Rapid colorimetric detection of Hg²⁺ ion by green silver nanoparticles synthesized using Dahlia pinnata leaf extract

2.1 Materials

Fresh leaves of D. pinnata (Figure 1) were collected from a local nursery for preparing leaf extract. Pure silver nitrate and mercury chloride required for this work were purchased from Merck India Ltd., Mumbai, India. Other metal salts used in this experiment were also the products of Merck India Ltd.

Figure 1:

Dahlia pinnata leaves.

2.2 Methods

2.2.1 Preparation of silver nanoparticles:

To prepare the leaf extract of D. pinnata, the collected leaves (weight around 50 g) were cleaned, chopped and crushed inside a grinder. Then it was filtered and centrifuged at 2000 rpm for obtaining clear extract soup of D. pinnata leaves. A stock solution (20 mm) of AgNO₃ was prepared by dissolving around 0.34 g of silver nitrate into 100 ml of deionized (DI) water. To reduce the silver ions present in the AgNO₃ solution, an equal amount (i.e. 100 ml) of leaf extract was added drop wise to it and the half diluted reacting mixture (with resulting concentration of 10 mm) was kept at room temperature in darkness. After a couple of hours of incubation, the color of the mixture began to change from colorless to dark yellow (Figure 2) indicating the formation of nanosilver in the reacting mixture. When the reaction completed, the mixture was centrifuged at 10,000 rpm for 20 min to separate colloidal nanoparticles from other components of the mixture. To enhance the purity of the silver nanoparticles and remove the biomass completely, the precipitate formed at the bottom of the centrifuge tube after centrifugation was further re-dispersed in a small amount (approximately 10 ml) of DI water and centrifuged again at 5000 rpm for 10 min. After centrifugation, the soup was decanted and the pellet of colloidal particles formed at the bottom of the tube was collected carefully and dried inside a vacuum drier for 12 h to obtain dry powder of silver nanoparticles. The dry powder was later used for different characterizations and study of its hazardous metal ion sensing ability.

Figure 2:

Color change of reacting mixture with incubation time.

2.3 Study of sensing activity

To evaluate the metal ion detecting ability of green synthesized silver nanoparticles, five different metal salts, i.e. Cd(NO₃)₂, Cr₂(SO₄)₃, Pb(NO₃)₂, Zn(NO₃)₂ and HgCl₂ with the required amount were dissolved in DI water to prepare the stock solution (1 mm) of each salt for colorimetric study. When 1 ml solution of each metal salt was added to 1 ml light brown suspension of Ag nanoparticles, a distinct color change was observed only in the case of HgCl₂ where the light brown suspension turned colorless instantly. No such specific colorimetric change was noticed for the other metal ions after addition to the suspension. To determine the minimum detectable concentration of Hg²⁺ (for visual detection by color change), various concentrations of mercury ions were exposed to the suspension of silver nanoparticles and the UV-Vis spectra of the mixture was recorded by a Perkin Elmer UV-Vis spectroscope (λ-35, USA). Later, the biogenic silver nanoparticle based Hg²⁺ ion detection was performed at a wide pH range (between pH 3 and 8), taking the concentration of Hg²⁺ as 10 μm, i.e. the minimum detectable concentration as derived from UV-Vis spectra.

3.1 Determination of Ag nanoparticle formation by UV-Vis spectroscopy

D. pinnata is a herbaceous plant, the leaves of which are a potent source of functional organic molecules [24]. Hence, the leaf extract of this plant is expected to reduce silver ions, if exposed. When the leaf extract of D. pinnata was added to the AgNO₃ solution, the color of the mixture started to change from colorless to dark yellow after a couple of hours of incubation. The color intensified with incubation time and turned into dark brown after 8 h of observation, indicating the reduction of Ag⁺ ions and formation of silver nanoparticles gradually in the medium. The color change is expected to arise due to surface plasmonic resonance of Ag nanoparticles [33]. The colloidal mixture was scanned using a UV-Vis spectrometer and the peak absorbance noticed near 460 nm further confirmed the formation of silver nanoparticles. The UV-Vis spectra were obtained at regular intervals (every 2 h) to have an insight of nanoparticle formation in the reacting mixture (Figure 3). The peak value of absorbance near 460 nm was observed to be increasing with incubation time without any shifting and its variation with time is shown in Figure 3 (inset). From Figure 3 (inset), it is clear that the maximum absorbance increases linearly up to 8 h of reaction time denoting production of a greater number of Ag nanoparticles in the medium. Beyond 12 h of observation, the formation rate saturates, suggesting the completion of reaction.

Figure 3:

Time dependent UV-Vis spectra of silver nanoparticle formation; inset figure shows the variation of maximum absorbance with reaction time.

3.2 Structural analysis by XRD and TEM

The XRD pattern of the biogenic silver nanoparticles (Figure 4) consists of seven noticeable peaks at 2θ=27.75°, 32.15°, 38.15°, 46.25°, 54.65°, 57.25° and 76.83° which can be assigned to (220), (122), (111), (231), (331), (241) and (311) planes of face centered cubic structures of silver, respectively, (correlated to JCPDS: File No. 04-0783). In addition, absence of any other peaks in the curve also indicates the purity of the biosynthesized silver nanoparticles.

