Abstract
Here, we are reporting two methods for detection of Cu+2 ion and Hg+2 ions using biosynthesized silver nanoparticles. The detection of Cu+2 ion was based on changes in absorbance resulting from complex formation of the metal ion. Various concentrations of Cu+2 ion were used to test the linearity and sensitivity of the method. A new peak at around 770 nm, in addition to the peak of the AgNP at 406 nm, was observed in each case (above 20 µM). With the increase of concentration of Cu+2 ion solution, the absorbance at 406 nm peak decreased and that of 770 nm increased gradually. The calibration curve obtained from the ratio of the absorption coefficients of these two peaks (Ex770/406) versus concentration of Cu+2 ions enables one to estimate quantitatively the amount of Cu+2 ions present in water in µM levels. This AgNP was further functionalized with 3-mercapto-1, 2-propanediol (MPD) for detection of Hg+2 present in water by colorimetric method. As soon as Hg+2 solution was added in MPD-functionalized AgNP (MPD-AgNP), a new peak at around 606 nm appeared along with the peak at 404 nm. The new peak might be due to the aggregations occurred by the recognition of heavy metal ion Hg+2 by MPD-AgNP through dipropionate ion. A calibration curve between the ratios of the absorption coefficients of these two peaks (Ex404/606) and concentration of Hg+2 was drawn for quantitative estimation of Hg+2 present in water at µM level.
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Introduction
Monitoring the levels of potentially toxic metal ions such as Cu+2, Hg+2 in aquatic ecosystems is important because these ions can have severe effects on human health and the environment (Campbell et al. 2003). Copper is a transition metal which plays significant roles in environmental and biological systems. It is widely used in the industrial and agricultural sectors, but it is also highly toxic to organisms, such as algae, fungi and many bacteria and viruses (Merian 1991). High concentrations of copper in drinking water are hazardous to human health (Barranguet et al. 2003). Some cases of liver damage of children have been proved to be associated with the excessive intake of copper (Zietz et al. 2003). Moreover, high concentrations of copper may destroy the biological reprocessing systems in water (Zhao et al. 2009). Mercury-based pollutants that arise mainly from coal-burning power plants are of great environmental concern because of high toxicity of many Hg compounds (Wang et al. 2004). Hg+2 is one of the most toxic metal ion and damages various human organs (Zahir et al. 2005; Zheng et al. 2003), resulting in serious symptoms and health deterioration. Therefore, the fabrications of sensitive and selective methods to monitor the presence of Cu+2 ions as well as Hg+2 are of great significance to protect public health and environment.
Many methods for detection of Cu+2 and Hg+2 ions have been reported till date. Most of them include chromogenic sensors (Banthia and Samanta 2005), Chemodosimeter-functionalized sensor (Kim et al. 2008a, b), fluorescent chemosensor (Zhang et al. 2011), atomic absorption spectroscopy (Gonzales et al. 2009), inductively coupled plasma mass spectroscopy (Backer et al. 2005) and inductively coupled plasma atomic emission spectroscopy (Liu et al. 2005) for detection of Cu+2 ions. Chemical sensors for the detection of Hg+2 include devices based on thin films of gold (Morris and Szulczewski 2002), polymeric materials (Tang et al. 2006), bio-composites (Ono and Togashi 2004), Chemodosimeter-functionalized sensor (Lee et al. 2009) etc. There is still a scope to develop new practical assays for determining Cu+2 and Hg+2 in real samples. In recent years, gold and silver nanoparticles have been widely used as colorimetric probes for chemical sensing and biosensing of various substances (Zhao et al. 2008) such as viruses (Niikura et al. 2009), cancerous cells (Midley et al. 2008), toxins (Uzawa et al. 2008), heavy metals (Barman et al. 2014a, b; Fan et al. 2009), pesticides (Barman et al. 2013; Maiti et al. 2013; Dasary et al. 2009), many inorganic and organic pollutants in water (Daniel et al. 2009; Xiao and Yu 2010). The methods of determination rely on their unique size and interparticle distance-dependent absorption spectra and color change of the solution. Inspired by the outcome of the abovementioned methods, we attempted a colorimetric application for detection of toxic metals like Cu+2 and Hg+2 ions in the solution. In this work, the synthesis of silver nanoparticles has been carried out using the juice extract of Citrullus lanatus (watermelon). The Citrullus lanatus is a good source of vitamin A and C. The presence of polyphenol in this juice is found to be responsible for the reduction of Ag+1 to Ag0 ions and also stabilization of AgNP (Maiti et al. 2014; Huang et al. 2010; Prakash and Prakash 2011).
