Label-free detection of sulfide ions based on fluorescence quenching of unmodified core–shell Au@Ag nanoclusters

Abstract

Based on the principle of fluorescence quenching, by the interaction between S²⁻ ions and the Ag atoms/ions on the surface of the core–shell Au@Ag NCs, we propose a simple label-free method for the detection of S²⁻ ions with high selectivity and sensitivity by using fluorescent core–shell Au@Ag NCs in aqueous media.

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The sulfide (S²⁻) ion and its compounds are reported to cause a variety of physiological and biochemical problems, such as the corrosion of metal surfaces, the degradation of concrete when oxidized to sulfate and toxicity to living organisms.^1,2 Moreover, the S²⁻ ion has recently emerged as a novel critical mediator in the cardiovascular system, the nervous system and various biological signaling functions.³ The combination of high toxicity and widespread occurrence has created an urgent need for effective monitoring and measurement of S²⁻ ions. Until now, a plethora of methods, including titration,^4,5 spectroscopy,^6–9 electrochemistry,^10,11 chromatography¹² and combinations thereof,¹³ have been applied to detect S²⁻ ions. However, these methods often require time-consuming analysis, complicated procedures, large sample volumes, and/or specialized skills. To overcome these drawbacks, various probe systems for the detection of S²⁻ ions have been developed.^14,15 Although these probe systems provided high selectivity, they fell short of the desirable criteria for S²⁻ ion detection such as low cost and high sensitivity. Thus, there is still an urgent need to develop sensitive and simple probes not only for qualitative analysis but also for quantitative analysis of S²⁻ ions from real samples at trace levels.

Fluorescent gold (Au) and silver (Ag) nanodots (NDs)/nanoclusters (NCs) have recently come to the forefront of the detection of S²⁻ ions as possible means to fulfill the above-mentioned requirements due to their intrinsic advantages such as ease in preparation and conjugation, biocompatibility, and large Stokes shifts.^16–20 For example, a simple and sensitive fluorescence assay has been reported for S²⁻ ion detection based on the fluorescence quenching of glutathione-stabilized Ag NCs.²¹ Recently, Chen et al. designed a fluorescent nanoprobe composed of DNA-templated Au/Ag nanoclusters to detect the spatial and quantitative distribution of S²⁻ ions in an aqueous system.²² However, such sensing systems require quite expensive reagents and long preparation times to synthesize the fluorescent probes prior to use due to their complex labeling or surface functionalization. Therefore, an economical and simple fluorescent sensing system for S²⁻ ions is highly required.

Compared with conventional fluorescent Au and Ag NCs/NDs, core–shell Au@Ag NCs possess some excellent characteristics, such as higher quantum yields,²³ larger diameters, size-tunable electronic transitions, strong fluorescence emissions, and other special chemicophysical properties.^24,25 These interesting chemicophysical properties appear on the combination of Au and Ag NCs and their fine structures, evolving new surface characteristics. As a consequence of their special structure and unique optical properties, core–shell Au@Ag NCs are at the center of significant research efforts into the development of alternatives that have both the desirable optical characteristics of Au/Ag NCs and the structural characteristics of other core–shell metal NCs. So far, to the best of our knowledge, there are only a few studies on the application of core–shell Au@Ag NCs as fluorescent probes in biological and chemical sensing.^26,27

Herein, we propose a simple label-free method for the detection of S²⁻ ions with high selectivity and sensitivity by using fluorescent core–shell Au@Ag NCs in aqueous solution. The principles of the proposed S²⁻ ion sensing concept is shown in Scheme 1. The detection could be realized using the naked eye with the help of 365 nm UV irradiation or can be concisely performed with fluorescence spectrometry, without the requirements for either complicated instrumentation or skilled personnel with knowledge of the electron or energy transfer involved in the electrochemical or conventional systems.

Scheme 1 Proposed graphic for the detection of S²⁻ ions with core–shell Au@Ag NCs.

Initially, the Au NCs were prepared by a bovine serum albumin (BSA)-directed synthesis and purified according to the literature.²⁸ Then the obtained Au NCs were used to act as a core to prepare the core–shell Au@Ag NCs (see the detailed synthesis of the core–shell Au@Ag NCs in the ESI†).

The UV/Vis absorption and the fluorescence spectra of the as-synthesized Au@Ag NCs are shown in ESI Fig. S1.† The Au@Ag NC solution shows a broad UV/Vis absorption with a shoulder at 370 nm (ESI Fig. S1,† curve a). Upon excitation at 370 nm, the fluorescence spectrum of the Au@Ag NCs shows a strong emission peak at 650 nm with a Stokes shift of 280 nm (ESI Fig. S1,† curve b). When the Au@Ag NCs were excited at varying wavelengths from 340 to 480 nm, the emission spectra were always centered at 650 nm (Fig. 1). Meanwhile, the decrease or increase of the excitation wavelengths from 370 nm causes a gradual decrease of the maximum fluorescence intensity. Thus, the as-prepared Au@Ag NCs should be uniform owing to the same emission peak wavelength in the fluorescence spectra for the excitations. As shown in the inset of Fig. 1, the bright yellow aqueous Au@Ag NC solution emits intense red luminescence under UV light.

