Document Type : Original Research Paper


School of Chemical Sciences, Goa University, Goa, India, 403206


The key to simple and rapid detection of a large volume of samples lies in the hands of solution-based nanomaterial sensors. Quantification of mercury in the river and coastal water is analytically challenging due to the potential interference of the matrix. In this endeavor, lysine-capped gold nanoparticles (Lys-AuNPs) based colorimetric sensors are demonstrated here towards efficient detection of trace amounts of mercury ions (Hg2+) in coastal and estuarine water. The colorimetric behavior of Lys-AuNPs is related to surface plasmon resonance (SPR)
During analysis, interestingly a decrease in the intensity of the original SPR peak at 530 nm was observed, with the concomitant appearance of a new peak at a longer wavelength due to agglomerated Lys- AuNPs. Developed sensors exhibit excellent performance in different environmental samples with high selectivity towards Hg2+ ions in the presence of other metal ions. For the analysis of coastal water samples, a low value of regression coefficient was observed due to the potential interference of salt in the sample. To overcome this, matrix-matching experiments were carried out. Developed Lys- AuNPs show good selectivity towards Hg2+ in matrixed matched diluted coastal water samples. With a sensitivity of 0.02 ppm, the sensor can be utilized to screen large numbers of coastal water samples for their Hg2+ content to satisfy coastal regulation norms. As a whole, this method is simple, sensitive, selective, cost-effective and can be used to screen large numbers of samples across the coastal area for monitoring Hg2+ concentration.


