCs 8,57,58 . The distance between the Au NCs could be significantly shortened after assembly to promote inter-cluster aurophilic interaction. The increase in intercluster aurophilic interaction could increase the PL of the NCs. The assembly of Au NCs could also reduce non-irradiative relaxation of excited electrons, which also enhances the PL of the NCs 59,60 . For example, the bridging Zn 21 between neighboring carboxylate anions (via either an intra-or inter-EL) could inhibit the vibration or rotation of the thiolate ligands, which is one of the non-irradiative relaxation pathways 8 . Experimentally the PL (at 610 nm) of the NC assemblies ( Figure 6) was significantly higher than that of discrete Au NCs. In order to rule out the possibility of PL enhancement through the formation of Zn 21 -thiolate (-SG) complexes, a control experiment was carried out by mixing Zn 21 and GSH in water. The resultant Zn-SG complexes did not exhibit any luminescence (data not shown). Two other experimental observations also provided supporting evidence on proximity-induced properties in the NC assemblies. First, as shown in the inset of Figure 6b, the PL enhancement effect also displayed a similar step-like trend with the increase of the Zn 21 concentration, which corresponds well with the step-like assembly of the Au NCs. Second, the PL enhancement could also be turned on or off corresponding to the assembly or disassembly of Au NCs in pH cycling (Figure 5b).

Discussion
The ion-induced assembly of NCs developed in this study is expected to be generic, and hence should be applicable to other charged metal NCs and divalent counter ions. For example, other divalent cations, such as Cd 21 , may be used to substitute for Zn 21 . Similar spherical NC assemblies were indeed obtained by using Cd 21 as the counter cations ( Supplementary Fig. S4). Similarly, the PL intensity of the NCs assimilated by Cd 21 was also significantly higher than that of discrete Au NCs (Supplementary Fig. S4). The ion-induced assembly of NCs could also be extended to other nanoions; e.g., red-emitting Ag NCs protected by GSH. As shown in Supplementary Fig. S5, the addition of Zn 21 to the Ag NC solution also induced the assembly of Ag NCs, leading to the formation of spherical NC assemblies. Enhanced PL was also detected for the Ag NC assemblies (Supplementary Fig. S5).
In summary, we have developed a facile and general method to assemble charged noble metal NCs in water based on the electrostatic interaction between oppositely-charged entities. Negatively-charged Au NCs were assembled into monodisperse and fractal-like spherical assemblies by divalent counter cations such as Zn 21 and Cd 21 , via the formation of inter-cluster electrostatic linkages (inter-EL) of the type -COO 2 ???M 21 ??? 2 OOC-. Owing to their high charge density, ultrasmall size, and structural uniformity, the Au NCs may be regarded as an ion-mimic nanomaterial, exhibiting ion-like properties of steplike assembly dictated by K sp and a high structural order in the assembly. The bridging effect of the divalent cations is essential to the formation of NC assemblies. A strong synergy between the NCs due to the close-packed order in the assembly resulting in the PL enhancement of NCs was also demonstrated in this study. The NC assemblies and the assembly methods in this study are of interest not only because they provide a simple and general method to generate a superstructure of functional noble metal NCs, which is of interest to both basic and applied research; but also because they exemplify a NC surface with charged hydrophilic ligands as three-dimensional nanoions, with the potential to mimic some of the useful behavior of real ions in various practical settings.
Synthesis of orange-emitting Au NCs. A more detailed description of the synthesis method can be found in the original work 8   Synthesis of red-emitting Ag NCs. Red-emitting Ag NCs were prepared by a cyclic reduction-decomposition method detailed elsewhere 10 . Briefly, 125 mL of 20 mM AgNO 3 and 150 mL of 50 mM GSH aqueous solutions were added to 4.85 mL of ultrapure water under vigorous stirring (,1200 rpm) to form Ag(I)-thiolate complexes. 50 mL of 112 mM NaBH 4 aqueous solution was then introduced to reduce the Ag(I)-thiolate complexes, giving rise to a deep-red solution of Ag NCs within 5 min. The As-formed Ag NC solution (deep-red) was allowed to decompose to Ag(I)-thiolate complexes (colorless) after ,3 h of incubation at room temperature (25uC). 50 mL of 112 mM NaBH 4 aqueous solution was then added to this colorless solution under vigorous stirring (,1200 rpm). The color of solution turned from colorless to light-brown within 15 min. This light-brown Ag NC solution was allowed to stand (without stirring) at room temperature (25uC) for 8 h to form red-emitting Ag NCs in aqueous solution.
Ion-induced assembly of orange-emitting Au NCs. ZnCl 2 and CdCl 2 aqueous solutions in specific concentrations were freshly prepared by dissolving calculated amounts of ZnCl 2 and CdCl 2 in ultrapure water respectively. The pH of each metal salt solution was adjusted to ,6.5 by adding 0.1 M NaOH. The As-prepared Au NC aqueous solution was diluted 4 times (43 diluted) and the pH was adjusted to ,6.5 before use. In a typical assembly (taking R [Zn21]/[-SG] 5 0.6 as an example), 0.25 mL of 1 mM ZnCl 2 was quickly added to 1 mL of the diluted Au NC solution, followed by mixing on a vortex mixer (1500 rpm for 30 s). The pH of the mixture was maintained at ,6.5. Subsequently, the mixture was kept still at room temperature (25uC) for 15 min to allow the assembly to complete. Assembly of Au NCs by Zn 21 in other concentrations and by Cd 21 solution were also carried out likewise, using different metal salt solutions before mixing.
Ion-induced assembly of red-emitting Ag NCs. The pH of Ag NC as well as 2 mM ZnCl 2 aqueous solution was brought to ,6.5 before use. Similar to the Zn 21 induced assembly of Au NCs, 1 mL of Ag NC and 0.25 mL of 2 mM ZnCl 2 aqueous solutions were mixed while pH was maintained at ,6.5, followed by still incubation at room temperature (25uC) for 15 min.
Dialysis of NC solution. 5 mL of NC solution was sealed in a dialysis bag made of a semipermeable membrane (MWCO 5 3500 Da, Spectra/PorH), followed by immersion into 5 L of ultrapure water. The dialysis process lasted overnight under moderate stirring (1000 rpm).
Estimation of the ionic strength of ZnCl 2 and NaCl in the solutions. The ionic strength of NaCl or ZnCl 2 solution was calculated by the equation, I 5 0.5 P c i z i 2 , where I is the ionic strength, c i is the concentration of a specific ion, and z i is the specific charge number of the ion.
Characterization. pH was monitored by a Mettler Toledo FE 20 pH-meter. UV-vis absorption and photoluminescence (PL) spectra were recorded by a Shimadzu UV-1800 spectrometer and a PerkinElmer LS-55 fluorescence spectrometer respectively. Transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and Energy-dispersive X-ray spectroscopy (EDX) elemental analysis were performed on a JEOL JEM 2010 microscope operating at 200 kV. The size and f-potential of NCs before and after assembly were measured by dynamic light scattering (DLS) and electrophoresis light scattering (ELS) on a Malvern Zetasizer Nano ZS, respectively. The samples were centrifuged by an Eppendorf Centrifuge 5424. The Au and Zn contents in the samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) measurement on an Agilent 7500A. The GSH and carboxyl group contents were calculated based on the Au content via the relation Au5GSH5COOH 5 150.8451.68 8 .