One-pot synthesis of biofunctional and near-infrared fluorescent gold nanodots and their application in Pb²⁺ sensing and tumor cell imaging

Abstract

Monodisperse and water-soluble Cys-Au dots with near-infrared emission were successfully prepared by a one-pot synthesis approach, and the as-prepared Au dots could be used for long-term tumor cell imaging and highly selective detecting Pb²⁺.

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Lead ions (Pb²⁺), highly toxic heavy metal pollutants, have attracted much attention due to their serious threat to human health and the environment.^1,2 Because of the biotoxicity properties of Pb²⁺, many approaches for Pb²⁺ detection have been reported, such as quantum dots (QDs), DNAzymes, functional gold nanoparticles, proteins, polymer, and organic dyes.^3–8 Many of these methods, however, are either constrained with respect to simplicity, selectivity and sensitivity, or economically limited in practical applications, which limit their universal application for routine detection. Therefore, it is significant to develop cost-effective and practical approaches for sensitive and selective Pb²⁺ detection.

Fluorescent gold nanodots (Au dots), with its ultrasmall size, good photophysical properties and excellent biocompatibility, have been recognized as promising candidates for biosensing and bioimaging in the past few decades.^9–15 Particularly, Au dots with red emission have great superiority in bioimaging application because red emission can improve the signal-to-noise ratio due to its maximal penetration in tissue and negligible tissue scattering. Hence, there is an increasing demand for the development of the monodispersed and red fluorescent Au dots for bioimaging applications. Unlike ultrasmall Au NCs (<1.5 nm), Au nanodots (1.5–3 nm)¹⁶ display visible fluorescence with large Stokes shifts and long lifetimes,^17,18 and these differences in optical properties make Au dots more preponderant than Au NCs in biological imaging. Up to now, several synthetic strategies have been proposed for the preparation of Au-dots including using template-assisted synthesis (dendrimers and proteins), chemical reduction of Au³⁺ ions, or etching of large gold nanoparticles into smaller ones.^19–21 Nevertheless, these synthetic strategies are not entirely satisfactory due to the relatively long reaction times or complicated procedures which greatly bound the investigation of practical applications. Recently, owing to the distinct and fascinating advantages of uniform heating, rapid reaction and environmentally friendly feature, microwave (MW)-assisted techniques have been successfully utilized to synthesize BSA-stabilized and HSA-protected Au dots.²² The reaction time can be shortened from dozens of hours to several minutes, thanks to the superheating and non-thermal effects of the MW energy. To the best of our knowledge, the development of a rapid, facile and green approach to synthesize water-soluble and environment-friendly Au-dots with good biocompatibility still are of extremely important significance.

Herein, we demonstrate a facile one-pot strategy to successfully synthesize the monodisperse and water-soluble Au dots with near-infrared emission stabilized by L-cysteine. The as-prepared Cys-Au dots exhibits orange red emission at 635 nm with a quantum yield (QY) of 4.1% and have the following obvious advantages: (1) they can be rapidly synthesized in 90 seconds; (2) they have a large Stokes shift (>280 nm) indicating that they could be an excellent fluorescent probe for biological sensing and imaging; (3) they have excellent water dispersibility and low cytotoxicity. Afterwards, we used the as-obtained Cys-Au dots as detecting probes for the sensing of Pb²⁺, based on the aggregation-induced fluorescence quenching mechanism. These easily prepared and water-soluble sensing Cys-Au dots offered high selectivity in discriminating Pb²⁺ from other metal ions as well as high sensitivity [ limit of detection (LOD): 4.1 nM, at a signal-to-noise ratio (SNR) of 3]. Furthermore, the Cys-Au dots are successfully applied to the optical imaging of tumor cells (HeLa and A549), suggesting that such synthesized Cys-Au dots have the available potentials for biolabelling and bioimaging.