Seawater contaminations by anthropogenic chemicals pose one of the worst problems to coastal and estuarine ecosystems around the world. When coastal water is used for recreational purposes, the toxicity of a metal present in it is attributable significantly to its labile fraction. These metal ions, especially heavy metal ions such as mercury, and Hg2+ are reported to be highly toxic even at very low levels in the aquatic environment. Mercury is reported to be among the first four toxic pollutants. Reported literature also supports the high amount of Hg2+ across the coast, [1-4]. Considering the immense potential of coastal areas for recreational activities and the threat due to reported Hg2+ level, its quantification is very important. To monitor our estuarine/coastal water quality for these toxic pollutants, analytical methods, which are simple, economical, having high throughput capability with minimal sample preparation, are needed for an hour. The developed method should work excellently as a screening tool where discrimination between permissible and non-permissible limits should be easily possible. Regulatory bodies across the globe have also specified guidelines for these toxic components in recreational water [5] whereas the major emphasis is given to microbial contamination. A guideline for inorganic contaminants such as metal ions is missing.  Reports by the United State Environmental Protection Agency (US-EPA) [6] for guidelines for heavy metals in seawater are among the rare guidelines in this area. The guidelines are summarized in Table 1. 
In the table, the criterion on maximum concentration (CMC) is an estimate of the highest concentration of a material in surface water to which an aquatic community can be exposed briefly without resulting in an unacceptable effect, whereas criterion continuous concentration (CCC) stands for the highest concentration for indefinite exposure. According to WHO guidelines for coastal water 2003 (WHO report, 2003) [7], environmental quality standards for chemicals in recreational waters should be based on the assumption that recreational water makes only a relatively minor contribution to intake. They assumed a contribution for swimming of an equivalent of 10% of drinking-water consumption. Since most authorities (including WHO) assume consumption of 2 liters of drinking- water per day, this would result in an intake of 200 ml per day from recreational contact with water. With guidelines for mercury ion for drinking water as 1ppb (European standards) [8], and 2ppb (for Hg2+ by WHO), the guidelines for recreational purposes can be relaxed tenfold i.e., 20 ppb for Hg2+. From Table 1, it is also evident that Hg is the highest priority contaminant among the listed ones. The significant reasons behind this include the high toxicity of organic mercury, the high solubility of inorganic mercury i.e.   69 g/L at 20 °C, and continuous conversion of organic, inorganic, and elemental Hg into each other forms [9].  The list of ill health effects associated with trace Hg2+ through oral or dermal exposure is endless which includes gastrointestinal problems, kidney damage, dermatitis, discoloration of the nails, corrosion of the mucous membranes, and sometimes corrosive burns. It should be noted here that the concentration of Hg2+ can be interpreted into its more toxic forms such as organic mercury which makes sensing inorganic mercury ions (Hg2+) more significant.
Monitoring Hg2+ in the complex matrix is a challenging task as speciation change of metal ions takes place during pre-treatment /extraction of metal ions. Thus, the analytical method for on-site determination is required. A variety of optical methods has been developed in the last few years for this where colorimetry and fluorimetry sensors are significant ones. These sensors became more attractive due to their simplicity, high sensitivity, selectivity, and capability for on-site analysis. To the best of our knowledge, there are very few reports on the applications of colorimetric sensors for the metal Hg2+ analysis in seawater, especially because of the lack of selective recognition ligands and interference due to matrix components [10-11]. Surface plasmon resonance (SPR), is advantageous due to the unique optical, electrical, and chemical properties of nanoparticles. Recently, the nanomaterial-based colorimetric assay has been widely used for the sensing and removal of toxic compounds from environmental and biological samples [12-14]. SPR or localized SPR (LSPR) has a strong absorption band at a wavelength of about 521 nm, so it shows a deep red color. The colorimetric performance of gold nanoparticles AuNPs is associated with their SPR, which keenly depends on the shape, geometry, inter-particle distances, particle size, and refractive index. Any variation in these factors changes surface-plasmon frequency, which affects the strength and color of plasmonic enhancement [15]. A solution containing smaller separated gold nanoparticles shows deep red color whereas larger particles or aggregates of smaller particles showed a purple to deep blue color.
In recent years, an increasing number of works focused on the development of novel nanoparticle-based sensors for Hg2+ detection, mainly motivated by the need for low-cost portable devices capable of giving a fast and reliable on-site analytical response, thus contributing to the analytical decentralization. Some of the significant work includes [16] where the colorimetric method for determination of Hg2+ and Ag+ ions based on stabilization of AuNPs by redox-formed a metal coating in the presence of ascorbic acid (AA) is reported. Chaudhary et al., 2015 [17] also reported AuNPs based system for Hg2+ ions but with modification with Lys and dithiothreitol (DTT). The detection limit of these assays is reported as 27 pM and 58 pM for deionized water and tap water, respectively.  Selvakannan et al., 2 003 [18] reported that capping aqueous AuNPs with the amino acid i.e., lysine helps in stabilizing the particles in solution electrostatically and also renders them water-dispersible.  Apart from the advantages known to us, these bio-inspired colorimetric sensing systems include cost-effective production, facile usage, and a visual color change for the detection method [19]. The above sensors allowed for reaching detection limits within the pM and nM range for different water samples whereas limited work is published on coastal water analysis [20-21]. Wang and Sun 2006 [22] have reported that optical limiting is observed in the presence of ionic salt where NaCl causes a slight decrease in UV-vis absorption signal as compared to KCl. This limits the applicability of the sensor in coastal water analysis.
Although most of these reported sensors proved their suitability in detecting and quantifying Hg2+ ions in synthetic solutions or spiked water samples, the state of the art in these technologies is still behind the standard methods of mercury quantification, such as cold vapor atomic absorption spectrometry and inductively coupled plasma techniques, in terms of reliability and sensitivity. This is mainly because the response of nanoparticle-based sensors is highly affected by the sample matrix. The developed analytical nanosystems may fail in complex real samples because of the negative incidence of the ionic strength and the presence of exchangeable ligands. Capping of lysine on AuNPs is reported to be serving both purposes. Among the reported work in seawater for Hg2+, Lin et al. 2010 [21] have reported tween 20-modified AuNPs for selective detection of Hg2+ and Ag+ in seawater. Here the surface of citrate-capped AuNPs was modified with Tween 20, which stabilizes the citrate-capped AuNPs against conditions of high ionic strength. 
From the reported data it is clear that AuNPs have been effectively used for sensing Hg2+ in various water samples, mainly drinking and tap water. The application of the above sensor for coastal water analysis is limited where a LOD of 0.1μM is reported by Lin et al., 2010. In this work, the use of surface-modified gold nanoparticles as an LSPR-based chemical sensor for the colorimetric detection of Hg2+ in coastal water samples is demonstrated. The method is based on the color change of amino acid-modified AuNPs from red to purple with the addition of Hg2+. This color change is caused due to the shift in the LSPR band due to the inter-particle coupling effect. The aim of our study is also to develop a simple analytical method with minimal sample preparation for on-site analysis. The usefulness of this method for the critical analysis of Hg2+ in coastal and estuarine is successfully demonstrated here.
The highlights of the above work are 
● Successful demonstration of a simple nanoparticle-based method for on-site analysis of Hg2+ in coastal and estuarine water.
● The method with minimal sample preparation where mere dilution is used as a sample preparation step.
● Limit of detection (LOD) of developed sensor for Hg2+ in coastal water is 0.02 ppm (0.0583mM), 
● Application of developed sensor method for sensing Hg2+ in a coastal area of Goa where hardly any work is reported. Considering the potential of Goa, India for recreational activities and related revenue generation this study is important.

