Step-by-Step Design and Synthesis of Au@SiO2@Phenyl-azathiacrown for SERS-Based Specific Quantification of Inorganic Mercury.

Direct SERS-based quantification of inorganic metal species has been a problem, because they have a small Raman cross-section or even no vibrational mode. Here, we report a new strategy for SERS-based quantification of such metal species, as exemplified by inorganic mercury (HgII) in waters. Step-by-step design and synthesis from azathioethers [3, 9-dithia-6-monoazaundecane (DMA) and 3,6,12,15-tetrathia-9-monoazaheptadecane (TTM)] to an azathiacrown [7-aza-1,4,10,13-tetrathiacyclohexadecane (NS4)] demonstrate an improved S-pulling effect and size-fit specificity towards HgII to form Hg-S bonds. Modification of NS4 on the surface of Au@SiO2 by using a 4-(bromomethyl)benzoic linker enabled direct SERS-based specific quantification of HgII for the first time, in which the ultrathin layer (ca. 2 nm) that covered the Au core (55 nm) could be a barrier preventing the Au core from having direct interaction with the HgII, and with phenyl serving as an internal standard (IS). The ratio of the Hg-S SERS band intensity at 270 cm-1 to that of IS [(γCC+γCCC) at 1046 cm-1] was practically proportional to the concentration of HgII, eliminating the inevitable uncertainties encountered in SERS-based measurements. Such a methodology is expected to pave a new way for SERS-based quantification of inorganic metal species when specific complexing substrates and suitable ISs are designed.

As an affordable molecular structure information tool, surface‐enhanced Raman scattering (SERS) on the roughened nanostructured surface of noble metals amplifies the orders of magnitude of Raman signals.1 One, thus, expects such a sensitive SERS to be applicable to the quantification of not only target molecules, but also small inorganic metal species.2 However, SERS‐based quantification is still a difficult task, especially in the direct and reliable quantification of inorganic metal species. They have small Raman cross‐sections or even no vibrational modes, providing almost negligible Raman signals, for example, inorganic mercury (HgII) in waters. To the best of our knowledge, almost all SERS‐based methods reported for the detection of HgII are mediated by Raman reporting molecules. Interactions between HgII and the SERS reporting molecules or the SERS reporting‐molecule‐modified pre‐adsorbed ligands lead to variation in their distance to the SERS‐active substrate, causing the SERS signals to turn on or turn off.2a, 3 In this way, HgII was detected indirectly through changes in the SERS intensity or frequency of the reporting molecules, with possible uncertainties. More reliable quantification of the SERS signal that came directly from HgII itself remains challenging, when comparing with techniques that use atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry.4

Herein, we report a new strategy for direct SERS‐based quantification of the inorganic metal species, as exemplified by the specific quantification of HgII, a never out‐of‐date star, because its irreplaceable usefulness, but terrible toxicity, has always attracted attention. Azathioether and azathiacrown, which contain S atoms to form the quasi‐covalent Hg−S bonds, were designed and synthesized step‐by‐step to obtain the direct Raman signal solely from the Hg−S bond. This was modified on the surface of Au@SiO2 nanoparticles within an effective distance5 of the enhanced electromagnetic field through a 4‐(bromomethyl)benzoic linker, achieving highly sensitive SERS of Hg−S. At the same time, the phenyl moiety in the linker served as an internal standard (IS) to normalize the determined Hg−S SERS signal, which might be influenced by possible instrumental variations, less uniform distribution of the electromagnetic hot spots on the surface of SERS‐active Au@SiO2 nanoparticles, and diverse adaptations of the localized near‐field microenvironment towards the samples of different physicochemical properties,6 so as to realize a reliable SERS‐based specific quantification of HgII (Scheme 1).

Scheme 1

Au@SiO2@phenyl‐azathioether/azathiacrown designed for the SERS‐based quantification of HgII.