Experimental
Chemicals
Silver nitrate, AR grade, was purchased from Sigma-Aldrich Chemical Ltd. Sodium hydroxide and 3-mercapto-1, 2-propanediol were purchased from Merck. The metal salt Cu (NO3)2 and Hg (NO3)2 were purchased from Beijing Chemical Reagent Company (Beijing, China). Double distilled de-ionized water was used in all experiments.
Preparation of juice extract
The Citrullus lanatus was collected from local market and washed with Double distilled de-ionized water. It was cut into pieces. The inner red portion of the pieces was pasted by mortar pestle. Then, it was filtered using whatman filter paper to get Citrullus lanatus juice.
Synthesis of silver nanoparticles
Five milliliter of double distilled de-ionized water was added to 5 ml of pure juice extract to make it 1:1 and it was cooled in ice cold water. The solution was made alkaline (pH 10) by adding NaOH. The whole apparatus was placed on a heating mantle. During heating 6 ml 3 × 10−3 (M) aqueous silver nitrate solution was added drop wise with continuous stirring from burette and finally it was heated for 20 min at 70 °C. The color of the solution gradually changed from light pink to reddish yellow. The reddish yellow color indicated the formation of AgNP (Scheme 1).
Detection of Cu+2 by sensing the colorimetric property of AgNP
The colorimetric detection of aqueous Cu+2 ion was studied using AgNP solution at room temperature. To demonstrate the effect of Cu+2 ion on AgNP, 500 µL portions of several concentrations of Cu+2 ion (0–200 µM) were added one at a time to 3 ml of AgNP and the resulting mixture was then allowed to stand for 10 min at room temperature, during which the yellow color changed to bluish. The intensity of this color gradually increased with the increase of Cu+2 ion concentration.
Detection of Hg+2 by sensing the Colorimetric property of functionalized AgNP
One ml 3 × 10−3 (M) 3-mercapto-1, 2-propanediol (MPD) was added to alkaline (pH10) 10 ml of as prepared AgNP sol to form MPD-functionalized AgNP (MPD-AgNP). This was used for detection of Hg+2 ions in water. Various concentrations of Hg (NO3)2 (0–600 µM) were prepared from stock solution by quantitative dilution. The colorimetric detection of Hg+2 in aqueous solution was studied at room temperature. Typically, 3 ml of MPD-AgNP solution was added to 100 µl of Hg (NO3)2 solutions of different concentrations with continuous stirring and thereby the yellow color subsequently changed to blackish.
Characterization
The absorbance spectra of the AgNP were analyzed using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high-resolution transmission electron microscope (HRTEM). Samples for the TEM studies were prepared by placing a drop of the aqueous suspension of particles on carbon-coated copper grids followed by solvent evaporation under vacuum. The crystalline nature of the AgNP was examined using X’ Pert Pro X-ray diffractometer operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation. FTIR measurements of AgNP and copper complex of AgNP were studied by Perkin Elmer Spectrum Version spectrophotometer.
Results and discussion
UV–Vis spectral studies
Optimization of synthesized AgNP
In the present work, the Citrullus lanatus juice extract was utilized for the synthesis of silver nanoparticles. For the preparation, we have tried three different compositions (extract:water) at pH 10. For 3:7 ratios, a broad absorption band was observed at 410 nm and a sharp but not smooth curve found in case of 7:3 composition at 406 nm. The Plasmon band appeared sharp and smooth at 406 nm when 5:5 composition was used (Fig. 1A). As the sharp, single peak represents the monodispersity of the sol, 5:5 (extract: water) composition was preferred by us. To investigate the effect of pH on the formation of AgNP, pH was varied by controlling the amount of NaOH. 5:5 composition extract having different pH was further tried for synthesis of AgNP. It was found that only at pH 10, a narrow and sharp peak was obtained with higher extinction coefficient. With the decrease of pH, the plasmon band gradually broadened with lower extinction coefficient. The sol was not formed at all at below pH 7 (Fig. 1B).