Chemicals and Instruments 
Sodium Citrate tribasic dihydrate, ( Molychem) Gold (III) chloride trihydrate, Cysteine, Lysine, Copper nitrate hexahydrate, Cobalt nitrate hexahydrate, Lead nitrate, Nickel nitrate hexahydrate, Ferric nitrate nonahydrate, Zinc hydrate hexahydrate, Calcium nitrate, Aluminium nitrate, Potassium nitrate, Mercuric nitrate monohydrate, Sodium chloride, Nitric acid, Hydrochloric acid (Avra). For recording spectra, a Shimadzu UV-1800 spectrophotometer is used. Scanning Electron Microscopic images were taken using the SEM instrument. 

Synthesis of lysine-capped gold nanoparticles and sensing of Hg2+
The AuNPs were prepared by the Turkevich method [15] in which the gold chloride salt is reduced using sodium citrate tribasic dihydrate. 12 ml of preheated sodium citrate solution (1% W/V) was added to 100 ml of boiling water containing 8.5 mg of gold chloride salt. The solution was vigorously stirred for 30 minutes until the solution turned a deep red color. Finally, the solution was cooled to room temperature and the volume of the gold nanoparticle solution was adjusted to 100 ml by using double distilled water. AuNPs were characterized by UV-visible spectrometry. These stabilized colloidal AuNPs exhibited characteristic SPR peaks at 519 nm and appeared red. 1 ml of AuNPs solution prepared as mentioned above was mixed with 1 ml of lysine solution (0.4 mM) at room temperature. The resultant solution was mixed to get Lys-AuNPs solution. Similarly, cysteine-capped AuNPs (Cys-AuNPs) was also prepared and used for detecting Hg2+ in a water sample. (Spectra in Supplementary information; SI-1)
In a typical experiment, 1.0 mL of Lys-AuNPs was mixed with 0.4 mL of Hg2+ solutions (in deionized /tap/river water) at different concentrations to give a final Hg2+ concentration in the range of 0.02 ppm to 1 ppm. Finally, the color change from red to blue was detected by the naked eye and/or by recording the UV- vis absorption spectra in the wavelength range 200 to 800 nm.
Similarly, the detection of Hg2+ by using Cys-AuNPs was also performed in the studied range. For understanding morphological changes, SEM analysis of citrate-capped AuNPs, lysine-capped AuNPs, and cysteine-capped AuNPs with and without the addition of mercury was carried out. (Spectra in Supplementary information; SI-2)
The selectivity of the Colorimetric Sensor
To investigate the selectivity of the method, several metal ions such as Ni2+, K+, Co2+, Cd2+, Fe3+, Al3+, Cu2+, and Ca2+ including Hg2+ were added to the prepared lysine-capped gold nanoparticle (Lys- AuNPs). The corresponding color change was observed and the UV- visible absorption spectra were recorded. In addition, controlled experiments were performed in the absence of lysine and for high reproducibility; all experiments were performed in triplicate.

Sample collection and preparation 
For the collection of water samples, the sampling sites were selected along the Goa coast. Sampling was carried out by the grab method in the post-monsoon period. Samples of water were analyzed on the same day (within 2 hours) without any prior acidification. The analyzed water samples include water from different sampling locations across the Zuari river/estuary / coastal area of goa i.e. S-1 to S-15. A figure representing details of the sampling location is given below (Fig 1). In practice, for sample pre-treatment, water samples were centrifuged (4470 RPM for 10 minutes), filtered (0.45 μm pore size filter paper), and diluted (if required) before analysis. The pre-treated sample is then reacted with Lys- AuNPs. 

Real Samples Analysis 
The proposed method was applied for the determination of Hg2+ in different water samples. In the case of nanoparticle-based colorimetric sensing for coastal water samples, matrix effects are reported to be more serious. The constituents of the coastal water (Na+ and Cl-) pose significant interferences.  Thus, reporting the concentration of analytes without separation from the matrix may include huge errors [26]. During coastal water analysis, we observed that color is highly dependent on the concentration of the matrix. Therefore, experiments were conducted to minimize the effect of the matrix by decreasing the concentration of Na+ and Cl-.  Dilution seems to be an economical and effective approach for it and thus optimized. 
For optimization, initially artificial seawater and natural seawater with various dilutions were reacted with Lys-AuNPs. Optimum dilution was calculated where the matrix gave negligible effect. Then, the samples were spiked with different concentrations of Hg2+, and recovery studies were performed. Similar studies were carried out with Cys- AuNPs which gave a high positive error in the presence of NaCl (as for the colorimetric response of Cys-AuNPs, the presence of NaCl is essential). Thus, for coastal water analysis, Lys- AuNPs with optimized dilution for minimal matrix interference were finalized. The main objective of matrix matching is to obtain optimum color and ʎ max in the adsorption spectrum.

