Weihua Dingab,
Saipeng Huangab,
Lingmei Guanab,
Xianhu Liuab and
Zhixun Luo*a
aState Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. E-mail: zxluo@iccas.ac.cn
bGraduate University of Chinese Academy of Sciences (GUCAS), Beijing 100049, P. R. China
First published on 16th July 2015
Ligand-protected metal nanoclusters (NCs) having hydrodynamic diameters and fluorescent properties have reasonable potential for ion detection and bio-imaging applications by taking advantage of their smaller sizes and degradability as compared with larger nanoparticles. By controlling the pH value, here we have synthesized a fluorescent silver nanocluster, Ag–glutathione, which is found to be a versatile chemosensor operative for the detection of both manganese (Mn2+) and iodine (I−) ions. Mn2+ and I− are further distinguished by the ratiometric absorption spectrometry method. Care was taken for sufficient spectral analyses, based upon which we have fully demonstrated the chemosensing mechanisms of Ag@glutathione (Ag@SG) NCs applied in ion detection. Having expounded this issue, we further investigated the potential application of Ag@SG NCs in bio-imaging. It is interesting to find that the as-prepared Ag@SG NCs are nontoxic and available for fluorescence imaging of MCF-7 cells, with on/off alternation of fluorescence emission in the presence of Mn2+ or I− ions. The low cytotoxicity, good penetrability and on/off fluorescence property of Ag@SG NCs in MCF-7 cells suggest promising biological applications.
For this purpose, many strategies have been used in the past, including chromatography, electrochemical analysis, spectrometry with artificial chromophore and fluorophore, etc.3 Among these available detection methods, fluorescence chemosensors such as conjugated fluorescent polymers have stimulated increasing research interest owing to their high sensitivity and simplicity with rapid response.4 Recently, with the development of nanotechnology, nanoscaled fluorescent chemosensors have become the most applied analytical tools to detect and trace ions in aqueous solution.5–9 It has been recognized that metal nanoclusters (NCs; small nanoparticles having sizes between 1 and 10 nanometers) encapsulated in different scaffolds such as thiols and even DNA, etc., are effective for ion detection as the ligand protection provides enhanced stability frequently under a superatom cluster concept.10–13 Increasing advances in this field enable a promising future for the design of NCs-based chemosensors having notable optical, electrical and chemical properties for bioimaging and ion detection.14–26
However, a common view in this field is that chemosensors are usually valid for detection of either a cationic ion or an anionic ion, thus a pending question remains as to what happens if both cationic and anionic ions coexist in a complex unknown solution. Also, only rarely have fluorescent chemosensors been applied in living cells to our best knowledge. It is highly desirable to develop eco-friendly and nontoxic fluorescent chemosensors with high sensitivity for ion detection. Facile synthesis procedure and high selective capability are expected in order to enable detection of multiple ions simultaneously. In addition, the predominant sensing mechanism varies depending on the type of the sensor, and is accommodated strictly by the altering of the fluorescence property of the NCs through specific interactions with either the metallic core or the protecting ligand.27–31 These issues are still open questions and need to be further explored.
Thanks to the biocompatible glutathione (HSG) which does not exhibit cytotoxicity itself, the Ag–glutathione complex also does not show obvious cytotoxicity with concentrations up to mmol L−1.2,32–36 Motivated by this, here we have synthesized eco-friendly silver NCs, and performed an in-depth study of ion detection utilizing the glutathione-protected silver (Ag@SG) NCs as a fluorescence chemosensor. It is interesting to find that the as-prepared Ag NCs chemosensor is effective for the detection and differentiation of Mn2+ and I− ions. Together with an insight into the sensing mechanisms, we demonstrate feasible applications in living cells with on/off controllability in the presence of predetermined ions.
The Ag@SG NCs were synthesized according to the following procedure (Scheme 1). Firstly, an aqueous solution of AgNO3 (84.9 mg, 0.5 mM) and glutathione (GSH; 615.0 mg, 2.0 mM) were mixed together in 25 mL water with vigorous stirring, and the solution was kept at ∼0 °C in an ice bath. Next, an aqueous solution of NaOH (0.5 M) was added into the above solution until the white precipitate completely disappeared (pH = 6). To this clear solution, freshly prepared ice-cold aqueous NaBH4 (189.0 mg, 5.0 mL) was slowly added dropwise under vigorous stirring. Then, the reaction was allowed to proceed under constant stirring for 7 h, and the clusters were precipitated with methanol. After filtration, a brown precipitate of Ag@SG NCs was obtained, which was then washed with excess methanol.
