Simple and rapid preparation of orange-yellow fluorescent gold nanoclusters using DL-homocysteine as a reducing/stabilizing reagent and their application in cancer cell imaging

Abstract

A simple, convenient and fast preparation for successful fabrication of water-soluble DL-homocysteine capped Au nanoclusters (hcy-AuNCs) was demonstrated. This preparation allowed the generation of water-soluble Au NCs within a short time of 15 min. The resulting hcy-AuNCs were characterized by photoluminescence, UV-Vis absorption, X-ray photoelectron spectroscopy, and transmission electron microscopy (TEM). The mean diameter of hcy-AuNCs was found to be 1.6 ± 0.2 nm. The Au NCs exhibited an orange-yellow fluorescence emission at 560 nm with a large Stokes shift of 120 nm, a quantum yield of 3.01% and a good stability over the physiologically relevant pH range and ionic strength. Furthermore, cytotoxicity studies showed that the Au NCs exhibited negligible effects in altering cell proliferation or triggering apoptosis. Cancer cell imaging of HeLa cell lines indicated that the obtained Au NCs could serve as a promising fluorescent bioprobe for bioimaging. This strategy, based on the one-step preparation using DL-homocysteine as a reducing/stabilizing reagent, may offer a novel approach to fabricate other water-soluble metal nanoclusters for application in biolabelling and bioimaging.

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1. Introduction

Noble metal nanoclusters (NCs) typically smaller than 2 nm have attracted substantial research interest in the fields of chemistry, materials, biology, and medicine due to their unique optical, catalytic and magnetic properties, which are different from bulk metal and metal nanoparticles larger than 3 nm.^1–5 The NCs exhibit molecule-like properties such as discrete electronic states⁶ and size-dependent fluorescence.^7–10 Among these nanoclusters, gold nanoclusters (Au NCs) with different fluorescence wavelength are of great interest for scientific research.^11,12 Au NCs are considered as promising candidates for biolabelling and bioimaging due to their intense fluorescence, ultrafine size and good biocompatibility,^12–16 so there is a growing need for the development of a facile method for the fabrication of fluorescent Au NCs for biomedical applications.

Over the past few decades, several preparative methods for Au NCs have been developed such as chemical reduction of Au³⁺ ions in the presence of stabilizing agents,^2,5,14,17,18 template-assisted synthesis^19–22 and core etching of gold nanoparticles or gold NCs into smaller NCs.^23–25 However, most of these methods involved multiple reaction steps and needed some non-biocompatible precursors or etching reagents, resulting in limited application. Therefore, it is highly desirable to develop facile methods for directly producing water-soluble Au NCs.

Recently, there has been increasing interest in one-step methods for the preparation of Au NCs due to their facile operational procedure.^{14,15,26–31} The group of Ma fabricated intense red fluorescent gold nanoclusters basing on simply placing histidine (His), HAuCl₄ and 11-mercaptoundcanoic acid (MUA) together at room temperature over 3 days.¹⁵ Tatsuma's group developed a one-step preparation of glutathione (GSH)-protected Au, Ag, Cu, Pd and Pt cluster by mixing GSH, metal salts and ice-cold methanol for a certain time, then under vigorous stirring, an ice-cold NaBH₄ aqueous solution was added at once and aged for 1 h.²⁶ Chou's group prepared Au NCs emitting red fluorescence by mixing insulin and HAuCl₄ in Na₃PO₄ buffer with continuously stirring at 4 °C for 12 h.¹⁴ In addition, small molecules such as GSH,^26,27,32 N,N-dimethylformamide (DMF)³³ and amino acid^34–36 have been used to assist the preparation of Au NCs. And most of them do not need additional reducing or stabilizing reagents, especially amino acids are expected to be the favorable reagents for the preparation of Au NCs due to the advantages of low cost, good biocompatibility and chiral structure.

Herein, by employing DL-homocysteine (hcy) as a reducing/stabilizing reagent for the first time, we demonstrate a simple and rapid one-step strategy to successfully prepare the water-soluble, stable and orange-yellow emitting Au NCs. The reason to choose hcy as a reducing/stabilizing reagent is based on the fact that hcy is another kind of amino acid which contains an amino group, a carboxylic group and a thiol group, respectively. These groups would act as reducing and stabilizing function. Also, the amino and carboxylic groups allow the Au NCs to be functionalized by EDC/NHS chemistry and be conjugated with other molecules.³⁷ The obtained Au NCs possess a large Stoke's shift (120 nm), good biocompatibility and desirable imaging effects. The resultant Au NCs were applied as imaging nanoprobes for bioimaging.

