Recent development in deciphering the structure of luminescent silver nanodots

After decades of effort, luminescent silver clusters have been developed from solid phase-protected, unstable clusters into stable species in aqueous solution.^1–5 Protection groups have played a critical role in the stabilization of such silver clusters. When it is stable in aqueous solution, the supramolecular structure between a silver cluster and its protection group is named a “silver nanocluster” or “silver nanodot.” To emphasize that the two parts of this superstructure are indispensable to each other to preserve the stability and luminescence of the silver species, just acting as a single nanosized dot, we called this supramolecular structure a “silver nanodot.”^6,7 Stable silver nanodots exhibit excellent photoluminescent brightness and photostability, which are crucial as signal reporters for optical probing and imaging. However, research in luminescent silver nanodots has encountered a bottleneck issue: a lack of the knowledge about the nature of the silver nanodots. It is essential to determine what factors lead to stable luminescent silver nanodots in aqueous solution and how to improve their chemical and photophysical properties. From this perspective, we will review recent developments in deciphering the structure of silver nanodots and possible solutions to this problem.

From solid matrix-protected silver clusters to stable silver nanodots. Even though bulk silver is stable, few-atom silver clusters are vulnerable.⁸ Sophisticated protections are required to achieve stable silver clusters. Solid matrices have been the first choice to stabilize vulnerable silver clusters from further agglomeration and against oxidation by environmental oxygen. Noble gas matrices, for example, freeze silver clusters when co-condensing a noble gas-Ag mixture in the gas phase onto a cryogenic substrate.^9,10 Glass¹¹ and zeolites^12–14 are also used as platforms for the production of silver clusters. A significant advance in silver clusters was achieved by the discovery of stable luminescent silver nanodots in aqueous solution at ambient conditions.⁷ Screening of protection groups based on short peptides, poly(acrylic acid)-based polymers, and single-stranded DNAs has further improved their stability in aqueous solution, resulting in a series of spectrally pure silver nanodots with emissions ranging from blue to near-IR.^5,15–18 Silver nanodots show excellent photostability, sustained emission rates, and brightness at a single molecule level, illustrating great potential as biological imaging agents.^18–24 

Considerable difference in photophysical properties between silver clusters and silver nanodots. The difference in protection scaffolds between silver clusters and silver nanodots also results in a substantial difference in their photophysical properties (Table I). Silver clusters in noble gas matrices show strong absorption, mostly in the UV region and subsequent emission ranging from violet to near-IR, resulting in large Stokes’ shifts. Interestingly, all display blue and green emissions but only Ag₃ reportedly exhibits orange, red, and near-IR emissions. Silver clusters in zeolites are similar to those in noble gas matrices, except that most of their emissions locate in the region from yellow to near-IR.^12–14,25

Photophysical properties of silver nanodots (relatively stable silver clusters) depend on their protection scaffolds as well. Red-only emission and a large Stokes’ shift were found among polymer- or peptide-stabilized silver nanodots. However, the photophysical properties of single-stranded DNA (ssDNA)-encapsulated silver nanodots are significantly different from the rest of the silver clusters and nanodots. ssDNA protection not only decreases Stokes’ shifts and increases the photoluminescence quantum yield of silver nanodots from a few percent to as high as 60%^26,27 but also finely tunes the emission wavelength by adjusting the ssDNA sequence and the silver/DNA ratio to yield spectrally pure emissions in the visible and near-IR regions.^17,18,28 Moreover, the difference was also observed in their luminescence lifetime. Most silver nanodots exhibit monoexponential nanosecond luminescence lifetimes, typically between 2 ns and 4 ns. Some polymer- and peptide-protected silver nanodots show biexponential lifetime decays, with a lifetime as short as a few tens of picoseconds.^15,22,27 The top-down synthesis of silver nanodots by oxidizing silver nanoparticles only in the presence of a certain type of protection group yields luminescent silver nanodots, indicating that protection groups are critical for the generation of silver nanodots.²⁹ 

Early investigation of the structure of luminescent silver clusters. Silver clusters embedded in solid matrices provide early platforms to study the nature of silver clusters.^30–32 The mass of a silver cluster of interest has been selected by coupling a mass-selective quadrupole mass filter when co-condensing rare gas matrices and silver atoms from the gas phase onto a cryogenic substrate (Fig. 1).^9,10 Such studies have revealed that small clusters in noble gas matrices, such as Ag₁, Ag₂, Ag₃, Ag₄, and Ag₈, can be photoluminescent.^1,30,33–36 Emissions in all the visible and near-IR regions have been observed from the Ag₃ cluster.³⁶ However, both the Ag₁ and Ag₂ clusters show only blue and green emissions.⁶ The photophysical resemblance between silver clusters in noble gas matrices and silver nanodots under the protection of a peptide may suggest that both have similar core structures as silver species,¹⁵ but they are quite different from DNA-protected silver nanodots.¹⁷ 

