Water-dispersible near-infrared Ag₂S nanoclusters with tunable fluorescence for bioimaging application

Abstract

Water-dispersed NIR-emitting ultrasmall-sized Ag₂S nanoclusters (NCs) are prepared directly in the aqueous phase via a facile one-step microwave synthesis. Through fine adjustment of the experimental conditions, Ag₂S NCs with tunable multicolor emission are successfully realized from the visible region to the NIR region. The resultant Ag₂S NCs are promising biological probes for cellular imaging.

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Near-infrared (NIR) fluorescence is ideal for biological imaging owing to high tissue penetration capability, minimal biological autofluorescence background and negligible tissue scattering in this region.^1,2 Semiconductor nanocrystals (quantum dots, QDs) as a new generation of fluorescent probes are highly promising for biological and biomedical applications, due to their exceptional optical properties such as strong photoluminescence, superior photostability, and narrow and tunable emission spectra.^3–6 To date, various kinds of NIR-emitting QDs have been developed, such as CdTe/CdSe, CdTeSe, CdHgTe and PbS.^7–10 However, the promise of their practical application, especially for in vivo bio-applications, is heavily hindered by the presence of toxic element (Cd, Hg, and Pb) which could cause acute and chronic toxicity. Ag₂S QDs with narrow bandgap (0.9–1.1 eV) are a new type of NIR nanomaterials with low toxicity.¹¹ They are usually prepared in organic phase, and additional phase transfer process is needed to render them water dispersible for biological applications.^12–15 Furthermore, optical properties and stability of the modified Ag₂S QDs are prone to deteriorate during treatment. Recently, protein is used as a scaffold to guide the formation of fluorescent Ag₂S QDs directly in aqueous solution.^16,17 However, the as-synthesized Ag₂S QDs are embedded in the template molecules, leading to a relatively large hydrodynamic diameter (>3 nm). Thus, their usability as a fluorescent label is limited due to their large size, which would affect the recognition between QD-labeled bioprobes and target molecules due to steric hindrance and the movement of the bioprobes.¹⁸ It would be of great interest to develop a facile route for preparing water-soluble NIR fluorescent Ag₂S nanomaterials with ultrasmall size.

Metal nanoclusters (NCs) have attracted much recent attention because of their intriguing properties and promising applications in the field of catalysis, photoelectronics, sensing, bioimaging and etc.^19–22 Distinctive from large nanocrystals, metal NCs display molecule-like electronic properties, giving birth to new properties such as fluorescence.²³ Molecular Ag₂S NCs are of fundamental importance for understanding of their photophysical property as well as promising application for bioimaging by virtue of their low toxicity and ultrasmall size.²⁴ Several synthetic strategies have been developed for the preparation of Ag₂S NCs such as single source precursor and reverse-micelle methods.^25,26 Nevertheless, most of these methods suffer from non-aqueous solvent, high temperature, and/or sophisticated procedures.

Microwave dielectric heating possesses attractive advantages such as rapid temperature elevation, homogeneous heating and high reaction selectivity.^27,28 Through one-pot microwave-assisted reaction, we previously obtained highly fluorescent AgNCs protected by D-penicillamine (DPA), which exhibited supramolecular structure dependent photophysical features.²⁹ Inspired by this work, we herein report the synthesis of ultrasmall-sized NIR-emitting Ag₂S NCs with tunable emissions, excellent aqueous dispersibility, storage stability, and favorable biocompatibility. Additionally, we demonstrate that the resultant Ag₂S NCs are promising biological probes for cellular imaging.