Fluorescent Cys-Au dots can be easily prepared in aqueous solution by the MW-assisted synthesis (Fig. 1A). According to the previous report, the fluorescent gold nanodots was possibly generated by the aggregation-induced emission (AIE) of Au(I)–thiolate complexes, and the formation of the complexes involved two steps. The first step was the reduction of Au(III) to Au(I) by the thiol group of Cys,^23,24 immediately formed insoluble aggregates of Au(I)–thiolate complexes. The second step, the insoluble aggregates of Au(I)–thiolate complexes was dissolved to be oligomerization of Au(I)–thiolate complexes after the addition of NaOH. Then, after heating, strong luminescence would be generated when the complexes slowly aggregated by collision and fusion.²⁴ To characterize the nanostructures of such synthesized Au dots, a typical transmission electron microscopy (TEM) image in Fig. 1B-a shows that the Au dots are monodispersed and uniform. Moreover, the well-resolved lattice planes of approximately 0.23 nm spacing in the HRTEM image (inset in Fig. 1B-a) demonstrates the excellent crystalline structure of the Au dots, which is consistent with the previous report.²⁵ In addition, the selected area electron diffraction (SAED) pattern also indicates that the well organized Au dots are formed. The diameter histogram in Fig. 1B-b, calculated by measuring more than 100 particles in the TEM image, shows the Au dots have an average size of 1.96 nm. The fluorescence excitation and emission were also investigated to confirm the formation of Au dots. As shown in Fig. 1B-c, the as-prepared Au dots, with a quantum yield (QY) of 4.1% (calibrated with rhodamine B as the reference), exhibit strong orange red fluorescence with an excitation/emission maximum at 355/635 nm. The fluorescence lifetime of the Cys-Au dots was 2.51 μs (Fig. S1†). In order to achieve more information about the Cys-Au dots, infrared spectroscopy was performed. Detailed information was presented in the ESI (Fig. S2†).

Fig. 1 (A) Schematic representation of the synthetic strategy for Cys-Au dots and their fluorescence quenching upon interaction with Pb²⁺. (B-a) TEM image of Cys-Au dots. The top and bottom insets display the HRTEM image and SAED pattern of Au dots. (B-b) The size distribution of the prepared Cys-Au dots. (B-c) Fluorescence excitation (blue line) and emission (red line) spectra of the Cys-Au dots, the inset is the photograph of the Cys-Au dots under 365 nm UV light illumination. (B-d) XPS spectrum of Au 4f for Cys-Au dots.

In contrast to organic fluorophores, this Au dots exhibits a large Stokes shift (280 nm), which avoids crosstalk between the excitation and emission signals, indicating that they could be an excellent fluorescent probe for biological sensing and imaging. In order to obtain additional information about the Au dots, X-ray photoelectron spectroscopy (XPS) of the Cys-Au dots were conducted and the results were displayed in Fig. 1B–d. The binding energy (BE) of the Au 4f_5/2 and Au 4f_7/2 peaks is 87.7 and 84.0 eV, respectively. It has been reported that the Au (4f) peak-positions of Au dots in XPS are located between those of Au(I)–thiolate complexes (86.0 eV) and Au (0) film (84.0 eV).^26,27 The XPS spectra for Cys-Au dos were consistent with those of Au dots reported in the literature, which all located between those of Au(I)–thiolate complexes and Au(0) film, suggesting that both Au(0) and Au(I) exist in the Au dots. Additionally, a systematic study was performed to explore the optimal conditions (e.g. reaction time, the concentration of Cys, HAuCl₄ and NaOH, respectively) for preparing the highly fluorescent Cys-Au dots. As shown in Fig. S3,† an optimal composition of 90 seconds reaction time, 40 mg mL⁻¹ Cys, 20 mM HAuCl₄ and 1.0 mM NaOH generates the highest red-orange emission of the Cys-Au dots. Moreover, the as-prepared Au dots are pH responsive (Fig. S4†) and the maximum fluorescent intensity was achieved when the pH value was 4.5 in the HOAc–NaOAc buffer solution. Consequently, subsequent sensing experiments were all performed at pH 4.5.