Photophysical study of the silver nanodot structure. Due to their lack of crystal structures, investigation of silver nanodots was mainly based on their photophysical properties. Upon the reduction of silver ions with borohydride in the presence of ssDNA molecules, red emission species usually appear. The red emitters are considered fully reduced species. Ripening from red emitters results in typically blue,²¹ green,¹⁷ or yellow silver nanodots.^17,18 For example, the spectrum shifts gradually to the blue emission species in a multi-step, intermediate-involved process (Fig. 2). Reactive oxygen species expedite the spectral shift by quenching the red emission and facilitating the formation of the blue.³⁷ Similarly, a yellow silver nanodot has been produced by superoxide oxidation. It is likely that the oxidizing agents degrade large-size silver clusters to silver ions and smaller, mostly non-luminescent clusters. The deposit of silver ions on these small clusters at critical locations induces the formation of more stable charged species, and some of these are blue emitters or yellow emitters. These experiments indicate that the size and the charge of the silver nanodots are indeed the factors that determine their photophysical properties.

Current effort on decoding the link between stable silver clusters and their luminescence. The discrepancy between the photophysical properties of silver clusters in the gas phase and aqueous solution has prompted scientists to attempt to decipher the nature of silver nanodots. Note that the as-prepared silver clusters and silver nanodots are preliminarily a mixture. However, the detection limit of a fluorometer can be as low as 460 aM. The spectrally pure species might be chemically impure. Obviously not all the stable silver clusters are luminescent,³⁸ thus the first effort of such a mechanism study was to purify the silver nanodots, for example, with high-performance liquid chromatography (HPLC) or gel electrophoresis, and then characterize them with various analytical methods, such as TEM and mass spectrometry (Fig. 3).^20,39,40 Petty et al., who reported the analysis of near-IR silver nanodots by a combination of HPLC and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) in which the HPLC purified-silver species was analyzed by ICP-AES, showed that the number of silver atoms in the ssDNA-silver complex was 10.²⁰ The above results indicate that the number of silver atoms in a single luminescent silver nanodot must be less than or equal to 10 no matter how efficient the HPLC purification was. However, the exact structures of silver nanodots are still under debate. Another comparative study based on HPLC and mass spectrometry reported that the silver nanodots consisted of 10 to 21 silver atoms, depending on the emission wavelength.^39,41 The discrepancy in the number of silver atoms in a single DNA-silver complex might be ascribed to the presence of non-functional silver in the complex. As shown in Fig. 4, luminescent bright silver species and dark non-luminescent silver species stay in a single ssDNA. Two isomers of these species likely present quite similar physical properties which causes particular difficulty to purify. Such silver atoms are not part of luminescent clusters but can be detected with mass spectrometry.

Computational simulation has been another approach to decipher the silver nanodots’ structure.^42,43 Recently, the absorption spectra of silver clusters have been reported by simulation,⁴⁴ but the structure of luminescent nanodots were rarely reported. It was reported that a charged core,⁴⁵ such as $Ag42+orAg5+$⁠,^42,43 was observed, which is in line with the fact that the positively charged silver clusters exhibit much better stability compared to the neutral.^8,46 Larger silver clusters up to Ag₁₅ with an Ag₈ core were also reported to be luminescent.⁴⁷ However, more sophisticated solutions are needed to interpret the discrepancy between simulated conditions and the experimental results.^48,49

The amount of silver atoms determines the cluster size and photophysical properties of such clusters. For example, lowering the silver content in a ssDNA-silver mixture may result in a transition from a red emitter to a green emitter.¹⁷ A combination of mass spectrometry and simulation indicates that ssDNA-stabilized silver nanodots may consist of almost equal amounts of neutral and cationic silver.⁵⁰ These silver atoms construct a rod-like, rather than planar or globular, superstructure, as evidenced by its photophysical simulation.⁵¹ Gwinn et al. suggested that the shift in the emission wavelength of the ssDNA-stabilized silver nanodots might be ascribed to changes in the rod length of the silver cluster,⁵⁰ whereas Neidig et al. believed that cooperative effects of both Ag−DNA ligation and variations in cluster size regulate the photoluminescence of silver nanodots after analyzing the corresponding silver nanodots with extended X-ray absorption fine structure (EXAFS). Their results showed Ag–Ag bonds and Ag−N/O ligations to DNA in the silver nanodots with a silver atom number less than 30.⁵² A report from Petty et al. supports the presence of cationic silver in the cluster core of silver nanodots as well.⁵³ An octahedral $Ag62+$ capped with four Ag⁺ has been proposed to be the nanodot structure. The protective ssDNA nucleobases coordinate the capping shell. Recently the silver quantity in zeolites was controlled by exchanging sodium in zeolites with silver ions, and the corresponding zeolite-stabilized silver complexes were transformed to luminescent silver clusters after heat treatment.^14,25 A trinuclear silver cluster associated with green emission and a tetranuclear silver cluster related to yellow emission were observed directly with STEM.²⁵ Further investigation of silver clusters in such a platform by a fine adjustment of the silver contents in zeolites revealed that an improved degree of order of Ag₄ clusters within zeolites accounted for the increase in the quantum efficiency of Ag-containing FAUY zeolites rather than the contribution by changes in the nuclearity of clusters or the proportion of silver atoms involved in cluster formation.¹⁴ It also indicated that both the zeolite framework and cations such as sodium ions contributed the photophysical properties of silver clusters.