2. Experimental

2.1 Reagents

All chemicals used were of analytical grade and were used as-received without further purification. Rhodamine 6G and gold(III) chloride tetrahydrate (HAuCl₄·4H₂O) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DL-Homocysteine (hcy) was purchased from Sigma-Aldrich (Milwaukee, USA). In all preparations, high-purity deionized water (18.2 MΩ) from a Dura series (The Lab Corporation, USA) was used.

2.2 Preparation of hcy-stabilized gold nanoclusters (hcy-AuNCs)

All glassware used in the experiment was cleaned in a bath of freshly prepared aqua regia (HCl : HNO₃, 3 : 1 by volume) and rinsed thoroughly in water prior to use. A typical preparation is described as follows. 5 mL HAuCl₄ (0.04% by mass) was refluxed in oil bath at 120 °C, then with rapidly stirring, hcy (150 μL, 0.1 M) was rapidly added to it. The reaction was stopped after stirring for 15 min. As-prepared Au NCs were purified by dialysis, using dialysis tube with a molecular weight cut-off of 1 kDa to remove impurities. And the Au NCs solution was stored at 4 °C for later use.

2.3 Characterization of hcy-AuNCs

UV-Vis absorption spectra of hcy-AuNCs were recorded with a spectrophotometer UV-2300 (TECHCOMP, Shanghai, China), and fluorescence spectra were taken on a spectrofluorometer F-2500 (Hitachi, Japan). Transmission electron microscopy (TEM) was performed on a FEI Tecnai G-20 (FEI, Eindhoven, Netherlands), which was operated at an accelerating voltage of 200 kV. TEM samples were prepared by spraying a dispersion of Au NCs onto a Cu grid covered by a holey carbon film. Dynamic light scattering (DLS) and zeta-potential experiments were performed using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK). X-Ray photoelectron spectroscopy (XPS) measurements were carried out on a K-Alpha XPS spectrometer (ThermoFisher, E. Grinstead, UK), using Al Kα X-ray radiation (1486.6 eV) for excitation. The cellular fluorescence images were recorded by TCS SP5 II confocal laser scanning microscope (Leica, Germany).

2.4 Cell culture and viability

Human cervical carcinoma (HeLa) cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS), 100 U of penicillin, and 100 U of streptomycin in a humidified incubator at 37 °C and 5% CO₂. Prior to being tested, cells were seeded in 96 well plates at an initial density of 1 × 10⁵ cells per mL for 24 hours. Dilution of Au NCs was then added to DMEM medium and incubated for another 12 h, 24 h and 48 h, respectively. Afterwards, cells were washed twice with PBS followed by addition of 100 μL fresh medium and 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diph-eny-2h-tetrazoliumbromide (MTT) stock suspension (5 mg mL⁻¹) to each well. They were incubated for 4 h at 37 °C and 5% CO₂. After removing all from the wells, we added 150 μL dimethylsulfoxide (DMSO) to each well, mixed the solution thoroughly and incubated the plates at 37 °C for 10 min. The absorbance at 490 nm was measured with an enzyme mark instrument. The viability of the HeLa cells exposed to the AuNCs was expressed as a relative percentage as normalized to the untreated control which was set as 100%. All the records were explained as average data with error bars.

2.5 Cell imaging

The cellular uptake of Au NCs was studied by TCS SP5 II confocal laser scanning microscope. Typically, HeLa cells were cleaved by trypsin, seeded, and grown onto 18 mm glass coverslips in a 6-well culture plate for 24 h. After an appropriate incubation time (3 hours) with 50 μM of Au NCs, cells were washed three times with PBS, fixed with 4% p-formaldehyde for 30 min, and mounted on microscope slides for fluorescence imaging.