First, we used a simple sulfhydryl‐bearing compound, 2‐mercaptoethanol (HSCH2CH2OH, 2‐ME), to confirm the SERS signal of Hg−S in an Au@SiO2 nanoparticle sol‐based manner (Figures S1 and S2 a in the Supporting Information). The Raman band of Hg−S at 260 cm−1 was observed and was significantly enhanced by four orders of magnitude when Hg(SCH2CH2OH)2 was physically mixed with the Au@SiO2 nanoparticles (Figure S3 a). Then, we started to synthesize 3,9‐dithia‐6‐monoazaundecane (DMA, 1) (Scheme 1 and Figure S4) and conjugated with 4‐(bromomethyl) benzoic acid (4) to obtain 8‐[N,N‐bis(2‐(ethylthio)ethyl)aminomethyl] phenylacetic acid (phenyl‐DMA, 5) (Scheme 1 and Figure S5). Phenyl‐DMA was then linked with 3‐aminopropyltriethoxysilane (APTES, 6) and anchored onto Au@SiO2 nanoparticles (Scheme 1 and Figure S6) to obtain Au@SiO2@phenyl‐DMA (7) (Scheme 1). The zeta potential (ξ) of Au@SiO2, determined by using DLS experiments, changed from −23.0±0.2 to 44.8±0.9 mV after phenyl‐DMA modification (Table S1). Moreover, their TEM and STEM images indicated that Au@SiO2 had a 55 nm Au core covered by an SiO2 shell (ca. 2 nm) (Figure 1 a1–a5). It should be pointed out that the ultrathin SiO2 shell can be a barrier to prevent the Au core from direct interaction with HgII. The size of Au@SiO2@phenyl‐DMA (Figures 1 b1–b2) did not increase obviously, owing to the length of phenyl‐DMA (0.85 nm, as calculated by using the Molecular Mechnics MM2 method in ChemBio3D Ultra 14.0 software). STEM images (Figures 1 b3–b6) of Au@SiO2@phenyl‐DMA, especially the superimposed STEM image of Si and S (Figure 1 b7), clearly showed the modification of S‐containing phenyl‐DMA on the surface of Au@SiO2. Au@SiO2@phenyl‐DMA was subsequently used for the SERS‐based quantification of HgII. The Hg−S SERS band was observed as a shoulder at 275 cm−1, together with the Hg−N band at 236 cm−1 (Figure 2 a) that was verified by using the experiment of HgII with 2‐aminoethanol (H2NCH2CH2OH, 2‐AE) (Figure S2 b). A 15 cm−1 shift of the Hg−S SERS band compared to that (260 cm−1) in the case of 2‐ME was attributed to the interaction between Hg and N in DMA that competed and, thus, perturbed Hg−S bonding. Moreover, other inorganic metal species such as CdII, ZnII, CuII, PbII, MnII, NiII, FeIII, and AgI, which are similar in physicochemical properties considering their interactions with S and N atoms, and possibly coexist with HgII in water samples, might be trapped together by DMA. We investigated their SERS behavior, and the observed SERS bands of Cd−N (263 cm−1), Cu−N (258 cm−1), and Fe−N (222 cm−1) as well as Ag−N (240 cm−1) and Ag−S (238 cm−1) overlapped with those of Hg−N and Hg−S (Figure 2 a), indicating that AgI, CdII, CuII, and FeIII interfere with the determination of HgII when using Au@SiO2@phenyl‐DMA (Figure S2 c and S2 d).

Figure 2

SERS spectra. a) Au@SiO2@phenyl‐DMA‐(HgII, AgI, CdII, CuII, FeIII, MnII, NiII, PbII, and ZnII); b) Au@SiO2@phenyl‐TTM‐(HgII, AgI, CdII, CuII, FeIII, MnII, NiII, PbII, and ZnII); c) Au@SiO2@phenyl‐NS4‐(HgII, AgI, CdII, CuII, FeIII, MnII, NiII, PbII, and ZnII). The Au@SiO2@phenyl‐DMA, Au@SiO2@phenyl‐TTM, and Au@SiO2@phenyl‐NS4 used were 3 mg mL−1 and the metal species HgII, AgI, CdII, CuII, FeIII, MnII, NiII, PbII, and ZnII used were 1 μg mL−1 each. Laser: 785 nm; power: 3 mW; exposure time: 10 s. All the data presented were those after subtracting the background determined when using Au@SiO2 NPs alone. Each point is the mean of five duplicate experiments.

Higher bond dissociation energies of Ag−S (217 kJ mol−1), Cd−S (208), Cu−S (274), Fe−S (323), and Hg−S (217) compared to those of A−N (170 kJ mol−1), Cd−N (160), Cu−N (192), Fe−N (184), and Hg−N (177) (Table S2) implied that stronger interactions of these inorganic metal species with S than with N. Therefore, we considered introducing more S atoms into the DMA complexing moiety to synthesize 3,6,12,15‐tetrathia‐9‐monoazaheptadecane (TTM, 2) (Scheme 1) so as to pull them away from the N in TTM. This was an effective way to weaken the coordinative bond formed between the metal species and N, as evidenced by the results obtained using the synthesized Au@SiO2@phenyl‐TTM (Table S1 and Figure 1 c; its synthesis and characterization are described in Figures S7–S9). The SERS bands at 263 cm−1 (Cd−N), 260 cm−1 (Cu−N), and 222 cm−1 (Fe−N) as well as Hg−N and Ag−N disappeared compared to those observed in the case of Au@SiO2@phenyl‐DMA; however, Ag−S (232 cm−1) and Hg−S (270 cm−1) were still there with merely 6 and 5 cm−1 shifts, respectively (Figure 2 b). These observed phenomena indicated that the interactions between the metal species and N became negligible, confirming our consideration that more S atoms in TTM pull the metal species away from N. Elimination of the interferences from CdII, CuII, and FeIII towards HgII was achieved. However, the broad bands of Ag−S and Hg−S partly overlapped, inferring that AgI still interfered with the determination of HgII. In this situation, we had to use Cl− to get rid of AgI from the sample before SERS measurements (Figure S10), as AgI could easily be removed by forming an AgCl precipitate (K sp=1.8×10−10); whereas, HgII formed more soluble Hg−Cl complexes, for example, the stability constants of HgCl+ is 5.5×106 and HgCl2 1.6×1013,7 along with the increase in Cl− concentration, avoiding its co‐precipitation with AgCl.