The concentration ratio R_[DPA]/[Ag] is critical for the final product. In the synthetic process of AgNCs, the concentration ratio R_[DPA]/[Ag] is two. Besides as the effective ligand for stabilization of AgNCs, the SH moieties of excessive DPA play a crucial role in reducing Ag⁺ species. Then the hydrogen bond interaction of carboxyl groups between the adjacent Ag–S slabs and the argentophilic interaction were involved to form AgNC supermolecular structures. For the preparation of Ag₂S NCs, the ligand DPA acted as two roles: both the protective agents and the S²⁻ sources. Firstly, small thiolated Ag clusters protected by DPA are formed. Since the concentration ratio R_[DPA]/[Ag] is less than 1, thiolated Ag clusters cannot be reduced by DPA. Instead, the thiolated Ag clusters induce the scission of the C–S bonds of DPA to release S²⁻ ions under microwave irradiation, due to the electron withdrawal effect of metal clusters.^26,30 The Ag₂S nuclei further coalesce to produce stable Ag₂S NCs with ultrasmall size. Thus, the NIR Ag₂S NCs are directly synthesized in aqueous phase through a facile and rapid one-step microwave-assisted method.

In a typical synthesis (details in the ESI†), DPA (0.0191 g) was dissolved in 38.4 mL water, followed by adding AgNO₃ aqueous solution (1.6 mL, 0.1 M). The mixture was stirred for 20 min at room temperature, and then was subjected to microwave irradiation for 3 min. The colourless reaction mixture turned to a clear reddish-brown solution, indicating the formation of Ag₂S NCs (denoted as A2). Then additional amounts of AgNO₃ aqueous solution was added to as-prepared A2 and stirred for 8 h. In this process, the size of A2 further grew larger, leading to Ag₂S NCs A3 and A4 with longer emission wavelength. Otherwise, the Ag₂S NCs A1 with shorter emission wavelength was synthesized when the ratio of AgNO₃ to DPA was adjusted to 1. As comparison, the hot plate was used as the heating resource to synthesize Ag₂S NCs. With the elevated temperature, cloudy white Ag(I)SR thiolates aggregates appeared after stirring for about 5 minutes. The reaction mixture slowly turned from yellow to deep reddish brown, indicating the formation of Ag₂S NCs. The evolved fluorescent emission spectra are shown in Fig. S1.† The emission wavelength is gradually red-shifted over the reaction time. The PL intensity of the products is much lower, only half of that using microwave method. Compared with the conventional heating method, microwave irradiation with the ability of rapid temperature elevation and homogeneous heating, effectively suppressed the formation of the by-products Ag(I)SR thiolates aggregates. The reaction time can be controlled more conveniently. Overall, microwave dielectric heating possesses attractive advantages such as shortening the reaction time, improving reaction selectivity and therefore the yield and reproducibility.

Due to the ultralow solubility product constant (K_sp = 6.3 × 10⁻⁵⁰) and fast crystal growth, it is challenging to synthesize ultrafine Ag₂S NCs in aqueous solution. Taking advantage of microwave dielectric heating, ultrasmall Ag₂S NCs with tunable size can be obtained by varying the molar ratio of AgNO₃ to DPA. As shown in Fig. 1A and B, transmission electron microscopy (TEM) revealed that the resultant Ag₂S NCs appeared as spherical particles with good monodispersity. Upon careful measurement from the high-resolution TEM (HRTEM) image in Fig. 1B inset, the lattice spacing of 0.238 nm was identified, which corresponds to the (−103) facet of monoclinic α-Ag₂S (JCPDS 65-2356), demonstrating the excellent crystalline structures of the as-prepared Ag₂S NCs. The size distribution histograms in the Fig. 1A inset and S2,† calculated by measuring several hundred particles in the TEM images, showed that the A1 and A2 have average sizes of 1.63 ± 0.58 nm and 1.95 ± 0.53 nm, respectively.

Fig. 1 (A) The representative TEM images and size distributions (inset) of the as-prepared Ag₂S NCs A1; (B) the representative TEM images of the as-prepared Ag₂S NCs A2, inset: HRTEM image of Ag₂S NCs showing lattice fringes consistent with the (−103) diffraction plane of monoclinic α-Ag₂S; (C) XRD patterns of the sample A2 (a) before and (b) after thermal treatment at 180 °C under Ar flow for 1 h; (D) FTIR spectra of pure DPA and DPA-capped Ag₂S NCs.