Statistical Analysis 
Statistical Analysis was conducted to determine the difference between metal ion concentrations among the inter-day and intra-day analysis. The coefficient of variation was also calculated for real sample analysis to check the precision of Hg 2+ analysis in coastal water. 

Proposed mechanism of Hg 2+ analysis
The colorimetric Hg2+ sensing principle can be explained by a two-step mechanism. In the first step, lysine was immobilized on the AuNP surface and formed a shell around the particles. Then, Hg2+ addition to this solution induces aggregation of Lys-AuNPs and results in a rapid color change from red to purple because the distance between the Lys-AuNPs decreases than the average particle diameter. Thus causing the broadening and shifting of the surface plasmon resonance (SPR) peak [15]. 
From Fig 2 (b) it is clear that AuNPs or Lys-AuNPs when characterized by UV-visible spectrometry show a characteristic SPR peak at 519 nm and appear red. But after the addition of Hg2+ solution, the intensity of the peak decreases, and the peak emerges at a longer wavelength. The peak shows a bathochromic shift. The role of lysine in the SPR signal is that it contains two amino groups which can interact strongly with Hg2+ ions and forms a bridge. This bridging leads to the aggregation of AuNPs leading to a shift in wavelength. 
Colorimetric detection of Hg2+ using lysine-capped gold nanoparticles
For the calibration curve, different concentrations of Hg2+ were added to the Lys-AuNPs solution. With the increase in Hg2+ concentration from 0.02 to 1 ppm (0.02 ppm =0.058µMnM), the SPR band around 643 nm was gradually enhanced, demonstrating a sensitive response to Hg2+
The high correlation coefficient (R2) of 0.9873 in Fig 2. shows the good linear relation between  Hg2+ concentration and response for the selected method at maximum wavelength. Also, the increase in intensity has been observed concerning an increase in concentration as shown in the supplementary information, SI-3 for different Hg2+ concentrations. Significant analytical features of merit for the given method are tabulated in Table 2.

The selectivity of the Colorimetric Sensor
The selectivity of this colorimetric sensor in the presence of Ni2+, K+, Co2+, Cd2+, Fe3+, Al 3+, Cu2+, and Ca2+ was carried out.  Controlled experiments were also performed in the absence of lysine. From the spectra obtained the results demonstrated that only Hg2+ could cause the significant color change of the Lys-AuNPs, and Ni2+, K+, Co2+, Cd2+, Fe3+, Al 3+, Cu2+, Ca2+ species had no obvious effect on the SPR band and the color of the colloid. With the increase in Hg2+ concentrations, there is a significant increase in agglomeration of AuNP-lys was also observed. The larger atomic weight and lower electronegativity of Hg2+ might be the prime reason behind preferential sensing. The above results indicated that this developed method could effectively distinguish the inorganic mercury, while other metal ions had no disturbance.