Figure 1

TEM and STEM images. a1, a2) TEM and a3) STEM images of Au@SiO2, a4–a6) corresponding elemental mapping (Au−M, Si−K, S−K signal), and a7) the superimposed STEM of Si−K and S−K. b1, b2) TEM and b3) STEM images of Au@SiO2@phenyl‐DMA, b4–b6) corresponding elemental mapping (Au−M, Si−K, S−K signal), and b7) the superimposed STEM of Si−K and S−K. c1, c2) TEM and c3) STEM images of Au@SiO2@phenyl‐TTM, c4–c6) corresponding elemental mapping (Au−M, Si−K, S−K signal), and c7) the superimposed STEM of Si−K and S−K. d1, d2) TEM and d3) STEM images of Au@SiO2@phenyl‐NS4, d4–d6) corresponding elemental mapping (Au−M, Si−K, S−K signal), and d7) the superimposed STEM of Si−K and S−Kl.

Crown molecules can recognize metal ions of different diameters by their cavity size.8 Combining this size‐fit property and the S‐pulling effect on the soft metal species, as demonstrated in the case of TTM, we designed and synthesized 7‐aza‐1,4,10,13‐tetrathiacyclohexadecane (NS4, 3) (Scheme 1), an azathiacrown that contains four S atoms, to further improve the binding‐selectivity towards HgII. It was modified onto the surface of Au@SiO2 following the same procedures as in the cases of Au@SiO2@phenyl‐DMA and Au@SiO2@phenyl‐TTM to obtain Au@SiO2@phenyl‐NS4 (Table S1 and Figure 1 d; its synthesis and characterization are described in Figures S11–S13). The TEM and STEM images (Figures 1 d1–d6) as well as the superimposed STEM image of Si and S (Figure 1 d7) confirmed the modification of phenyl‐NS4 (1.1 nm as calculated with MM2) on the surface of Au@SiO2. In addition, the ξ value of Au@SiO2@phenyl‐NS4 increased to 11.7±1.5 from −23.0±0.2 mV for Au@SiO2 (Table S1). Specific recognition of HgII was realized at the SERS band of 270 cm−1 with four orders of magnitude signal enhancement (see Figure S3 b), whereas the SERS band of Ag−S at 232 cm−1 and those of other metal species were not detected, as shown in Figure 2 c. The formed HgII chelate was verified by using mass spectroscopy (Figure 3). HgII was coordinated in the chemical form of Hg(OH)2 [phenyl‐NS4‐Hg(OH)2, m/z 710] with the precise isotope distribution pattern based on the theoretical estimation using IsoPro 3.0 software. This was in agreement with the fact that the predominant species of HgII in natural water is Hg(OH)2 (stability constant is 1.0×1022) with an approximate linear configuration.9 Moreover, theoretical minimized‐energy calculations with MM2 indicated that the distance between the diagonal sulfur atoms in the phenyl‐NS4 cavity was 5.878 and 7.666 Å for 4S to 11S and 7S to 14S, respectively (Figure S14 a). After coordinating to Hg(OH)2, the distance between 4S and 11S decreased to 4.832 Å for 4S−Hg−11S with a bond angle of 148.3°, and that of 7S to 14S decreased to 4.900 Å for 7S−Hg−14S with a bond angle of 154.9° (Figure S14 b). These configuration changes were for size‐fit recognition, considering that the radius of Hg2+ is 1.02 Å and each Hg−S bond length was 2.510 Å in the octahedral configuration, in which Hg(OH)2 with the HO−Hg−OH bond angle of 154.1° was almost vertically coordinated into the cavity (Figure S14 b). It is worth pointing out that the approximate linear configuration and vertical‐style insert of Hg(OH)2 into the azathiacrown was crucial for HgII‐specific coordination and recognition, regardless of the fact that HgII might associate with other anions in some real water samples when concentrations of the anions were abnormally high, such as Cl− in seawater. For AgI, however, a more energetic cost (47.81 kcal mol−1) compared to that in the case of HgII (22.50 kcal mol−1) was needed to form 4S−Ag−11S with a bond angle of 126.0°, and 7S−Ag−14S with 119.7°. The rigid azathiacrown NS4 had to be distorted seriously in order to reach the smaller distances of 4S−Ag−11S (4.199 Å) and 7S−Ag−14S (4.076 Å), owing to the shorter Ag−S bond length 2.354–2.359 Å (Figure S14 c), resulting in an unstable state.