3. Results and discussion

3.1 Preparation of hcy-AuNCs

Fluorescent Au NCs can be prepared in one step by reducing gold salt with DL-homocysteine. Actually, in this study, both amino acids (e.g. homocysteine and cysteine) were tested. It was found that the solubility of cysteine in pure water was lower than that of DL-homocysteine, and cysteine was easily oxidized and the nitrogen gas purging system was required in the process of Au NCs preparation. Therefore DL-homocysteine was finally selected as a reducing/stabilizing reagent for facile preparation of water-soluble Au NCs. A rational selection of time and temperature was vital to prepare Au NCs with intense fluorescence. Accordingly, the influence of the reaction time on the fluorescence intensity was investigated (Fig. S1†). As the reaction time reached to 15 min, the fluorescence intensity was the most intense. Thus, 15 min was chosen as the optimal reaction time. In addition, reaction temperature was another significant factor for the preparation of nanoclusters. In this work, different temperatures were tested to prepare Au NCs. From Fig. S2,† we can see that the fluorescent intensity of the Au NCs produced at 120 °C was the most intense, so the optimal temperature to prepare the Au NCs was 120 °C. At the optimal time and temperature, the various amounts of DL-homocysteine (0.1 mol L⁻¹) were added in the HAuCl₄ solution (5 mL, 0.04% by mass) and 150 μL of DL-homocysteine was finally chosen (Fig. S3†). After reaction, an orange-yellow fluorescence was observed via irradiation of the light green product with a UV lamp. The obtained light green Au NCs solution was used for subsequent characterization and imaging applications.

3.2 Characterization of the hcy-AuNCs

The fluorescence spectra of Au NCs was recorded and showed an excitation and emission maximum at 440 nm and 560 nm (Fig. 1a), respectively. The obtained Au NCs possess a large Stokes shift (ca. 120 nm) and an emission band in the orange-yellow spectral region, which was observed from the photos of the Au NCs solution under UV light irradiation and by the naked eye (Fig. 1b). The emission spectrum does not change with the excitation wavelength varying from 360 to 460 nm (Fig. 1c), proving that the optical signal is a real luminescence from Au NCs rather than mere light scattering. The UV-Vis spectra of Au NCs solution missed the characteristic surface plasmon absorption of gold nanoparticles at 520 nm.^38,39 Instead, it exhibited absorptions around 200–400 nm (Fig. 1d). For comparison, the absorption of pure DL-homocysteine solution was recorded and the absorption only at 200–250 nm was observed (Fig. 1d). The absorption around 240–400 nm in the UV-Vis spectra of Au NCs featured the absorption of gold particles or clusters with dimensions less than 2 nm and implied their intrinsically molecule-like properties.^6,36,39,40

Fig. 1 (a) Fluorescence/excitation spectra of hcy-protected gold nanoclusters in water. Excitation spectrum (black line) recorded at λ_em = 560 nm and fluorescence (red line) by excitation at λ_ex = 440 nm. (b) Photographs of hcy-protected gold nanoclusters solution under visible light and under UV light. (c) Photoluminescence spectra of the hcy-protected gold nanoclusters at different excitation wavelengths. (d) UV-Vis absorption spectra of hcy-protected Au nanoclusters in water (red line), the HAuCl₄ solution (blue line), and the DL-homocysteine solution (black line).

In the transmission electron microscopy (TEM) image of hcy-AuNCs in Fig. 2, the nanoclusters are well dispersed, and aggregates are absent. The mean diameter of hcy-AuNCs was determined as 1.6 ± 0.2 nm, as judged from over 100 individual particles. A dynamic light scattering (DLS) measurement of the gold nanoclusters was also performed. It was observed that the hydrodynamic diameter of the hcy-AuNCs was within 2.3 ± 0.32 nm (Fig. S4†). Obviously, the diameter of the hcy-AuNCs obtained from DLS is larger than that measured by TEM, which might be mainly due to the fact that the hcy shell contributes to the hydrodynamic diameter in the DLS experiment. Their smaller size in comparison to other luminescent nanomaterials such as semiconductor QDs, rare earth up-converting nanoparticles and dye-doped silica nanoparticles makes these hcy-AuNCs attractive as fluorescence probes in biological research.^41–43

Fig. 2 Typical TEM image of as-prepared hcy-AuNCs.

Using Rhodamine 6G (QY = 95% in ethanol) as the reference, the quantum yield of our luminescent Au NCs in aqueous solution (pH 7.0) was measured to be 3.01%. Although the quantum yield of hcy-AuNCs was lower than values reported for semiconductor quantum dots and most organic dyes, their brightness is sufficient for their application as fluorescent probes in cellular imaging or biosensors (see below).