Fig. 1 Fluorescence spectra of the Au@Ag NCs at different excitation wavelengths from 340 to 480 nm. Inset (from left to right): photographs of an aqueous solution of the Au@Ag NCs taken under visible light and 365 nm UV light, respectively.

The surface of the NCs possesses a small amount of Ag⁺ cations or Ag atoms, which should have strong and specific interactions with the S²⁻ ions due to the very low solubility product (K_sp) value of Ag₂S, namely 8.0 × 10⁻⁵¹ M².^29,30 Therefore, the introduction of S²⁻ ions could generate Ag₂S layers on the Au@Ag NC’s surfaces through the interaction between S²⁻ ions and the Ag atoms/ions. Interestingly, the generation of Ag₂S layers effectively quenches the fluorescence of the Au@Ag NCs. For example, after the addition of 700 μM S²⁻ ions to the 200 μM Au@Ag NC aqueous solution, the red fluorescence of the Au@Ag NCs was almost completely quenched within seconds (ESI Fig. S2†). The fluorescence quenching is attributed to the formation of a Au@Ag/Ag₂S nano-composite. Furthermore, the fluorescence intensity of the Au@Ag NCs is dramatically affected by the amount of S²⁻ ions added. Thus, one can use the interaction between S²⁻ ions and the Ag atoms/ions to design a simple fluorescent assay for S²⁻ ions according to the protocol depicted in Scheme 1.

Since the resulting Au@Ag NCs contained BSA, the solution’s pH value is a critical factor for the S²⁻ ion assay, as evident from Fig. S3 in the ESI.† The maximum response is obtained at pH 7.4. It is known that sulfide exists in three species (H₂S, HS⁻, and S²⁻) in solution defined by its pK_a,³¹ suggesting that the more charged, less protonated species (i.e. HS⁻, and S²⁻) exist at pH 7.4. On the other hand, the zwitterionic BSA molecule is more negatively charged with elevated pH value,³² whereas the Au@Ag NCs are very stable in the form of the core–shell structure themselves as well as in aqueous dispersions under ambient conditions. Therefore, pH 7.4 was chosen to provide the method with some advantages such as ultrasensitivity and convenience compared to the recent reports.^32–34

To observe the morphology of the prepared Au NCs and to identify the formation of the Au@Ag NCs and the resulting Au@Ag/Ag₂S nano-composite in the presence of S²⁻ ions, transmission electron microscopy (TEM) was performed. The TEM images of the NCs show that the average diameter is ∼1.0 nm and ∼1.8 nm for the purified Au NCs (Fig. 2a) and the as-synthesized Au@Ag NCs (Fig. 2b), respectively, demonstrating that growing Ag layers have coated the Au NC surfaces to form Au@Ag NCs. The HRTEM images of the Au NCs and the Au@Ag NCs are shown in insets of Fig. 2a and b, respectively. One can observe the heterojunction structure from the different lattice spacing of Au and Ag. The as-synthesized Au@Ag NCs could be further characterised by cyclic voltammetry. The cyclic voltammogram of a Au@Ag NC film modified Pt electrode in 0.1 M PBS (pH 7.0) within the potential range of −0.1–0.5 V exhibits a typical pair of oxidation–reduction peaks of Ag (ESI Fig. S4†).

Fig. 2 TEM images of (a) BSA-capped Au NCs, (b) Au@Ag NCs, and ((c) and (d)) Au@Ag NCs in the presence of 100 and 600 μM S²⁻ ions, respectively. Inset (a) and (b): HRTEM images of Au NCs and Au@Ag NCs, respectively.

Upon deposition of the Ag shell to the fluorescent Au NC surface, the diameter of the NCs increased by ∼0.8 nm, demonstrating that the Ag shell was ∼0.4 nm thick. Simultaneously, the fluorescence intensity of the Au@Ag NCs is greatly enhanced by the deposition of the Ag shell to the fluorescent Au NC surface. When S²⁻ ions are introduced, the S²⁻ ion-induced Au@Ag/Ag₂S nano-composite is formed, and the diameter of the Au@Ag/Ag₂S nano-composite increased to 3–4 nm (Fig. 2c), and the fluorescence of the Au@Ag NCs is dramatically quenched in the presence of S²⁻ ions (ESI Fig. S2†). It is worth noting that the size of the nano-composite becomes larger with increasing S²⁻ ion concentration and the results are shown in Fig. 2d.