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Scheme 1 A schematic depicting the synthesis procedure of glutathione-protected silver nanoclusters in this study. |
Ion detections were conducted in a quartz cuvette (1 cm beam path). Briefly, small amounts of concentrated salt solutions were added to the Ag@SG NCs water solution, which did not give rise to obvious pH value changes. In all titration experiments, the metal cations (Fe3+, Co2+, Cd2+, Ca2+, Cr3+, Cu2+, Pb2+, Ni2+, Zn2+ and Mn2+) and anions (F−, Cl−, Br−, I−, NO2−, CO32−, AcO−, H2PO4−, HCO3−, SO42−, HPO42−, HSO3−, and OH−) were referred to normal ionic compounds as largely used in this field.37–41
The in vitro cytotoxicity was measured using the methyl thiazolyl tetrazolium (MTT) assay in MCF-7 cell lines. Cells in log-growth phase were seeded into a 96-well cell-culture plate at 1 × 104 per well in 100 μL DMEM (Dulbecco's modified Eagle's medium). Ag@SG NCs were added to the wells (100 μL per well) of the treatment group, and the final concentration ranged from 0 to 0.5 μg mL−1. The cells were incubated for 24 h at 37 °C under 5% CO2. A combined MTT/PBS solution (100 μL, 0.5 mg mL−1) was added to each well of the 96-well assay plate, and incubated for an additional 2 h. An enzyme-linked immunosorbent assay reader (μQuant, Bio-Tek, USA) was used to measure the OD570 (absorbance value) of each well referenced at 690 nm. The formula used to calculate the viability of cell growth is: viability (%) = (mean absorbance value of treatment group/mean absorbance value of control) × 100.
Prior to the bioimaging experiment, MCF-7 cells were cultured in DMEM. Cells were incubated with 0.25 μg mL−1 Ag@SG NCs at 37 °C for 2 h. After washing with PBS three times to remove remaining Ag@SG NCs, the cells were then incubated with an appropriate concentration of Mn2+ and I− solution for 30 min at room temperature. The incubated cells were washed with PBS and mounted onto a glass slide. Fluorescence images of the mounted cells were obtained using a confocal laser scanning microscope at 488 nm excitation.
The Ag@SG NCs exhibit an intense fluorescence emission peak centered at ∼630 nm (Fig. 1b, excited at 400 nm). As a comparison, also provided in Fig. 1b is the excitation spectrum which is consistent with the aforementioned UV-visible absorption behavior. In addition, the monodispersity of the Ag@SG NCs product was checked by polyacrylamide gel electrophoresis analysis (Fig. S1b, ESI†), which indicates a decent grade of purity of the as-prepared clusters. XPS (Fig. S3a and 3b, ESI†) survey spectrum of the Ag@SG NCs confirms the presence of Ag, S, C, N, O elements, and the Ag 3d5/2 peak at 368.2 eV is close to the Ag(0) value, which matches a previously published report;42 the presence of S (2p3/2) at 162.3 eV suggests that the ligand is chemically bonded to a silver cluster in the form of thiolates,43 which is in agreement with the IR spectrum (Fig. S4, ESI†) where the 2523 cm−1 mode corresponding to the S–H stretching of a HSG molecule disappears, evidencing that the formation of Ag@SG NCs is stabilized by S–Ag bondings.42 In addition, the Auger electron peak of silver (Fig. S3c, ESI†) confirms the presence of Ag(I); in other words, Ag(0) and Ag(I) coexist in Ag@SG NCs.
Following the successful synthesis of Ag@SG NCs, we performed a study of ion detection by observing the fluorescence changes of NCs when added to aqueous metal ions. As shown in Fig. 2a, the obtained Ag@SG NCs display strong fluorescence emission at ∼630 nm; however, the fluorescence emission intensities change in the presence of different cationic metal ions. The cations Fe3+, Co2+, Cd2+, Ca2+, Cr3+, Cu2+, Pb2+, Ni2+ and Zn2+ were found to enhance the nascent fluorescence intensity of NCs to different extents; however, the addition of Mn2+ to Ag@SG NCs exclusively results in a remarkable fluorescence quenching.
To further investigate the selectivity of Ag@SG NCs for Mn2+, we conducted a competitive experiment endeavoring to evaluate the practical effect in detecting Mn2+ ions in the presence of other metal ions. As shown in Fig. 2b, the coexistence of 0.1 equivalent of other metal ions in 1.0 mM Mn2+ ions also leads to an obvious fluorescence quenching effect. Therefore it is possible to use Ag NCs to detect Mn2+ ions up to millimoles even in the presence of other interference metal cations (up to 0.1 mM in this study), revealing that Ag@SG NCs have high selectivity toward Mn2+ among the other metal ions in water.
