One-Step Synthesis of Water-Soluble Silver Sulfide Quantum Dots and Their Application to Bioimaging.

This work reports a simple water-phase microwave method for the synthesis of water-soluble red emission Ag2S quantum dots at low temperatures without the need for an anaerobic process. It is worth noting that the prepared water-soluble Ag2S quantum dots enjoy positive water dispersion stability. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) results showed that the prepared Ag2S quantum dots had promising biocompatibility and low cytotoxicity. In addition, we further applied the low-toxicity near-infrared Ag2S quantum dots for cell imaging, demonstrating a promising biological probe for cell imaging.

Introduction

Quantum dots have been attracting increasing attention recently. The general morphology of quantum dots is spherical or quasispherical with diameters less than 20 nm.[1] Quantum dots with near-infrared (NIR) emission properties can be used to label different biomolecular drugs, to monitor various cellular processes, etc.[2,3] In this context, the widely studied near-infrared quantum dots, such as PbS, PbSe, PbTe, InP, and CdHgTe, contain highly toxic components, which limit their application in vivo. Therefore, whether there are toxic heavy metals in the prepared fluorescent quantum dots is the first concern of researchers.

At present, silver sulfide quantum dots (Ag2S QDs) enjoy almost non-existent toxicity and attractive fluorescence properties, which can cover the area between infrared and NIR, making them ideal objects for metal ion detection.[4] As a kind of near-infrared fluorescent agent, a silver sulfide quantum dot has exerted tremendous fascination on biomedical labeling and imaging in recent years.[5−10] Although great progress has been made in the synthesis and application of nano-silver sulfide quantum dots in the past decade, there are still challenges in the biological system. The physical and chemical pathways of traditional Ag2S quantum dot synthesis usually require harsh conditions and toxic reagents, including modified particle size engineering surface ligands. Ag2S quantum dots obtained from these methods are mostly hydrophobic due to the action of organic ligands, and a surface ligand exchange process is an effective method to achieve phase conversion.[11] Further phase transfer process reduces the quantum yield, which greatly limits further application. Therefore, it is indispensable to synthesize stable NIR nanomaterials in a water environment. Currently, several methods for synthesizing water-soluble NIR fluorescent Ag2S quantum dots have been reported to eliminate further phase transfer.[12,13] However, the synthesized Ag2S quantum dots are embedded in template molecules, resulting in a large hydrodynamic diameter (>3 nm). Therefore, due to its large size, its availability as a fluorescent label is limited.[14] Researchers currently expect to obtain water-soluble Ag2S quantum dots with small particle sizes, which enjoy excellent near-infrared luminescence potential, favorable water solubility,[4] biocompatibility, low toxicity, and easy one-pot preparation.[15]

In a variety of imaging technologies, fluorescence bioimaging has advantages in in vivo analysis and real-time visualization due to its fast feedback, high sensitivity, and no ionizing radiation.[16] It is of great significance for understanding cellular functions, pathologic diagnosis, and drug discovery and to monitor the distribution of biological molecules in cells.[17] Cell imaging is important to understand the spatial distribution of biological molecules in a cell; therefore, quantum dots as biomarkers have captured widespread attention. Silver sulfide quantum dots as new types of cover area infrared and NIR quantum dots have successfully demonstrated in vivo imaging[7] in recent years. The application of nanomaterial Ag2S quantum dots in photoelectron biosensors and catalysis has attracted much attention.[18,19] The advantages of in vivo bioimaging of Ag2S quantum dots lie in their biocompatibility chemistry and high quantum yield (QY) of light stability, and their absorption into mammalian skin or tissues is almost zero.

In this paper, we developed a simple solution method for the synthesis of water-soluble Ag2S quantum dots at low temperatures without the need for a traditional anaerobic process. d-Penicillamine (DPA) as a sulfur source and a ligand protectant is a kind of amino acid, which is obtained from penicillin; it is a detoxification medicine used for heavy-metal-ion poisons of molecular thiol and amino complexation metal ions and produces water-soluble complexes.[20,21] Ag2S quantum dots with rosy water dispersion, superior stability, and outstanding biocompatibility was synthesized using microwave water-phase heating.