The first crystal structure of a luminescent silver cluster shows an octahedral $Ag64+$ core (Fig. 5(a)).⁵⁴ A mixture of silver ions, triphenylphosphine, and 3,4-difluoro-benzenethiol were reduced with borohydride in organic solvents and the product was subsequently recrystallized. The corresponding crystal contains cube-like Ag₁₄(SC₆H₃F₂)₁₂(PPh₃)₈ clusters with an octahedral $Ag64+$ core. The Ag–Ag distance in this core has a distribution between 2.813(1) Å and 2.852(1) Å, slightly shorter than the Ag–Ag distance of bulk Ag metal (about 2.889 Å). It seems that the shell silver species bearing charges and coordinate groups is essential to stabilize the core structure. Similar metal core-shell structures have been found in ultrastable silver nanoparticles (superclusters).^55,56 An inner icosahedron Ag₁₂ core (Fig. 5(b)) is encapsulated solely by a Ag₂₀ dodecahedron (Fig. 5(c)), forming a $Ag3214+$ cluster core (Fig. 5(d)). In addition to the p-mercaptobenzoic acid stabilization, this core is further protected with silver complexes, resulting in six Ag₂S₅ capping units (Fig. 5(e)). It is believed that the clusters polymerize to form larger plasmonic silver nanoparticles when such capping units are removed.⁵⁵ Other structures of silver clusters based on small organic molecules, such as dithiophosphate protected Ag₂₁, Ag₂₉, Ag₄₄, or Ag₄₂ clusters, have been reported recently.^57,58 The photophysical properties of these silver clusters are significantly different. Only the small silver cluster such as the $Ag64+$ core shows photoluminescence. Unfortunately, such a silver cluster emitter was prepared in an organic solvent with small organic thiol protection groups and showed unsatisfied stability. As reported, the protection group likely participates in the luminescence of silver nanodots.⁵⁹ The structure of stable luminescent silver nanodots in aqueous solution is still a mystery. However, current crystal structures of silver clusters do not indicate the nature of the stable silver nanodots.

Both organic ligands and cations can stabilize silver clusters. Current results suggest that silver clusters likely have a core structure of $Agnm+$ (n > m), especially in zeolites, glass, and other solid matrices. As outstanding optical probes, silver nanodots stabilized with ssDNA remain mysterious species. It is essential to determine what factors lead to the formation of stable luminescent silver nanodots in aqueous solution and how to improve their chemical and photophysical properties. As mentioned above, there are major differences in photophysical properties among silver clusters and silver nanodots, for example, in lifetime, luminescence quantum yield, emission wavelength, and Stokes’ shift, which may indicate that they have completely different structures and luminescence mechanisms. Coordination of silver atoms with ligands might be the preliminary explanation, for example, the binding to the N3 nitrogen of cytosine. We still need to know why and how the N3 nitrogen is so critical and how silver nanodots are stabilized.

Another challenge is how to make silver nanodots more stable in aqueous solution. Even though peptide-protected silver nanodots have shown excellent chemical stability in live cells, the low K_sp of silver halide as well as high affinity of silver ions for some biological molecules induce low chemical stability in most silver nanodots in physiological conditions, which hinders the wider biological application of silver nanodots, especially those under ssDNA protection. Without fully understanding the nature of silver nanodots, it is difficult to improve the stability of silver nanodots in physiological solutions.

The last but the most important question is how the silver nanodots emit light. What core structure of silver nanodots is responsible for photoluminescence? Which factor determines the color of the silver nanodot? What causes the photophysical difference between silver clusters in the gas phase and aqueous solution? Previous studies have shown that silver clusters in noble gas matrices show multiple emission regions with a single core structure, for example, Ag₃. This may suggest that “nearby” cationic silver in the matrices not only stabilizes the silver cluster but also vary energy gaps between the ground state and excited states of silver clusters, resulting in various emission bands. Without solid matrix protection, silver clusters become vulnerable. The capping of a silver cluster core with cationic silver may significantly stabilize the cluster when silver clusters are in aqueous solution. Sophisticated tuning of ligands is then required to construct a coordinate sphere that perfectly wraps the cationic silver-capped silver cluster, leading to the synthesis of a series spectrally pure silver nanodots. However, we cannot exclude scenarios in which ligands coordinate the core silver cluster and adjust its energy gaps between the ground state and excited states.

This work was supported by the Korean National Research Foundation (Grant Nos. 2013R1A1A2061063 and 2015R1A2A1A15055721). S. Choi thanks the NRF (Grant No. 2015R1D1A1A01057710).