Detection of Cu+2 by sensing the colorimetric property of AgNP
For detection of Cu+2 ion present in aqueous solution, coordination complex formation method has been tried and consequent color changes have been noticed. Several concentrations (0–200 µM) of Cu (NO3)2 solutions were used for the purpose of complex formation. Different concentrations of Cu (II) solution were added one at a time to the AgNP solution and corresponding changes of the absorption coefficients were noted. In each case (above 20 µM), we found a new peak at around 770 nm in addition to the peak of the AgNP. More interestingly, with the increase of concentration of Cu+2 ion solution, the absorbance of 406 nm peak decreased and that of 770 nm peak increased gradually (Fig. 2A). The change of the UV–Visible absorbance ratios (Ex770/406) was found to be linear with the concentration of Cu+2 ion. The calibration curve obtained from this ratio of the absorption coefficients of these two peaks (Ex770/406) versus concentration of Cu+2 ions enables one to estimate quantitatively the amount of Cu+2 ions present in water in µM levels (Fig. 2B). Riboflavin, present in Citrullus lanatus Juice, contains polyphenol which might be responsible for the stabilization of AgNP. (Huang et al. 2010; Prakash and Prakash 2011) and the peptide linkage (–CONH–) for formation of coordination complex with Cu+2 (Garland 1973; Szilágyi et al. 2005).
The formation of the complex is evidenced from the color change (detectable by human eye) and the appearance of a peak at around 770 nm (Barman et al. 2014a, b; Li et al. 2013; Frederix et al. 2003). We repeated the experiment taking other heavy metal ions like Fe+2, Pb+2, Hg+2, Zn+2, Cd+, Ni+2 one at a time in place of Cu+2 ion. Fe+2, Pb+2and Hg+2 destroyed the sol completely whereas in case of Zn+2, Cd+2, Ni+2, though no precipitation occurred, but they could not produce any significant color change and thus no change in the UV–Visible absorption spectrum (Fig. 2C). These findings tested the specificity of this assay for detection of Cu+2 ion.
Detection and estimation of Hg+2 ions using MPD-AgNP
The detection of minimum concentration of Hg+2 in aqueous solutions by color changes has been performed by utilizing the chelating ability of MPD. Several concentrations of Hg (NO3)2 solutions (20–120 µM) were added to MPD-functionalized AgNP and the corresponding changes of the absorption coefficient were observed. In presence of Hg+2 ion, a new peak at around 606 nm appeared (above 40 µM) in addition to the peak found at 404 nm and with the increase of the concentration of Hg+2 ion, the absorbance of the 606 nm peak increased and that of AgNP peak decreased gradually (Fig. 3B). This phenomenon suggested that with the increase of the concentration of Hg+2 ion, complex formation enhanced with MPD-AgNP might be through the O atom available in MPD. The absorption coefficient ratio (the ratio of the absorption coefficient of MPD-AgNP peak at 404 nm and peak of sol in presence of Hg+2 at 606 nm Ex404/606) measured the changes in absorption peak explicitly by the addition of Hg+2. The change in coefficient confirmed that the ratio increased with the increase of concentration of the Hg+2. A calibration curve obtained from the ratio of the absorption coefficients of these two peaks (Ex404/606) versus concentration of Hg+2 ion may be applicable to estimate the amount of Hg+2 present in water in µM level (up to 120 µM) (Fig. 3C).
The experiment was repeated using other heavy metal ions like Fe+2, Cu+2, Pb+2, Mg+2, Zn+2 and Ni+2 one at a time in place of Hg+2 (120 µM to 3 ml of MPD modified AgNP). For Fe+2, Cu+2, Pb+2 precipitation occurred (Fig. 3D) and Mg+2, Zn+2; Ni+2 could not produce any significant change in color and UV–Visible spectrum (Fig. 3E) which suggested the specificity of this assay for detection of Hg+2 ions.
HRTEM analysis
Figures 4 and 5 are due to the TEM images of AgNP produced from Citrullus lanatus juice extract and the Cu-complex of AgNP, respectively. It was observed in Fig. 4 that the particles were mostly spherical and their sizes varied from 10 to 50 nm. Selected area diffraction (SAED) pattern illustrated the crystalline nature of AgNP (Fig. 4C).