The colloidal stability of the Au NCs was firstly examined by zeta potential measurement. It was found that the zeta potential values of the Au NCs synthesized at 110 °C, 120 °C, and 130 °C were 30.5 mv, 33.6 mv and 31.8 mv, respectively (Fig. S5†). Apparently, the zeta potential of the Au NCs synthesized at 120 °C is higher than those synthesized at 110 °C and at 130 °C, suggesting higher stability of the Au NCs synthesized at 120 °C. The colloidal stability of hcy-AuNCs synthesized at 120 °C was also characterized quantitatively by measuring their fluorescence intensity in PBS over a wide pH range and in KCl solution with different ionic strengths. As shown in Fig. 3, the fluorescence intensities of Au NCs have less changeable between pH 3 and 9, are very stable in high-ionic strength solutions, implying that these nanoclusters possess a good colloidal stability over the entire physiologically relevant pH range and ionic strength. For comparison, the TEM images of hcy-AuNCs at different pH values were taken. It was found that all TEM images were similar to that taken at pH 7.0 (Fig. 2), which further indicated the high stability of the Au NCs. Such a good colloidal stability is particularly beneficial for biological applications.

Fig. 3 Effects of (A) pH of buffer solution, (B) KCl solutions with different ionic strengths on the colloidal stability of hcy-protected gold nanoclusters.

To analyze the valence states of gold in the obtained Au NCs, we carried out X-ray photoelectron spectroscopy (XPS) measurements to investigate the oxidation states of their surfaces. The Au4f XPS spectrum (Fig. 4) shows the binding energy (BE) of Au4f_7/2 and Au4f_5/2 at 84 eV and 87.8 eV, respectively. The BE of Au4f_7/2 falls between the Au0 BE (84.0 eV) of a metallic gold film and the Au⁺ BE (86.0 eV) of gold thiolate, suggesting that both Au0 and Au⁺ exist in the luminescent Au NCs. This phenomenon supports the hypothesis that a fraction of the gold atoms in luminescent Au NCs exist in the Au(I) oxidation state, consistent with previously reported literature.^16,44–46

Fig. 4 XPS spectra showing the binding energy of Au4f of hcy-protected gold nanoclusters.

3.3 The test of cytotoxicity

Although ultrasmall Au NCs are generally considered nontoxic owing to their minimal metal content and the chemical inertness of Au,⁴⁷ it is important to test their cytotoxicity to explore potential applications as optical probes in the life sciences. The cytotoxicity of the obtained luminescent Au NCs was assessed via MTT assay after incubation with HeLa cells for 12 h, 24 h and 48 h, respectively. It was observed that the cell viability was greater than 75% even after 48 h incubation with hcy-AuNCs in the concentration range of 0–400 μg mL⁻¹ (Fig. 5), indicating that these Au NCs indeed have a low cytotoxicity.

Fig. 5 Viability of HeLa cells after 12 h, 24 h and 48 h incubation with different concentrations of hcy-protected gold nanoclusters in the cell medium as determined by an MTT assay. The error bars represent variations among six independent measurements.

3.4 Cellular imaging applications

To further investigate the potential application for the presented Au NCs as luminescent probes in biological imaging, we then tested the imaging capability of the obtained luminescent Au NCs by using HeLa cells. After incubation of 3 h with Au NCs, cells were imaged by using confocal microscopy (Fig. 6). The emission luminescence from the Au NCs can be clearly observed, and the luminescence signals suggest that the luminescent Au NCs were bound to the cell membrane and that some of them had been internalized by the HeLa cells.

Fig. 6 Confocal fluorescence images (A), bright field images (B) and the overlay of fluorescence and bright field images (C) of HeLa cells upon 3 h incubation with hcy-AuNCs. Fluorescence images were taken with 405 nm excitation. Scale bar: 100 μm.

4. Conclusion

The proposed new one-step strategy using DL-homocysteine as a reducing/stabilizing reagent for the preparation of water-soluble Au NCs is much simpler and rapider than previous etching-based strategies. The obtained Au NCs possess a small diameter (less than 2 nm), a fluorescence emission at 560 nm, a quantum yield of 3.01%, a good stability over the physiologically relevant pH range and ionic strength, and a negligible effect in altering cell proliferation or triggering apoptosis. The application of Au NCs as fluorescent nanoprobes in bioimaging has been demonstrated by imaging with HeLa cells. The imaging of the experimental results suggests that the luminescent Au NCs could be potentially employed for cellular imaging. This one-step strategy using DL-homocysteine as a reducing/stabilizing reagent may offer a simple and rapid approach to fabricate other water-soluble metal nanoclusters for application in biolabelling and bioimaging.

Acknowledgements

The authors thank the National Natural Science Foundation of China (NSFC, contact no. 20835003, 21075087 and 21175097) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for financial support of this study.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra13288b

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