Considering the promise of the Au@Ag NC sensor system for application in biological and environmental fields, the selectivity of the photoluminescent sensor for S²⁻ ions was evaluated. The high specificity of Ag⁺–S²⁻ interactions^22,29 provided the excellent selectivity of this method towards detecting S²⁻ ions over other environmentally relevant metal ions. Under the optimal conditions [phosphate buffer (5 mM, pH 7.4)], we tested the selectivity of the Au@Ag NC (40 μM) probe toward S²⁻ ions (100 μM) against one of the following ions and their corresponding species, (for simplicity, SCN⁻, acetate (Ac⁻), PO₄³⁻, NO₂⁻, CO₃²⁻, HCO₃⁻, HPO₄²⁻, F⁻, Cl⁻, I⁻, ClO₄⁻, SO₃²⁻, SO₄²⁻, and S₂O₈²⁻ were denoted; each 1000 μM, and Br⁻ was 700 μM). Fig. 3 shows the fluorescence spectra as well as relative fluorescence intensity [(F₀ − F)/F₀] of the Au@Ag NC solutions containing 100 μM of S²⁻ ions, 700 μM of Br⁻ or 1000 μM of other ions, respectively. The fluorescence intensities of the Au@Ag NCs in the absence and presence of other ions are denoted by F₀ and F, respectively. The results demonstrate that S²⁻ ions can significantly decrease the fluorescence intensity of the Au@Ag NCs in the presence of all the potential competitors tested. In contrast, no clear reduction is observed with any other ions. It is worth noting that Br⁻ does not show large interference on the biosensor for S²⁻ ion detection due to the difference of the K_sp between Ag₂S and AgBr. The significant interference from I⁻ may be attributed to the formation of AgI, whose K_sp is 8.3 × 10⁻¹⁷ M².³⁵ To minimize the interference from I⁻ ions, further investigation demonstrates that S₂O₈²⁻ as a I⁻ masking agent is able to capture I⁻ from the AgI precipitation,²² and thus S₂O₈²⁻ ions (0.5 mM) were added to the phosphate buffer as a masking agent; I⁻ reacted with S₂O₈²⁻ to form I₂ and SO₄²⁻.^22,36 As expected, the interference from I⁻ ions for the Au@Ag NC probe toward S²⁻ ions is negligible in the presence of S₂O₈²⁻ (Fig. 3b). It is to say that the interference from I⁻ ions could be effectively eliminated by the addition of S₂O₈²⁻ (Fig. 3a and b). Therefore, this probe offers the advantages of simplicity, selectivity, reproducibility and good stability, which make it a valuable alternative to other optical sensing systems employed to date for the detection of S²⁻ ions at trace levels. The above results validate that the method meets the selectivity requirements of the S²⁻ ion assay in environmental fields.

Fig. 3 (a) Fluorescence emission spectra of the Au@Ag NCs (40 μM) in the presence of various ions in 5 mM phosphate buffer (pH7.4). (b) Selectivity of the fluorescence assay for S²⁻ ions over other ions. The concentration of S²⁻ ions was 100 μM. The concentration of the other ions was 1000 μM except for Br⁻ ions which was 700 μM. To detect I⁻ ions, 0.5 mM S₂O₈²⁻ was also added. The excitation wavelength was 370 nm and the emission at 650 nm was monitored. The inset is the visual picture showing fluorescence changes (from left to right) corresponding to the different fluorescence intensity [(F₀ − F)/F₀] values.

The fluorescence at 650 nm of the Au@Ag NCs decreased upon increasing the concentration of the S²⁻ ions from 0 to 700 μM. As indicated in Fig. 4a, the sensitivity and linearity of the Au@Ag NC–S²⁻ system was evaluated by varying the S²⁻ ion concentrations in the presence of phosphate buffer (5 mM, pH 7.4). With the increase of the concentrations of the S²⁻ ions, the fluorescence emission intensity of the Au@Ag NCs at 650 nm decreased gradually. A linear region was obtained in the plots of the value of [(F₀ − F)/F₀] for the Au@Ag NCs versus the concentrations of S²⁻ ions, 0–700 μM (Fig. 4b).

Fig. 4 (a) Fluorescence emission spectra of Au@Ag NCs (200 μM) in the presence of different concentrations of S²⁻ ions (0–700 μM, top to bottom, excitation at 370 nm). (b) Relative fluorescence intensity [(F₀ − F)/F₀] of Au@Ag NCs (200 μM) in the presence of different concentrations of S²⁻ ions (0–700 μM). The inset is the visual image of the solutions corresponding to S²⁻ ion concentrations (from left to right) of 0, 50, 100, 200, 300, 400, 500, and 600 μM.