Due to the coordination complex formation with Cu+2 ion, the morphology of the AgNP changes appreciably (Scheme 2) which is evident from the TEM micrographs (Fig. 5). Different morphologies such as hexagonal, triangular, and heptagonal, along with the spherical structures (Fig. 5) are visible. The formation of complex was further confirmed from the FTIR spectra.
Figure 6 shows the TEM images of Hg+2 complex with functionalized AgNP. The strong binding of the Hg+2 metal ions with the functionalized AgNP through the O atom of MPD facilitated the aggregation of the silver nanoparticles (Scheme 3) and hence polydispersed silver nanoparticles having various morphologies like hexagonal, tetrahedron or dumb bell shaped along with spherical particles were observed which are shown in the TEM micrographs (Fig. 6A, B). Selected area diffraction (SAED) pattern illustrated the crystalline nature of the complex formed between MPD-AgNP and Hg+2.
FTIR analysis
Representative FTIR spectra of pure Citrullus lanatus juice and the synthesized AgNP are shown in Fig. 7A, B, respectively. The spectrum of Citrullus lanatus juice shows broad peak between 3300 and 3600 cm−1 which may be due to hydroxyl (OH) stretching bands of polyphenols present in riboflavin as well as NH bands present in peptide linkage (Renugadevi et al. 2010). AgNP spectrum shows slightly narrower peak in this region which suggests the involvement of the polyphenols (Song et al. 2009) in the reduction. The peak at 1636 cm−1 may be due to the presence of carbonyl group (C = O) associated with NH moiety in Riboflavin. When Cu+2 ion forms complex with modified AgNP the peak at 1636 cm −1 shifts to 1628 cm−1 (Fig. 7C). This shift indicates the involvement of peptide linkage present in Riboflavin during complex formation.
XRD analysis
The XRD analysis was performed to confirm the crystalline nature of the biosynthesized AgNP. The AgNP shows peaks at 38.20°, 44.10°, 64.59° and 77.80° which are found normally. The findings indicate that the synthesized AgNP is crystalline and essentially fcc in nature. XRD of the Copper complex of AgNP (Fig. 8) shows peaks at 32.81°, 41.61°, 46.87°, 55.20°, 57.58°, 81.84° in addition to the above peaks.
Conclusions
Here, we have reported methods for detection of Cu+2 ion and Hg+2 ions using biosynthesized silver nanoparticles. The detection of Cu+2 ion present in aqueous solution was based on changes in absorbance resulting from complex formation of the metal ion. Various concentrations of Cu+2 ion were tried to test the linearity and sensitivity of the method. A new peak at around 770 nm, in addition to the peak of the AgNP at 406 nm, was observed in each case (above 20 µM). With the increase of concentration of Cu+2 ion solution, the absorbance at 406 nm peak decreased and that of 770 nm increased gradually. The calibration curve obtained from the ratio of the absorption coefficients of these two peaks (Ex770/406) versus concentration of Cu+2 ions enables one to estimate quantitatively the amount of Cu+2 ions present in water in µM levels. This AgNP further functionalized with 3-mercapto-1, 2-propanediol for detection of Hg+2 present in water by colorimetric method. A new peak at around 606 nm appeared along with the peak of AgNP when solution of Hg+2 was added in functionalized AgNP (MPD-AgNP). The appearance of the peak might be due to the recognition of heavy metal ion Hg+2 by MPD-AgNP through dipropionate ion. A calibration curve between the ratios of the absorption coefficients of these two peaks (Ex404/606) and concentration of Hg+2 was exploited for quantitative estimation of Hg+2 present in water at µM level.
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Acknowledgments
We are thankful to Central Research Facility at IIT, Kharagpur, India for HRTEM and XRD studies.
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The authors declare that there is no competing interest.
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Maiti, S., Barman, G. & Konar Laha, J. Detection of heavy metals (Cu+2, Hg+2) by biosynthesized silver nanoparticles. Appl Nanosci 6, 529–538 (2016). https://doi.org/10.1007/s13204-015-0452-4
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DOI: https://doi.org/10.1007/s13204-015-0452-4