Results and Discussion

Preparation and Characterization of Water-Soluble Ag2S Quantum Dots

Microwave reactor heating has advantages of a uniform and faster heating reaction with higher selectivity,[22,23] as reported previously,[24] via a one-step microwave-assisted method by the d-penicillamine protection of high fluorescent silver nanoclusters; its physical properties show a light-dependent supramolecular structure inspired by the previous work. We report that excellent water dispersion stability is obtained by the superior biocompatibility of the Ag2S QDs synthesis process at low temperatures without the need for a traditional anaerobic process. The synthesis process is shown in Figure . d-penicillamine is selected as a sulfur source and a ligand, silver nitrate as a silver source, and microwave heating is adopted in the aqueous phase. The synthesis process, DPA, and Ag+ concentration ratio of the resulting product are vital. As an effective ligand to stabilize Ag2S quantum dots, the −SH group in excess DPA plays an important role in Ag+ reduction. In the preparation of Ag2S quantum dots, the DPA ligand plays a dual role of a protecting agent and a S2– source. Small sulfide silver clusters were formed under the protection of DPA. Under microwave radiation, the sulfur silver clusters induced the C–S bond of DPA to break and release S2– ions. Ag2S nuclei are further combined to produce stable small-size Ag2S quantum dots. Therefore, NIR Ag2S quantum dots are synthesized directly in the aqueous phase by a simple and rapid one-step microwave-assisted method.

Figure 1

Schematic representation of the Ag2S quantum dot synthesis process.

Figure shows Ag2S QDs at about 60 °C (microwave low fire) under the synthesis conditions of fluorescence spectra. Interestingly, after microwave heating for 2 min, a colorless and transparent mixture reacted and turned into a reddish-brown solution. At this time, no fluorescence could be observed in the reacted mixture. Then, the nanoparticles became fluorescent after the mixture was placed in a refrigerator for 12 h. This may be due to the continuous growth of the nanoparticle size after being placed in the refrigerator. Figure a shows that the synthesis of water-soluble Ag2S QDs in the near-infrared range has strong fluorescence; the fluorescence spectrum of the maximum emission wavelength at 657 nm, corresponding to an excitation wavelength of 450 nm, and water-soluble Ag2S QDs under 365 nm ultraviolet irradiation can be observed in bright red fluorescence. Figure b shows the transmission electron microscopy (TEM) image of the synthesis of water-soluble spherical Ag2S QDs, with outstanding monodispersity. Moreover, through the measurement of hundreds of spherical particles in the TEM image, the size distribution diagram, as shown in the illustration, shows that the particle size of Ag2S quantum dots is distributed within the range of 0.51–4.98 nm, with an average size of 2.5 nm.

Figure 2

(a) Fluorescence spectra of highly fluorescent Ag2S quantum dots with excitation at 450 nm (black line) and emission at 667 nm (red line). The inset shows highly fluorescent Ag2S quantum dots under visible light (left) and a UV lamp (right) at 365 nm. (b) TEM image and size distribution of highly fluorescent Ag2S quantum dots.

The elemental composition and surface groups of water-soluble Ag2S quantum dots were analyzed by X-ray photoelectron spectroscopy (XPS). Figure S1 shows the X-ray photoelectron spectroscopy full-range survey spectra of water-soluble Ag2S quantum dots, which were observed in the full spectra at 162.2, 241.6, 285.1, 370.7, 402.1, and 532.9 eV. These six main peaks correspond to S 2p, S 2s, C 1s, Ag 3d, N 1s, and O 1s, respectively, indicating that water-soluble Ag2S quantum dots are mainly composed of sulfur, silver, carbon, nitrogen, and oxygen elements. As shown in Figure , the high-resolution Ag 3d spectrum has two typical peaks at 367.8 and 373.8 eV, which can be classified as the binding energies of Ag 3d3/2 and Ag 3d5/2, respectively. The binding energy of d-penicillamine S 2p3/2 was 162.0 eV, which was consistent with the S value of the chemisorption[4] C 1s high-resolution spectrum at 284.7, 285.9, and 288.5 eV of the three peaks, respectively, belonging to the DPA CH3–, −CH–, and −COOH groups, suggesting that Ag2S quantum dots on the surface terminate with −COOH groups. The N 1s spectrum was decomposed into three peaks at 399.1, 401.6, and 406.8 eV, which were, respectively, attributed to the presence of −NH, N–O, and NH3+. Therefore, it was observed that there are carboxyl and amino groups on the surface of Ag2S quantum dots, which enables the prepared Ag2S quantum dots to enjoy excellent water dispersability and stability in the water system, which is conducive to the application of Ag2S quantum dots in biological imaging.[25]

Figure 3

High-resolution images (a) Ag 3d, (b) S 2p, (c) C 1s, and (d) N 1s of the as-prepared Ag2S quantum dots.