As shown in Fig. 1C, powder X-ray diffraction (XRD) patterns of the resultant A2 revealed only weak and undistinguishable diffraction peaks due to the small sizes and amorphous surface ligands. After a thermal treatment at 180 °C under Ar flow for 1 h, improved XRD patterns were obtained, which agreed well with the standard diffraction data of bulk monoclinic α-Ag₂S, as shown as black bars in Fig. 1C (JCPDS 65-2356). It is worth noting that the diffraction peak from (−103) is even stronger than those from (−121). This result suggests that abundant (−103) planes exist in the present Ag₂S NCs, which is in good agreement with the observation of (−103) planes in the HRTEM measurements (Fig. 1B).

When continuingly increasing the ratio of AgNO₃ to DPA, only bulky deposition was obtained. Therefore, an alternative method was employed to control the growth of larger Ag₂S NCs. Owing to the ultrasmall size, a large fraction of atoms are located on the surface, which are coordinatively unsaturated.³¹ Upon exposure of A2 to additional amount of Ag⁺ ions, epitaxial growth would occur on the specific crystallographic faces of Ag₂S NCs. This was clearly observed in the HRTEM image of the resultant A4 (Fig. S3† inset). As expected, the TEM images and the size distribution histograms in the Fig. S3† revealed that the as-prepared A3 and A4 were spherical in shape and had average diameters of 1.98 ± 0.58 nm and 2.33 ± 0.85 nm, respectively.

As the optical properties of NCs are size-dependent, the Ag₂S NCs with tunable emission wavelength in the NIR region are obtained, which are requisite for multicolour imaging in vivo. The absorption and PL spectra of a series of Ag₂S NCs with different sizes are shown in Fig. 2. The absorption spectra of sample A1 display a clear peak at 546 nm, while other samples are less structure and gradually red-shift. Correspondingly, the emission peak exhibited a significant red-shift (163 nm) from 639 nm (A1), 670 nm (A2) and 743 nm (A3) to 802 nm (A4). The PL QYs are measured to be 1.53% (A1), 2.7% (A2), 1.76% (A3) and 0.72% (A4), respectively. The as-prepared NCs are transparent under ambient light conditions, suggesting excellent aqueous dispersibility without further treatment (Fig. 2C). Under UV irradiation, the obtained Ag₂S NCs aqueous solution displayed distinct red emission and gradually became darker as the emission wavelength gradually shifted out of the visible region. The PL decay spectrum of Ag₂S NCs is shown in Fig. S4.† The curve can be fitted into a biexponential function with lifetimes of 1148.6 ns (64%) and 224.8 ns (36%), and calculated averaged lifetime is 816.0 ns, comparable with previous report.¹⁶ They feature good storage stability, which are physically stable for six months directly at ambient conditions under room light while retaining high fluorescence (Fig. S5†). Moreover, the proposed synthetic strategy is reproducible with a relative standard deviation (RSD) of 5.1% for three parallel batches.

Fig. 2 (A) Absorption and (B) emission spectra of the as-prepared Ag₂S NCs. (C) Photographs of the as-prepared Ag₂S NCs in aqueous solution under room light (top) and 365 nm irradiation (bottom).

To gain further insight about chemical and surface properties, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were exploited to characterize the as-prepared Ag₂S NCs. FTIR spectrum of Ag₂S NCs is similar to the pure ligand DPA though the broad spectral features (Fig. 1D). The surface-capping agents on NCs surfaces contain carboxylate (COO⁻) and primary amino (NH₂) groups, which was verified by the presence of characteristic peaks for the stretch modes of COO⁻ (1382 and 1494 cm⁻¹) and those for the N–H stretch (3376 cm⁻¹) and the N–H bending (1614 cm⁻¹) of NH₂.³² DPA molecules anchor on the surface of Ag₂S NCs via Ag–S bonding, revealed by the disappearance of the S–H stretch mode at 2527 cm⁻¹.³³

XPS survey spectrum suggested that all the expected elements C, O, N, S, and Ag are present in the purified products (Fig. S6†). The binding energy of S 2p_3/2 is observed at 162.0 eV, which corresponds to the typical value of chemisorbed S species, further confirming the formation of Ag–S bonds (Fig. S7†).¹⁴ The high resolution XPS spectrum of Ag 3d showed binding energies of 368.1 eV and 374.2 eV, which are assignable to Ag 3d_5/2 and Ag 3d_3/2, respectively. The deconvolution of the C1s spectrum of the NCs indicated the presence of three types of carbon bonds: COO⁻ (287.8 eV), CH (285.4 eV) and CH₃ (284.5 eV). The N1s peaks at 399.3 eV and 401.1 eV are attributed to NH and NH₃⁺, respectively.²⁴ Thus, the surface of the Ag₂S NCs is terminated with carboxyl and amino groups, which endows the as-prepared NCs with excellent aqueous dispersibility and stability in aqueous systems, and facilitates their applications in biological imaging.

