AgNPs Functionalized with Dithizone for the Detection of Hg2+ Based on Surface-enhanced Raman Scattering Spectroscopy

Mercuric ion (Hg2+), a poisonous metal ion that remained in water ecosystems, can severely damage the human central and peripheral nervous system and kidneys. Consequently, rapid and highly sensitive methods to determine trace Hg2+ are meaningful to discuss. In recent years, methods for detecting heavy metals by complexation reactions have emerged one after another. We have proposed a novel approach of surface-enhanced Raman scattering (SERS) for the quantitative analysis of Hg2+ in water samples using dithizone (DTZ) as a Raman reporter. DTZ-modified silver nanoparticles (AgNPs) produced a strong SERS signal. In the presence of Hg2+, the DTZ can capture Hg2+ composing a stable structure, resulting in DTZ leaving the surface of the AgNPs, with an accompanying decrease in the signal. The proposed SERS assay showed a linear range of 10–4–10–8 M, with a limit of detection of 9.83 × 10–9 M. The sensor has low detection cost, rapid detection speed, and uncomplicated sample pretreatment. Furthermore, this method can be successfully utilized to detect Hg2+ rapidly in water samples, which sheds new light on the detection of Hg2+ in the environment.


Introduction
Heavy metals have a considerable impact on soil, plants, aquatic animals, and humans [1,2]. Hg 2+ is one of the heavy metal ions, which is highly toxic in organisms and ecosystems even at a low concentration of 5 μg/L [3]. Mining activities, municipal waste, oil and coal combustion, cement production, consumer product emissions, waste-water emissions and so on are the major sources of mercury ion emissions into the environment, which has caused pollution due to improper treatment methods [4,5]. Hg 2+ poisoning can cause neurological, renal, immune, cardiac, sports, reproductive, and genetic diseases in humans [6][7][8]. Therefore, it is of great significance to study the widespread distribution of Hg 2+ in the aquatic environment and bioaccumulation in the food chain [9][10][11].
At present, many analysis techniques have been utilized in the detection of Hg 2+ such as atomic absorption spectrometry, inductively coupled plasma-mass spectrometry (ICP-MS), high-performance liquid chromatography, electrochemistry, colorimetry, and fluorescence spectroscopy [12][13][14][15][16][17][18][19]. However, these methods have limitations [20,21], such as complex sample preparation, expensive equipment, and time-consuming procedures. Therefore, a simple, highly selective, and sensitive method for measuring Hg 2+ must be developed to overcome these shortcomings. A nondestructive optical inspection is an attractive option, it can respond quickly in natural environments and organisms.
Surface-enhanced Raman scattering (SERS) is a nondestructive and rapid analysis technique that has a wide range of applications [21][22][23][24], especially the analysis of trace poisonous substances, such as heavy metal ions, dyes, and pesticides [25][26][27][28]. In recent years, using SERS to detect Hg 2+ has received widespread attention. The substrates which can enhance the signal of Raman, such as silver nanoparticles (AgNPs) and gold nanoparticles, are stable, easy to synthesize, and can be easily modified with different reagents, which improve the method of cognition Hg 2+ [29,30]. Various methods have been put forward for detecting Hg 2+ , based on differing SERS probes. Qi et al. studied a T-Hg 2+ -T mismatch base pair pattern for Hg 2+ detection [31]. Hao et al. utilized acriflavine to detect Hg 2+ quantitatively, since the interaction between acriflavine and AgNPs. Luo et al. introduced safranine T as a SERS probe, allowing trace analysis of Hg 2+ with high selectivity [17]. Although these methods achieve the advantages of rapid and sensitive detection of Hg 2+ , they are susceptible to biological and environmental influences.
Herein, for the first time, we propose a method for the detection of Hg 2+ with DTZ. In this work, indirect SERS detection of Hg 2+ is carried out based on dithizone (DTZ) as a Raman probe. The quantitative decrease of the SERS signal of the probe molecule of DTZ was observed when DTZfunctionalized AgNPs mixed with a certain amount of Hg 2+ , which suggests that the phenomenon of decreased SERS signal intensity should chiefly be attributed to the interaction between Hg 2+ and DTZ [32,33]. A linear correlation of Raman intensity with Hg 2+ concentrations was from 10 -4 -10 -8 M with a limit of detection was 9.83 × 10 -9 M. Eventually, the SERS method with DTZ as Raman reporter is beneficial for the rapid and sensitive determination of Hg 2+ in water. This quantitative detection strategy of the proposed method is shown in Fig. 1.