As shown in Figure a, although the FT-IR spectra of Ag2S quantum dots have wide spectral characteristics, they are similar to those of the pure DPA ligand. In the FT-IR spectra, the characteristic peak at 2531 cm–1 can be classified as the N–H stretching vibration peak of NH2, the characteristic peak at 1678 cm–1 can be classified as the N–H bending vibration peak of NH2, and the peaks at 1431 and 1309 cm–1 can be classified as the stretching vibration peaks of COO–. The FT-IR spectra also further confirmed that the protectants on the surface of Ag2S quantum dots contain COO– and −NH2 groups. This result is consistent with the results of XPS, but compared with pure DPA, it is found that the stretching vibration peak of the S–H bond at 2531 cm–1 of Ag2S quantum dots disappears, indicating that the d-penicillamine molecule binds through the formation of the Ag–S bond.[26] As shown in Figure b, the XRD results demonstrate that the X-ray diffraction peak is weak and arduous to distinguish due to the influence of the crystal size and amorphous surface ligands. However, the morphology of the XRD pattern is consistent with that of the Ag2S crystal, which is indicative of the product is Ag2S nanocrystals.

Figure 4

(a) FT-IR spectra of water-soluble Ag2S quantum dots protected by d-penicillamine and pure d-penicillamine. (b) XRD spectra of water-soluble Ag2S quantum dots protected by d-penicillamine.

Optical Properties of Water-Soluble Ag2S Quantum Dots

As shown in Figure S2a, the intensity of the absorption peak of water-soluble Ag2S quantum dots decreases with the increase of the dilution time, and the position of the absorption peak appears to be blue-shifted. As shown in Figure S2b, the emission spectrum of the synthesized water-soluble Ag2S quantum dots increases with the excitation wavelength from 360 to 620 nm, and the emission peak intensity of the synthesized water-soluble Ag2S quantum dots is the highest when the excitation wavelength is 470 nm. With the increase of the excitation wavelength, the emission spectrum does not demonstrate a red shift or a blue shift, indicating that the synthesized water-soluble Ag2S quantum dots possess certain stability and fewer internal defects. Then, the fluorescence stability of Ag2S quantum dots was studied and their photobleaching properties were investigated. As shown in Figure , the prepared Ag2S quantum dots showed an indistinguishable photobleaching phenomenon after continuous irradiation with a 365 nm ultraviolet lamp for 1 h. The results showed that the fluorescence intensity remained stable as the original fluorescence intensity during the continuous irradiation process.

Figure 5

(a) Fluorescence spectra of water-soluble Ag2S quantum dot photobleaching time monitoring. (b) The photobleaching performance of the Ag2S quantum dot solution under continuous illumination with a 365 nm UV lamp for 1 h.

Since the optical properties of water-soluble Ag2S quantum dots are related to their size, we attempted to synthesize Ag2S quantum dots with a tunable emission wavelength in the near-infrared region, as this is an indispensable condition for polychromatic imaging in vivo. As shown in Figure , the fluorescence emission peak of quantum dots can be adjusted from 675 to 719 nm by adjusting the heating temperature and growth time. In addition to regulating the heating temperature and growth time, ligand concentration is another important factor in regulating the size and fluorescence properties of synthetic nanoparticles, according to previous reports.[27]

Figure 6

(Left) Emission spectra of the as-prepared Ag2S quantum dots. (Right) MTT toxicological evaluation on HeLa cells exposed to Ag2S QDs at various concentrations from 0 to 200 mg/mL for 24 h.

Cell Imaging Applications of Water-Soluble Ag2S Quantum Dots

First, the cytotoxicity of water-soluble Ag2S quantum dots was studied by the MTT method. As shown in Figure , HeLa cells were exposed to different concentrations (0–200 mg/mL) of Ag2S quantum dot solutions to detect the effect of water-soluble Ag2S quantum dots on cell viability. The results demonstrated that more than 80% of the cells could retain their activity after being exposed to different concentrations of Ag2S quantum dots. The results manifest that Ag2S quantum dots possess low cytotoxicity and positive biocompatibility. The promising biocompatibility of water-soluble Ag2S quantum dots prompted us to further study its performance in cell imaging. As shown in Figure , Hoechst 33342 dye was used for the nuclear staining of HeLa cells. The reason why Hoechst 33342 dye was chosen was that it could be used for marking living cells, and it had the ability to cross the cell membrane and bind to living cells. It is worth noting that after 24 h of culture, strong red fluorescence appeared in the cells, indicating that a large number of Ag2S quantum dots were internalized. HeLa cell leakage did not weaken the cell activity in the water-soluble Ag2S quantum dots and maintained their normal shape. These Ag2S quantum dots were distributed in the cell cytoplasm, but no obvious aggregation was observed in the nucleus.