The correlation coefficients (R²) of the linear plots were 0.994, and the limit of detection (LOD) for S²⁻ ions, at a signal-to-noise ratio of 3, was estimated to be 0.31 μM (∼10 ppb), which was much lower than the maximum level (15 μM, 500 ppb) of S²⁻ ions in drinking water permitted by the World Health Organization (WHO).^37,38 This approach provided a sensitivity that was much better than those reported for S²⁻ ions sensors based on optical sensors.^2,33,34

To further explore the mechanism of the fluorescence quenching, UV/Vis absorption and fluorescence spectra of Au@Ag NCs were investigated in the absence and presence of S²⁻ ions. When aliquots of S²⁻ ion solution were successively added to the Au@Ag NC solution, the fluorescence intensity decreased gradually with a distinct change (Fig. 4a) in the profile of the fluorescence spectra (blue shift, peak shape and position), which is similar to the observation by Tian et al.³⁹ and Shang et al.⁴⁰ The blue shift may arise from the formation of a metal complex^41,42 or reduced polarity in the local environment of the emitting species.⁴⁰ These results indicate that higher concentrations of S²⁻ ions can quench the fluorescence of the Au@Ag NCs more effectively (Fig. 4a). The first excitonic absorption peak of the original Au@Ag NCs was shifted towards the shorter wavelength in the presence of S²⁻ ions (ESI Fig. S5†). Meanwhile, the fluorescence spectra of the Au@Ag NCs with 600 μM S²⁻ ions at different excitation wavelengths from 330 to 410 nm are reported. As shown in the ESI, Fig. S6,† the maximum fluorescence was still obtained at the excitation wavelength of 370 nm, but the fluorescence peaks of all of the spectra shifted to ca. 630 nm compared with 650 nm for pure Au@Ag NCs. It can be concluded that there is a possibility of ground state interaction between them and the quenching may be because of the surface capping phenomenon and trapping of photogenerated holes of the Au@Ag NCs induced by the S²⁻ ions which is schematically depicted in Scheme 1.

For comparison, the Au NCs were also used to detect S²⁻ ions due to the similar quenching effect via the interaction between S²⁻ ions and the Au atoms/ions on the surface of the Au NCs. Although the K_sp of Ag₂S (8.0 × 10⁻⁵¹ M²) is not as low as the K_sp of Au₂S (1.58 × 10⁻⁷³ M²), the sensitivity for S²⁻ ion detection of the core–shell Au@Ag NCs is much higher than that of the Au NCs, while the response time of the Au@Ag NCs (several seconds) is much shorter than that of the Au NCs (120 min). Furthermore, compared with Au NCs, the Au@Ag NCs are much more stable in high ionic strength media and a wider range of pH values. The reason for the enhancement in the fluorescence intensity of the core–shell Au@Ag NCs and the stability of the Au@Ag NCs may be caused by the synergistic effect between Au and Ag. Therefore, there is a dramatic increase in the sensitivity for the detection of S²⁻ ions using the Au@Ag NC fluorescent probe. The results reveal that the Au@Ag NC probe offers advantages of simplicity, precision, and speed for determining the concentration of S²⁻ ions in the aqueous system.

In conclusion, a new, simple, rapid, label-free method has been developed to detect S²⁻ ions with very high selectivity and sensitivity using fluorescent core–shell Au@Ag NCs in aqueous media. The addition of S²⁻ ions to well-defined core–shell Au@Ag NCs generates Ag₂S layers on the surface, which effectively quenches the fluorescence of Au@Ag NCs. The strategy described in this report offers advantages over many existing sensing methodologies. First, our approach is convenient, requiring only the mixing of two solutions at room temperature to achieve semiquantitative detection through visual inspection in 365 nm UV light or quantitative detection by using fluorescence spectroscopy, in which any usual steps such as modification and separation are therefore successfully avoided. Second, the detection of S²⁻ ions using this approach is rapid, requiring only a few seconds to complete the whole testing process. Third, our approach is fairly practical, economical and simple, requiring only NCs to detect S²⁻ ions, not using other expensive agents (e.g. DNA) or nanomaterials. Although the method showed a remarkably high selectivity for S²⁻ ions over other ions, and detected S²⁻ ions at concentrations as low as ∼10 ppb, this strategy has its own limitations, for example, the Au@Ag NCs cannot be employed reversibly due to the irreversible ion-induced Ag₂S precipitation. In short, this process is notable as it involves green chemistry, and could be developed as a simple approach for the rapid routine monitoring of S²⁻ ions.

Acknowledgements

This work was supported by the Natural Science Foundation of China (No. 21345008), the Fundamental Research Funds for the Central Universities, the Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University, and the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1211).

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and Fig. S1–S6. See DOI: 10.1039/c3ra45019h

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