It is reported that cysteine could bind Pb²⁺ by coordination with its acidic (–COOH) and basic (–NH₂) functional groups.^28,29 Pb²⁺ coordinates with the N and O atoms of the ligand acidic (–COOH) and basic (–NH₂),³⁰ it was deemed to be functional as a bridge between Cys-coated Au dots, resulting in the aggregation of Au dots. Therefore, owing to the presence of the Cys ligand on the nanodot surface, the Au dots solution is highly sensitive to Pb²⁺ as expected.³¹ The TEM images in Fig. 2A revealed that the Au dots tend to aggregate in the presence of Pb²⁺, resulting the fluorescence quenching. Moreover, Fig. S5† shows the photographs of the Au dots solution before and after adding 20 μM Pb²⁺ under 365 nm UV light irradiation. It can be seen that the fluorescence of the Cys-Au dots was quenched almost completely upon addition of Pb²⁺. However, the fluorescence of the Au dots would recover to almost its original value when EDTA was added to form a more stable conjugate with Pb²⁺, which indicated that the response of the Au dots toward Pb²⁺ was indeed directly related to Pb²⁺/Cys interaction as designed in our scheme.

Fig. 2 (A) TEM images of the Au dots in the (a) absence and (b) presence of Pb²⁺, respectively. Insets are corresponding photographs of the Au dots under 365 nm UV light illumination. (B) (a) Fluorescence emission spectra of Cys-Au dots with the addition of different Pb²⁺ concentrations increasing from 0 to 25 000 nM (top to bottom, excitation at 355 nm). (b) Plot of the relative fluorescence intensity versus the Pb²⁺ concentration.

On the basis of the above results, the fluorescent Cys-Au dots were used to investigate the quenching behavior of Pb²⁺ on the fluorescence emission. As shown in Fig. 2B, the emission intensity is gradually reduced with increasing the Pb²⁺ concentration. An excellent linear correlation (R² = 0.9946) exists based on the quenching effects (I₀/I) on the quencher concentration of Pb²⁺ over the range from 12.5 to 1562.5 nM. The detection limit is found to be 4.1 nM using 3σ/S method, where σ is the standard deviation of the blank signal and S is the slope of the linear calibration plot.³² The LOD was lower than the maximum level (15 μg L⁻¹ or 75 nM) of Pb²⁺ permitted in drinking water by the US Environmental Protection Agency.³³ In fact, the determination of Pb²⁺ based on fluorescent gold nanoclusters has also been reported. For example, Yuan and co-workers reported a glutathione (GSH) functionalized gold nanoclusters for the determination of Pb²⁺ based on the aggregation-induced fluorescence quenching process.³⁴ Lin and his co-workers reported an aptamer/reporter conjugates based fluorescent method for Pb²⁺ detection.³⁵ Nevertheless, it may be still restricted owing to the time-consuming synthetic steps, complex pretreatment steps and long response times. As a new Pb²⁺ sensing approach, the sensitivity of the present method is comparable to the above mentioned fluorescent gold nanoclusters based detection technique. Furthermore, the Cys-Au dots shows obvious advantages such as time-saving synthetic steps (only 90 seconds) and short response times, indicating the Cys-Au dots should be an ideal candidate for sensitive detection of Pb²⁺.

Besides a good sensitivity, a highly special response to the analyte over potentially competing species is a requirement for an application in biomedical and environmental systems. Therefore, the selectivity of the current sensing system to Pb²⁺ over other environmentally relevant metal ions (K⁺, Ba²⁺, Mn²⁺, Cd²⁺, Zn²⁺, Al³⁺, Hg²⁺, Mg²⁺, Ca²⁺, Na⁺) was evaluated under the same conditions. As shown in Fig. 3, it was found that the fluorescence emission of the as-prepared Cys-Au dots was readily quenched in the presence of Pb²⁺ while other metal ions had minor or negligible quenching effects on the fluorescence intensity. These results revealed that the assay approach had high sensitivity and excellent selectivity toward Pb²⁺.