Figure 3

ESI–MS spectra of ethyl‐esterified phenyl‐NS4 before (a) and after (b) coordination with HgII. The inset shows the enlarged isotopic distribution of ethyl‐esterified phenyl‐NS4‐Hg(OH)2 around m/z 710.

The water certificated reference material (CRM) GSBZ 50 016‐90:202037 was used to validate the feasibility of our proposed strategy for a direct SERS‐based specific quantification of HgII in waters using Au@SiO2@phenyl‐NS4. For accurate quantification, a good IS should be placed in the same situation together with the targeted analyte. This is particularly important for SERS‐based quantitative analysis, because the intensity of the SERS signals suffers from the uncertainties arising from possible instrumental variations, distribution uniformity of the electromagnetic hot spots on the surface of the SERS‐active substrate, and their unequable adaptation microenvironment towards the samples of different physicochemical properties, as discussed above. The phenyl moieties that link NS4 and Au@SiO2 (Scheme 1) were fully exposed to the same near‐field microenvironment as the extracted HgII, and thus the area ratio (A Hg−S/A phenyl) of the Hg−S SERS band at 270 cm−1 to the typical phenyl band (γCC+γCCC) at 1046 cm−1 could normalize the uncertainties (Figure 4 a). The obtained results demonstrated the necessity of phenyl as an IS when the calibration curve was plotted with A Hg−S/A phenyl compared to A Hg−S alone (Figure 4 b). The dynamic concentration linear range of HgII against A Hg−S/A phenyl was from 0.4 to 2.0 ng mL−1 (higher concentrations were not tested) with a correlation coefficient of 0.991 and an RSD of 7.4 % at 1.0 ng mL−1 (n=5), whereas that of HgII concentration against A Hg‐S alone began to bend at 1.0 ng mL−1 with insupportable fluctuations. In this way, the limit of detection (3σ) reached 0.09 ng mL−1 and the limit of quantification (10 σ) was 0.31 ng mL−1, satisfactorily meeting the basic requirement for safe drinking water [the threshold values of HgII are 6 ng mL−1 (WHO),10 2 ng mL−1 (USEPA),11 and 1 ng mL−1 (SAC)12]. The determined concentration of HgII in the CRM was 11.5±1.0 ng mL−1 (n=5). It was well in accordance with the certificated value (11.9±1.2 ng mL−1), confirming an accurate SERS‐based quantification of HgII using the designed Au@SiO2@phenyl‐NS4. It was applied to measure HgII in fresh water samples collected from the Ting River, which flows from Fujian through Guangdong province, and in the seawater around Xiamen Island, southeastern China (Figure S15).

Figure 4

a) SERS spectra obtained in the presence of different concentrations of HgII using Au@SiO2@phenyl‐NS4. b) Fluctuation of the SERS intensity of IS phenyl, and the calibration curves of HgII concentration against the intensity ratio of Hg−S SERS band at 270 cm−1 to phenyl band at 1046 cm−1 (black line), as well as the intensity of Hg−S SERS band at 270 cm−1 alone (blue dash line). The HgII concentrations tested were 0.40, 0.50, 0.60, 0.70, 0.80, 1.00, 1.25, 1.50, 1.75, and 2.00 ng mL−1, whereas 3 mg mL−1 Au@SiO2@phenyl‐NS4 was used. Laser: 785 nm; power: 3 mW; exposure time: 10 s. All the data presented were those after subtracting the background determined when using Au@SiO2 NPs alone. Each point is the mean of five duplicate experiments.

In summary, we have demonstrated the step‐by‐step design and synthesis of Au@SiO2@phenyl‐azathioether/azathiacrown for direct SERS‐based specific quantification of HgII for the first time, in which the intensity ratio of the Hg−S SERS band (270 cm−1) to the IS phenyl band (1046 cm−1) directly reflected the HgII concentration, achieving a reliable SERS‐based quantification of HgII in various waters. Not limited to HgII, we believe that the methodology reported in this manuscript sets an example for the SERS‐based quantification of other inorganic metal species, whereby specific complexing substrates and suitable ISs are designed and synthesized. Moreover, not restricted to Au@SiO2 nanoparticles, the channel surfaces in a microfluidic chip that are chemically modified with SERS‐active nanostructures are expected to perform a more efficient SERS‐based quantification of HgII and other inorganic metal species in the near future.

Conflict of interest

The authors declare no conflict of interest.

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Supplementary

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