Water sample analysis 
Quantification of Hg2+ concentration in water samples collected from different sampling sites was carried out. In practice, to understand the matrix effect, the calibration curve was plotted in all matrices. Hg2+ solutions corresponding to standard calibration curves in all these matrices were prepared and analyzed. Table 2, represents a summary of the calibration curve for different water samples
The slopes of the calibration graphs prepared by the standard curve method for the tap, distilled and wastewater samples were found to be almost identical to that of the standard addition plot. Whereas for coastal seawater, the values for slope and correlation coefficient (R2) are relatively less. This indicates the interference of the salt matrix in coastal seawater analysis. To overcome this, matrix matching for coastal seawater samples is required before analysis. Considering this, the experiments were performed where the concentration of Hg2+ and Lys- AuNPs is kept constant and various dilutions (with distilled water) of coastal seawater samples have interacted. The various dilutions used for this study are; Neat sea water (ASW-1), 1:1 dilution of seawater (ASW-2), 1:2 dilution of seawater (ASW-3), 1: 3 dilution of seawater (ASW-4) and so on. The color change was detected by the naked eye. Also, the UV- visible absorption spectra were recorded in the wavelength range of 200-800 nm. The absorbance and extinction coefficient for each sample was calculated and compared with the reaction of Lys- AuNPs in distilled water. Fig 5, Represent matrix matching for artificial coastal water by dilution.
It was observed that with a dilution of seawater, the extinction coefficient changes and ultimately matches the value for the reaction of Lys- AuNPs in distilled water. With 1: 4 dilutions (i.e. ASW-4 in fig.), the extinction coefficient of Lys-AuNPs as well as absorbance value matches. This indicates that with 1: 4 dilutions, the matrix interference is negligible. Using matrix matching, samples from different locations (containing different Hg2+ concentrations) were analyzed (representative spectra are given in supplementary information (SI-4) Later, spiking studies were also carried out to compare the response of matrix-matched sample and Hg2+ with standard Hg2+ in the calibration curve. It was observed that for a few samples, the concentration is below the detection limit (BDL) of the developed method i.e., 0.02ppm whereas for a few samples the quantified amount was above BDL.
Recovery (%) for all spiked samples was carried out. Here, the recovery values calculated for the added standards are observed to be higher than 95%, thus confirming the accuracy of the procedure and its independence from the matrix effects. Also, high accuracy and precision of data can be confirmed by %RSD values which are less than 5.  From data in Table 3, for a few samples with actual concentration below BDL the % recovery values are around 100 % whereas, for a few samples, higher recoveries in the range of 100-110 were observed. The data obtained for these samples also matches that of data without spiking. Relatively high recovery values for sites 1 and 4 were observed which may be due to the presence of Hg2+ present in the seawater sample that may add to the signal and thus give a higher recovery value.  The results obtained for high heavy metal concentrations at these sites are also consistent with earlier reported studies by Sarkar, A., et al., 2008 [26]. The occurrence of Hg2+ at these sites may be due to the nearby hotels, restaurants, and port activities that discharge effluents into the water and thus pollute them. Also, an alarming signal for site S-1 is obtained indicating Hg2+ concentration more than the permissible limit. For a few samples, low recovery values were observed. This may be due to the presence of the interfering ions in the matrix that may not allow the analyte to react with lys-AuNPs. 
Statistical Analysis was conducted to determine the difference between metal ion concentrations among the inter-day and intra-day analysis. Inter-day and intra- day variations for samples were within the limit. The coefficient of variation was also calculated for real sample analysis to check the precision of Hg 2+ analysis in coastal seawater. It is clear from Table 4 that for real sample analysis, with different matric % RSD is within 5 % which confirms that data is precise.
Comparison of Colorimetric Determination of Hg2+ for Water Samples Using Lys-AuNPs as a Chemical Sensor 
Linearity range and LOD value obtained with the present work are compared with other NPs based sensors for determination of Hg2+ from a variety of samples (Table 4). 
With our method, the LOD obtained for coastal water is 0.02 ppb i.e., 0.058 μM which is much below the published work for coastal water analysis and guidelines for recreational water. The added advantage of our method is minimal sample preparation. The other reported methods are found to be tedious, time-consuming, and multiple steps procedures. With our sensor, the cost of one test performed using our colorimetric assay is very low (around 0.5 cents), which is calculated based on reagent prices. Because of its simplicity (single-step reaction), excellent sensitivity, and selectivity, our colorimetric assay is advantageous over several metal nanoparticle-based methods (Table 2) and feasible for practical applications for on-site analysis of coastal water. 

In this paper, a highly selective lysine-capped gold nanoparticles optical sensors method for ppb levels of Hg2+ ions in coastal seawater has been shown in the range of 0.02-1 ppm. The range is useful to discriminate between polluted and non-polluted recreational water samples as per international guidelines. This method does not require any costly and laborious sample preparation steps. This method is highly selective for Hg2+ ions, and almost all metal ions examined gave no response at that wavelength. The developed method has the potential for performing on microwell plates and thus can serve as a high throughput screening method. 
Unlike most of the chemosensors for Hg2+ which have been evaluated in organic media or organic–aqueous mixtures owing to their low water-solubility, the high-water solubility of the AuNPs-Lys probes allow this assay to be performed in aqueous media without the need for organic co-solvents. In all, the simplicity of the developed sensor makes it a potent screening technology for Hg2+ in seawater for the future.

This paper and the experimental work behind it would not have been possible without the exceptional support of my students Pratiksha Prabhu Desai and Trupti Naik who graduated from the School of Chemical Sciences, Goa University. We are also grateful to Goa University, India for providing instrumental facilities and financial assistance to undertake this study.

No potential conflict of interest was reported by the authors. 

Supplementary Information
Please find Supplementary Information on Journal website. 

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