Instrumentation
A JEM-2100 ultra-high-resolution transmission electron microscope (JEOL, Japan) was used to characterize AgNPs, and to determine the geometry and size of the AgNPs. A PerkinElmer Lambda 35 spectrophotometer (326 nm; Norwalk, CT, USA) was used to record the ultraviolet-visible (UV-Vis) absorption spectra. The SERS measurements were performed using a Renishaw InVia Reflex confocal microscope (Renishaw, UK). All measurements were performed using a He-Ne laser (532 nm, exposure time of 10 s). The laser spot diameter was 1 μm, and the laser power was 50 mW.

Synthesis and Characterization of AgNPs
AgNPs were prepared according to previous work [34]. In short, 36 mg AgNO 3 was added to 200 mL of water and boiled under constant stirring. Then, trisodium citrate (4 mL, 1%) solution was added to the above solution quickly and boiling continued for another 30 min. Let the flask chill to room temperature, a green-gray colloid was acquired. Finally, the colloidal AgNPs were stored at 4 °C for later use.

Preparation of Hg 2+ Standard Solution and Samples
HgCl 2 was dissolved in deionized water to make a 1 mM solution. A set of Hg 2+ solutions with different concentrations was obtained by diluting 1 mM HgCl 2 solution. Drain water from a tap was collected in Shenyang City, Liaoning Province, China. The samples passed a 0.22-μm filter membrane to remove impenetrable matter for the later experiments.

Detection for Hg 2+
DTZ solution (20 μL), AgNPs (60 μL), and Hg 2+ (20 μL) or spiked water solution of different concentrations were transferred to a centrifuge tube in turn, and SERS detection was performed after fully sonicating and mixing at room temperature. Using a He-Ne laser (532 nm), laser power was 50 mW. The intensity of SERS change of DTZ at 1590 cm -1 was elected as a basis for quantification. Each experiment was performed three times in parallel.

SERS Measurement of DTZ and DTZ-Hg 2+
The TEM image showed that the size of uniform spherical AgNPs is about 30 nm (Fig. 2a, b) shows the UV-Vis absorption spectrum of AgNPs at 420 nm, which demonstrated the successful synthesis of AgNPs. The UV-Vis  Figure 2c is X-ray photoelectron spectroscopy (XPS) for elemental and structural analysis. The image shows that Hg, O, N, and C elements exist in the system, which proves that AgNPs, DTZ, and Hg 2+ exist in the detection system. The SERS signal of DTZ, DTZ with Hg 2+ and ethanol are in Fig. 2d, in which the main SERS signals of DTZ were located at 510, 855, 994, 1156, 1219, 1310, 1377, and 1590 cm -1 [35]. It is found that the SERS peak at 1590 cm -1 attributed to the C = N vibration of DTZ, has conspicuous changes, so it is the most suitable for use as a basis for quantitative analysis. Comparing the blue and the red lines, after adding Hg 2+ , the SERS intensity of the DTZ molecules reduced significantly. This is substantially due to the change in the number of DTZ molecules adsorbed on the SERS active sites. The SERS signal of DTZ was enhanced dramatically because the DTZ molecules drew closer to the SERS active sites through the Ag-N bond. However, after the addition of Hg 2+ , the binding force between Hg 2+ and DTZ is stronger than that with AgNPs, which leads to the desorption of DTZ molecules from SERS active sites. As a result, fewer DTZ molecules were left on the AgNPs surface and the SERS signal intensity was reduced.

Optimization of the Analytical Conditions
To increase the sensitivity of this method, we optimized the experimental parameters. The volume of AgNPs will affect the analytical performance. Figure 3a shows the intensity of the SERS signal of DTZ at 1590 cm -1 . Upon the increase in the volume of AgNPs, the corresponding SERS signal increased. However, when the volume exceeded 60 μL, the SERS signal achieved a steady value, which proved that DTZ is completely adsorbed on the AgNPs. Therefore, 60 μL AgNPs were chosen for the detection of Hg 2+ . In this method, DTZ will connect AgNPs and Hg 2+ , so we explored the influence of DTZ concentration. As shown in Fig. 3b, the SERS signal reached a maximum value when the concentration of DTZ is 10 -4 M. Thus, we fixed 10 -4 M as the optimum condition. Mixing time plays a critical part in this detection process. Figure 3c shows that after mixing for 8 min the signal tends to be stable, demonstrating that DTZ was entirely integrated with Hg 2+ . So, we chose 8 min as the optimized mix time.