We have also conducted fluorescence titration experiments for the silver NCs when adding Mn2+ ions of varying concentrations (Fig. 2c). The fluorescence intensity at 630 nm decreases remarkably with increasing concentration of Mn2+ ions. Upon further increase of the Mn2+ concentration, fluorescence emission tends to be quenched completely. Further analysis depicted in Fig. 2d reveals that there is a good linear relationship (coefficient of determination R2 = 0.97) in the low concentration range of 0 to 1.5 mM, although a linear convergence beyond 2.0 mM. From the linear fitting curve (red) it is deduced that the limit of detection for Mn2+ ions is ∼9.2 μM indicating decent sensitivity of this method which is comparable to that of other reported chemosensors for detecting Mn2+ ions.44–47
Interesting results were noted when we further examined the selectivity of the Ag@SG NCs toward anions. Fig. 3a displays the fluorescence responses in water with F−, Cl−, Br−, I−, NO2−, CO32−, AcO−, H2PO4−, HCO3−, SO42−, HPO42−, HSO3−, and OH− among which only I− quenches the fluorescence of Ag@SG NCs while the other anions even show minor enhancements to their fluorescence intensities. Similar to the aforementioned case for Mn2+, we have also performed competition experiments for I− in the presence of the other anions, as shown in Fig. 3b. It is notable that the presence of other anions had no obvious interference effects compared with the experimental results when only adding I− itself. In the fluorescence titration experiment of Ag@SG NCs with I− (Fig. 3c), the fluorescence intensity decreased with increasing I− concentration, with a good linear relationship (R2 = 0.96) in the concentration range from 0.03 mM to 0.26 mM. The limit of detection for I− was calculated to be ∼5.9 μM (Fig. 3d), indicating good sensitivity to the detection of I− ions, which is comparable to that of previously published reports.40,41 It is additionally worth mentioning that, when S2− ions exist in the solution, Ag@SG NCs do not show good selectivity for I− ions, which is probably caused by the strong affinity of silver toward S2− ions.2
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Fig. 4 Fluorescence responses of (a) Ag NCs + I− upon the addition of metal ions and (b) Ag NCs + Mn2+ upon the addition of anions in H2O. |
With this in mind, we have studied the ratiometric absorption behaviors for Mn2+ and I−. Fig. 5 presents results of UV-visible titration experiments of Ag@SG NCs in the presence of increasing concentrations of Mn2+ and I− ions, respectively. It is notable that the position of the broad absorption band from 350 nm to 500 nm barely changes, but the intensity decreased gradually with increasing Mn2+ ion concentration (Fig. 5a). This result excludes the possibility of direct binding of Mn2+ to the Ag atoms due to the absorption band of Ag nanoclusters being very sensitive to the presence of adsorbed substances.48 However, the gradual addition of I− ions (Fig. 5b and c) differs distinctly from that of Mn2+. Besides the gradual intensity decrease of the wide absorption band from 400 nm to 500 nm, two iso-absorptive points are noted successively in different concentration ranges. The first isobestic point emerges at ∼398 nm with I− concentration increasing in the lower concentration range, while more gradual addition of I− ions leads to the second iso-absorptive point at ∼378 nm. In addition, the spectrum exhibits a new absorption peak at ∼420 nm in the higher I− concentration range. Exclusion experiments were conducted by observing UV-visible absorption spectra of aqueous HSG in the presence of I− ions (Fig. S5, ESI†); however, the addition of I− to HSG solution was found to bring negligible changes. Therefore, the likely formed new complexes in the solution can be associated with the reaction of Ag@SG NCs core and I− ions. These results indicated that Ag@SG NCs could be used to distinguish Mn2+ from I− by the ratiometric absorption spectrometry method.