Figure 4:

X-ray diffraction (XRD) pattern of green synthesized Ag nanoparticles.

The high resolution TEM images (Figure 5) reveal the size, shape and morphology of the biogenic Ag nanoparticles. It may be observed from the TEM images that the shape of nanoparticles is almost spherical with diameter nearly 15 nm. The interplanar spacing was manipulated from the lattice fringes (Figure 5B) to be around 0.28 nm which may correspond to the (122) planes of silver nanoparticles.

Figure 5:

(A) and (B) transmission electron microscopy (TEM) images of biogenic silver nanoparticles.

3.3 Analysis by FTIR spectroscopy

FTIR spectra of both the extract and the biogenic Ag nanoparticles were recorded in absorbance mode to detect the functional biomolecules that contribute to the reduction and stabilization of the silver nanoparticles during interaction in the medium. The spectrum of the leaf extract of D. pinnata reveals six distinct peaks, as shown in Figure 6. Two bands noticed at 1064 cm^-1 and 3265 cm^-1 can be assigned to the C-N bond present in amines and O-H stretching of aromatic compounds (like phenol, etc.), respectively [34]. A band observed at 2916 cm^-1 may be attributed to the C-H stretching of aldehydes, whereas the band at 1423 cm^-1 possibly indicates C-H bending of alkanes [12, 35].The remaining two bands at 673 cm^-1 and 1595 cm^-1 may correspond to the stretching vibration of halo-alkanes and bending of C-H bonds present in hydrocarbons, respectively [36]. The spectrum of biogenic silver nanoparticles appears to be broadened and most of the intense peaks disappeared, indicating a high level of purity of the samples. It is clear from this analysis that the aromatic compounds (like phenol, etc.) along with other hydrocarbons present in the leaf extract of D. pinnata probably reduced the silver ions and stabilized the colloidal particles during interaction in the reacting medium.

Figure 6:

Fourier transform infrared (FTIR) spectra of (A) leaf extract of Dahlia pinnata and (B) biogenic silver nanoparticles.

3.4 Hg²⁺ sensing activity of Ag nanoparticles

The suspension of green silver nanoparticles was evaluated here as a colorimetric sensor for different metallic ions by adding equal concentrations (1 ml, 1 mm) of the solutions of metal salts into a suspension of green synthesized Ag nanoparticles that showed decolorizing after addition of Hg²⁺ selectively (Figure 7 [inset]). Addition of other metal ions (Cd²⁺, Cr³⁺, Zn²⁺ and Pb²⁺) into the suspension displayed no specific color changes except broadening and a small shift of the absorbance peaks as shown in Figure 7. Prominently, the addition of Hg²⁺ ion into the suspension of nanosilver instantly turned it colorless from light brown and thus established the selective colorimetric detection of Hg²⁺ [37]. The minimum detectable concentration of Hg²⁺ ions for visual detection was determined by adding different Hg²⁺ concentrations into the suspension of Ag nanoparticles and following the change of absorbance in the UV-Vis spectra of the solution. From Figure 8, it is clear that the minimum concentration of HgCl₂ (10 μm) added to the suspension slightly reduced the intensity of absorption. The absorbance peak decreased gradually with increasing concentration of Hg²⁺ and finally disappeared (almost at 100 μm). The variation of peak absorbance with Hg²⁺ concentration is shown in Figure 8 (inset). The mechanism of Hg²⁺ detection by suspension of Ag nanoparticles can be explained by the electrochemical differences between these two metal ions: Hg²⁺ and Ag⁺. The reduction potential of Ag⁺ is +0.80 V (Ag⁺+e=Ag) whereas the standard reduction potential for Hg²⁺ is +0.92 V (2Hg²⁺+2e=Hg₂²⁺) [38]. According to the electrochemical series, the higher reduction potential of the metals signifies better oxidizing abilities [39]. Hence, Hg²⁺ can oxidize the colloidal silver during interaction and form Ag⁺ ions, resulting in the decolorization of the solution [40].

Figure 7:

UV-Vis spectra of Ag nanoparticle suspension with different metal ions; inset figure shows the selective colorimetric sensing of Hg²⁺ ion in aqueous solution.

Figure 8:

Change of absorbance with Hg²⁺ concentration; inset figure shows the variation of peak absorbance with Hg²⁺ concentration.

To sense the presence of Hg²⁺ in water at various physiological conditions, we performed the colorimetric detection of Hg²⁺ (taking minimum detectable concentration) by suspension of nanosilver in a wide pH range (pH=3–8) [41]. The pH of the solution was controlled by addition of required amount of diluted NaOH to the reacting solution and the UV-Vis spectra of the solution were recorded for different pH values. However, no significant change was observed in the efficacy of the suspension of Ag nanoparticles for detecting Hg²⁺ ions throughout the whole range of pH. Therefore, these stable biogenic silver nanoparticles can be used successfully for detection of hazardous Hg²⁺ ions in water and further protect the environment from hazardous mercury pollution [38, 42].