Selectivity for the AgNPs-DTZ-Hg 2+ System
To study the selectivity of this method, various other environment-related metal ions were appraised (such as Fe 3+ , Cu 2+ , Al 3+ , Na + , K + , Ni 2+ , Bi 3+ , and Pb 2+ ). Figure 4a shows the comparison to SERS spectra of the Hg 2+ and other ions, based on the low binding affinity of DTZ to other ions, the SERS signal of DTZ did not reduce significantly in the presence of the other ions, which indicated the DTZ sensors for Hg 2+ detection had an excellent selection. To clearly show the SERS signal intensity corresponding to the 1590 cm -1 peak of DTZ, Fig. 4b is presented in the form of a histogram. The Hg 2+ and mixed groups showed significantly lower column heights, which indicates that the AgNPs-DTZ system can specifically detect Hg 2+ , and this method has an anti-interference ability.

Quantitative SERS Detection of Hg 2+
To assess the sensitivity and potential quantitative analysis application of the proposed method, we measured different concentrations of Hg 2+ standard samples. Under the optimum conditions, the present method showed a linear relationship for Hg 2+ concentrations and the SERS peak in the range of 10 -4 to 10 -8 M with a limit of detection of 9.83 × 10 -9 M, which conforms to the equation Y = 1096.3961X + 15738.17866 (R 2 = 0.999). The relevance between the SERS signal intensity and Hg 2+ concentration is shown in Fig. 5. In the comparison, the experimental results of some other methods for measuring Hg 2+ are listed in Table 1, which showed that our method is simpler and faster. More importantly, the detection result is inferior to the limit of 10 ppb (4.98 × 10 -8 M) in drinking water recommended by the World Health Organization.

Reproducibility of the Hg 2+ Detection
To assess the reproducibility of the AgNPs-DTZ-Hg 2+ system, the SERS intensity of Hg 2+ at a concentration of 10 −5 M under optimal experimental conditions is detected. Randomly collected 20 different points for testing with the result is shown in Fig. 6a. It is worth noting that the SERS intensity at 1590 cm -1 is relatively uniform. From Fig. 6b, we find that the relative standard deviation of the 20 sets of data is less than 5%, which proves that the SERS sensor proposed here article has satisfactory reproducibility.

Analysis of Hg 2+ in Tap Water Samples
To demonstrate the practicality of the developed SERS method, we detected the Hg 2+ contents in tap water under the optimum conditions by the standard addition method. Different concentrations of Hg 2+ were tested 3 times and the results are shown in Fig. 7. We further evaluated a comparison of the developed SERS method and the conventional ICP-MS method and the related statistics are shown   Table 2, which indicates that the Hg 2+ concentrations detected by the SERS sensor are very consistent with those measured by the classic ICP-MS method. The recovery for Hg 2+ was 89-90%. The results showed that this method is reliable and applicable for the detection of Hg 2+ in tap water.

Conclusion
In short, we developed a time-saving, high-sensitivity SERS sensor for detecting Hg 2+ . This sensor works with excellent sensitivity via the special recognition of Hg 2+ by DTZ, which belongs to the following: first, DTZ has a high SERS signal that can utilize for Hg 2+ quantitative detection. Moreover, DTZ captures Hg 2+ and forms a steady structure that can enhance the specificity of this method. We have merely fabricated the SERS sensor by mixing AgNPs and DTZ to achieve a rapid and ultrahigh sensitivity to detect Hg 2+ , which can exhibit a limit of detection of 9.83 × 10 -9 M under the best conditions. More importantly, this sensor can detect Hg 2+ in a broad range of concentrations and the detection result is inferior to the limit of 10 ppb (4.98 × 10 -8 M) in drinking water recommended by the World Health Organization. Remarkably, we further demonstrate the SERS sensor is suitable for detecting Hg 2+ from tap water in reliable and quantitative manners. With these advantages, we anticipate this method will have fine potential in the detection of Hg 2+ in complex water environments.
Author Contribution All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Na Guo, Guangda Xu, Qijia Zhang, Peng Song, and Lixin Xia. The first draft of the manuscript was written by Na Guo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this published article.

Competing Interests
The authors declare no competing interests.