The as-synthesized Ag₂S NCs solutions in phosphate-buffered saline (PBS) were stable for over 1 week, and no flocculation or precipitation was noticeable. As shown in Fig. S8,† the absorption peak was the same as its original value after filtration through a 0.2 μm pore membrane, further demonstrating the good colloidal stability under physiological media. The good colloidal stability may be attributed to reduced interactions between DPA ligands due to steric effects of the methyl groups on their β-carbon atoms.³⁴ The hydrodynamic diameter and zeta potential of the Ag₂S NCs in PBS buffer are measured to be 2.7 nm and −4.6 mV by dynamic light scattering (DLS) (Fig. S9†). The different diameters measured by TEM and DLS may arise from different surface species of the as-prepared Ag₂S NCs in aqueous phase.

For a probe to be used in live cells, the biocompatibility of the NCs was evaluated firstly. We tested the viability of live cells upon exposure to the Ag₂S NCs for 24 h using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. As can be seen from Fig. S10,† no apparent loss of cell viability was observed with incubated Ag₂S NCs in the concentration range of 0–200 μg mL⁻¹, indicating the excellent biocompatibility and nontoxicity of the NIR nanomaterials.

The excellent biocompatibility of the water-soluble ultrasmall Ag₂S NCs prompted us to further investigate their performance in cell imaging. In the bright-field image, the HeLa cells incubated with the ultrasmall Ag₂S NCs do not weaken the cell activity and maintain their normal morphology (Fig. 3A). Remarkably, intensely red fluorescence was seen inside the cells after 24 h of incubation, which indicates that a large number of Ag₂S NCs were internalized (Fig. 3B). These NCs displayed a cytoplasmic distribution in the cells and did not show a significant accumulation in the nucleus (Fig. 3C). The near-neutral charge of zwitterionic ligand DPA protected Ag₂S NCs reduces the nonspecific adhesion of nanoparticles to the cell membranes and are feasibly rinsed with buffer, resulting in less cytotoxicity.³⁴ The red emitters within the cells are large aggregates but not individual particles. With regard to good colloidal stability of the as-synthesized Ag₂S NCs, these NCs are internalization by cells by specific endocytosis pathways that package many nanoparticles in endosomal vesicle.³⁵ Also, they may be capable of penetrating the plasma membrane due to the tiny size.³⁶ Further detailed investigations on the underlying uptake mechanism are still under way. Those results indicate that the as-prepared ultrasmall Ag₂S NCs with good biocompatibility and nontoxicity can be used in bioimaging and further biomedical applications.

Fig. 3 Confocal fluorescence microscopy images of HeLa cells treated with Ag₂S NCs: (A) bright-field images, (B) dark-field images, and (C) overlap of images of dark and bright field.

Conclusions

In summary, water-dispersed NIR-emitting Ag₂S NCs with ultrasmall size were prepared directly in aqueous phase through a facile one-step microwave synthesis. Significantly, the as-prepared Ag₂S NCs display excellent aqueous dispersibility, storage stability, photostability, and favorable biocompatibility. Through fine adjustment of the experimental conditions, Ag₂S NCs with tunable PL emission were successfully realized from visible region to NIR region (from 639 nm to 802 nm). These advantages may render the NIR-emitting NCs promising tools for in vitro and in vivo NIR-fluorescence imaging applications.

Acknowledgements

This work is supported by the National Natural Science Foundation of China with Grants 21190040.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Complete experimental details and additional characterization data. See DOI: 10.1039/c5ra18361h

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