Fig. 3 Digital photographs under UV illumination (A) and relative luminescence (I/I₀)at λ_ex = 365 nm (B) of aqueous Cys-Au dots solutions (5 mM) in the presence of 20 μM of different metal ions.

To study cell–cell interactions, cell differentiation and tracking, cell imaging is a useful technique by using fluorescent probes as markers. Because conventional fluorescent probes for cell imaging such as organic dyes have limited photostability, so it may be a disadvantage for long-term experiments in live cells with high sensitivity.³⁶ On the contrary, owing to the excellent photoluminescence properties, small size and good biocompatibility, the potential application of the as-prepared Cys-Au dots has also been demonstrated by applying them to the direct cellular imaging. First, an MTT assay was performed on HeLa and A549 cells to examine the cytotoxicity of the Cys-Au dots, respectively. As shown in Fig. S6,† cell viability was both greater than 90% after incubation with Cys-Au dots in the concentration range of 0–500 μM for 24 h, indicating that these Au dots indeed have a relative low cytotoxicity. Afterwards, the images of HeLa and A549 cells were obtained using a confocal fluorescence microscope after 2 h incubation with Cys-Au dots. As shown in Fig. 4, the orange red feature on the HeLa and A549 cell surface or in the inside indicated that the Cys-Au dots were taken up by endocytosis pathways.³⁷ An overlay of fluorescence and bright field images shows that the fluorescence signals are localized in the intracellular area, indicating a good cell-membrane permeability of Cys-Au dots. Furthermore, the as-prepared Cys-Au dots are extremely suitable for long-term cellular imaging. The red signals of the Cys-Au dots are still stable during 90 min continuous observation under laser-scanning confocal microscopy (Fig. 5A), owing to the excellent photostability. By contrast, the signals of the control groups utilizing the CdTe QDs or FITC as fluorescent labels almost disappear after irradiation for short time periods (Fig. 5B and C). According to the previous reports, fluorescence lifetime of CdTe QDs and FITC was at the level of ns (55 ns for CdTe QDs and 3.96 ns for FITC).^38,39 Therefore, the fluorescence lifetime of the as-prepared Cys-Au dots (2.51 μs) was really much longer than that of CdTe QDs and FITC, further indicating that Cys-Au dots were good candidate for long-term cellular imaging.

Fig. 4 (A) Fluorescence confocal images (a) and Bright field (b) of HeLa cells after incubation with Cys-Au dots for 2 h. (B) Fluorescence confocal images (a) and Bright field (b) of A549 cells after incubation with Cys-Au dots for 2 h. (c) in (A) and (B) are the overlay of fluorescence and bright field images for HeLa and A549 cells, respectively. Scale bar = 10 μm.

Fig. 5 Stability comparison of fluorescence signals from HeLa cells labelled with (A) Cys-Au dots, (B) CdTe QDs and (C) FITC, respectively. Scale bar = 20 μm.

In summary, monodisperse and water-soluble Au dots with near-infrared emission have been successfully synthesized and applied to the sensitive and selective detection of Pb²⁺ based on a mechanism of the aggregation-induced fluorescence quenching process. Owing to the low cytotoxicity, ultrasmall size, excellent water dispersibility and good cell membrane permeability, Cys-Au dots were also successfully applied to the optical imaging of tumor cells (HeLa and A549). Moreover, the as-prepared Cys Au dots could also be facilely employed for long-term fluorescence labelling without requiring additional modify procedures. Consequently, our study demonstrated that the present Cys-Au dots may serve as potential biological probes for various biosensing and bioimaging applications.

Acknowledgements

The authors gratefully acknowledge the support from the National Natural Science Foundation of China (21275087, 21305160), the Natural Science Foundation of Shandong Province of China (ZR2012BM008), and the Taishan Scholar Program of Shandong Province, China.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12198h

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