Figure 7

Confocal fluorescence microscopy images of HeLa cells treated with Ag2S quantum dots and Hoechst 33342: (left) Hoechst 33342 images, (middle) Ag2S quantum dots images, and (right) overlap images of Hoechst 33342 and Ag2S quantum dots.

Conclusions

Using silver nitrate as a silver source and d-penicillamine as a sulfur source and a ligand, water-soluble Ag2S quantum dots were synthesized by a simple microwave heating method at low temperatures without the need for a conventional anaerobic process. The prepared Ag2S quantum dots demonstrate bright red luminescence, with an average particle size of about 2.5 nm, and are uniformly dispersed in aqueous solution. The synthesized Ag2S quantum dots have excellent fluorescence performance, excellent stability, and biocompatibility; therefore, they can be applied to the bioimaging of HeLa cells. The results manifest that Ag2S quantum dots can maintain excellent resolution and high visibility in biological tissues by comparison with nuclear chromatids.

Experimental Section

Chemicals and Materials

d-penicillamine was purchased from Shandong West Chemical Industry Co., Ltd. Silver nitrate was purchased from Tianjin Fuchen Chemical Reagent Factory, and Hoechst 33342 was purchased from Shanghai Shengong Biological Reagent Co., Ltd. A phosphate-buffered saline (PBS) solution is prepared with KH2PO4 and Na2HPO4 (50 mm, pH = 5.0). All reagents and chemicals used in this work were used directly without further purification. The water source used throughout the experiment was pure water.

Sample Characterization

UV–vis absorption and fluorescence spectra were recorded by a UV–vis spectrophotometer (Hitachi U-2900, Hitachi, Japan) and a fluorescence spectrophotometer (Hitachi F-4600, Japan), respectively. A diluted Ag2S quantum dot solution was dropped into a 400 copper mesh and dried completely by a vacuum dryer. The morphology of Ag2S quantum dots was observed by a JEOL-2100F transmission electron microscope (TEM, Japan), and their average diameter was measured. The groups on the surface of Ag2S quantum dot products were further characterized by a Fourier infrared spectrophotometer (Thermo Fisher Scientific). X-ray diffraction measurements were made with an advanced X-ray diffractometer at a current of 40 mA and a voltage of 40 kV with a scanning range of 10–80° and a scanning rate of 3° min–1.

Preparation of Ag2S QDs

Water-soluble Ag2S quantum dots were prepared after slight modification according to the previously reported method.[28] In a typical synthesis process, d-penicillamine and AgNO3 were added to deionized water in different proportions, and the solution was vigorously stirred for 20 min to form a uniform mixture. The mixture was then heated in a microwave oven at a certain temperature for a certain period of time. The solution immediately changed from a colorless transparent liquid to a dark brown liquid. The product was placed in an environment of 4 °C and stored before use. The synthesized product was filtered by an ultrafiltration membrane (0.22 μm) to remove the unreacted raw material and impurities in the product.

Toxicity Analysis

First, the previously purified perfect Ag2S quantum dots were scattered in the PBS solution, and then according to the characteristics of HeLa, an appropriate number of cells were spread out in the middle of 96 orifice. Then, the cultured HeLa cells were exposed to the Ag2S quantum dot solution with different concentrations ranging from 20 to 200 μL and incubated for 24 h. Ag2S quantum dots were treated for 24 h, incubated with MTT for 4 h, and finally, the absorbance value at a specific wavelength was determined by an enzyme micrometer.

Bioimaging

During cell imaging, HeLa cells were stained with the Ag2S quantum dot solution in the PBS solution 37 °C for 2 h, and 5% carbon dioxide was added. Then, the Hoechst 33342 solution was added three times. Nuclear staining was conducted at 27 °C for 30 min, followed by three repetitions of centrifugal cleaning, and the final sample preparation was observed under a confocal microscope.