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Fig. 5 UV-visible titration spectra of Ag@SG NCs (10 μg mL−1) in the presence of increasing amounts of Mn2+ (a) and Ag@SG NCs (2.5 μg mL−1) in the presence of increasing amounts of I− (b and c). |
Fig. 7 provides a comparison of XPS patterns of Ag 3d core level for the Ag@SG NCs before and after reacting with Mn2+ and I− ions. For the case when adding I− ions (Fig. 7a), it is clearly seen that the peaks assigned to both Ag 3d5/2 (368.2 eV) and Ag 3d3/2 (374.1 eV) shift to the lower-energy side by up to 1.0 eV in the presence of I− ions. This is in sharp contrast to the situation when adding Mn2+ ions where a minor shift is towards higher energy (Fig. 7c). The low-energy shifts in the presence of iodide ions demonstrate the complexation of Ag with electron-rich groups or elements such as iodide. This is an important piece of evidence as fluorescence quenching often could be caused by energy transfer or static quenching by forming nonfluorescent complexes. Actually many studies of the interactions of halide ions with silver or other nanoparticles have been reported,49–51 and it is known that iodides have a stronger affinity for noble metal nanoclusters than the other counterparts.52–54 Further evidences are determined by noting the presence of AgnIx species via mass spectrometry (Fig. S6, ESI†). Combining with the ratiometric absorption behaviors for I− (Fig. 5b and c), the isobestic points indicate that there are multiple reaction pathways in a solution of Ag@SG NCs with gradual addition of I−. It is ascertained that I− ions in low concentration adsorb rapidly on the Ag@SG NCs surface owing to the high strength of the Ag–I bond or the low solubility of AgI, which leads to catalyzed etching likely via a reaction pathway analogous to that of a strong acid displacing a weak acid, as described in eqn (1):
Agn(SG)x− + yI− + yH+ → Agn−y(AgI)y(SG)x−y− + yHSG, or |
Agn(SG)x− + yI− + yH+ +yH2O → Agn−y(SG)x−y− + yAgI(H2O)0 + yHSG | (1) |
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Fig. 7 XPS spectra of Ag 3d core level for the Ag@SG NCs in the presence of I− (a), Ag@SG NCs itself (b) and in the presence of Mn2+ (c). |
In this equation, the formation of Agn−y(AgI)y(SG)x−y− or AgI(H2O)0 species adsorbed in the Stern plane of Ag@SG NCs may play a role in the stability by truncating the van der Waals attraction at distances of closest approach relative to the electrostatic repulsion interactions.55 Next, more I− ions chemisorbed on the surface of NCs may rearrange the surface charges,1 which will increase the weak intermolecular interactions among the newly formed iodide-interspersed silver particles and then lead to aggregation. At the same time, a small number of protection ligands means reduced sizes of the NCs, allowing for increased specific surface area and free surface energy, and hence incidental aggregation (eqn (2)):
Agn-y(AgI)y(SG)x−y− + Agn(SG)x− + zI− + γH+ → Ag2nIy+z(SG)δ(HSG)γ−, or |
Agn(SG)x− + yAgI(H2O)0 + yI− → Agn+yI2y(SG)x−y(HSG)y− + yOH−, or |
Agn−y(SG)y− + Agn−y(SG)y− + zI− + γH+ → Ag2n−2yIz(SG)δ(HSG)γ− | (2) |
Different from I− ions, the result that Mn2+ induces aggregation of Ag@SG NCs in solution could simply follow a well-known AIFQ (aggregation-induced fluorescence quenching) mechanism, which profits from the interaction of hard acid Mn2+ ions with the multiple hard base –NH and –O moieties present in several –SG ions. What is more, Mn2+ is a highly paramagnetic ion, which can quench the fluorescence of metal NCs via energy transfer and weak bonding (eqn (3)):
Agn(SG)x− + Agm(SG)y− + Mn2+ → [Agn(GS)x⋯Mn⋯(SG)yAgm]0 | (3) |
The final important result relates to the attempt to detect Mn2+ and I− in living cells. For this study, MCF-7 cells were incubated with 0.25 μg mL−1 Ag@SG NCs for 2 h firstly, and then treated separately with 1.5 mM Mn2+ and 0.3 mM I− for 30 min. As shown in Fig. 9a, the cells only incubated with Ag@SG NCs display strong fluorescence, while the strong red fluorescence is quenched in cells when exposed to aqueous solution of Mn2+ (Fig. 9b) or I− (Fig. 9c) ions. It is noted that the chemosensor of Ag@SG NCs is cell-permeable allowing for bioimaging in living cells and the decent fluorescence on/off property enables detection of intracellular Mn2+ and I− ions. Besides, the shape of the MCF-7 cells further indicated no cytotoxicity effect of the Ag@SG NCs and no aggregation on the cell membrane. Our biolabeling and chemosensing effect in living cells is comparable to that of recent reports in this field, such as HSG-protected silver nanoclusters used in A549 cell imaging by Le Guevel et al.,57 and Ag NCs were used to demonstrate cytocompatibility and show better inhibition effects through MTT assay by Li et al.58
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Fig. 9 Confocal fluorescence images of (a) only Ag@SG NCs, (b) addition of Mn2+ and (c) addition of I− in MCF-7 cells in the fluorescence (left), bright (middle), and overlay (right) field. |
Footnote |
† Electronic supplementary information (ESI) available: Chemicals, properties of the silver clusters, more spectral experimental details. See DOI: 10.1039/c5ra11124b |
This journal is © The Royal Society of Chemistry 2015 |