- Email: [email protected]
Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples
Accepted Manuscript Title: Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples Author: Buduru Prasad B.C. Sundher Raja Reddy N.V.S. Naidu PII: DOI: Reference:
S0925-4005(17)30048-5 http://dx.doi.org/doi:10.1016/j.snb.2017.01.041 SNB 21566
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-9-2016 2-1-2017 5-1-2017
Please cite this article as: Buduru Prasad, B.C.Sundher Raja Reddy, N.V.S.Naidu, Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples
Buduru Prasad*a, B.C. Sundher Raja Reddyb and N.V.S. Naidua
a
Department of Chemistry, Sri Venkateswara University,Tirupati-517502, Andra Pradesh, India Department of Geology, Sri Venkateswara University, Tirupati-517502, Andra Pradesh, India
b
*
Corresponding author, E-mail: [email protected]
1
Research Highlights
Gln-His-Ag NPs act as a probe for colorimetric sensing of Hg2+ ion.
The aggregation of Gln-His-Ag NPs induced by Hg2+ ion causes red-shift and color change.
The sensitivity of the probe was improved to 28-times in the presence of 0.2 M NaCl.
A good linear response for Hg2+ ion was found with a detection limit of 0.90 µM.
This probe showed good selectivity towards Hg2+ ion in the presence of other metal ions.
Abstract A simple, rapid and sensitive colorimetric method was developed for the detection of Hg2+ ion in water samples using glutamine (Gln) and histidine (His) functionalized silver nanoparticles (Gln-His-Ag NPs) as a probe. The functionalized Ag NPs were confirmed by UVvisible, FT-IR and transmission electron microscopic (TEM) techniques, respectively. The Hg2+ ion was induced quickly the aggregation of Gln-His-Ag NPs, yielding a color change from yellow to orange, which can be readily seen by naked eye. The recognition mechanism is attributed to unique supramolecular nanostructures of Gln-His-Ag NPs, which yields strong interaction (cooperative metal-ligand interaction) between Gln-His-Ag NPs and Hg2+ ion. A linear correlation was obtained in the range of 100 – 1000 μM with a detection limit of 25.48 µM. However, the sensitivity of the probe was greatly improved by the addition of 0.2 M NaCl as an ionic strength. The calibration graph was constructed between absorption ratio (A500/A402) and concentration of Hg2+ ion in the range of 1.0 – 500 µM with the detection limit of 0.90 µM, which is 28-times lower than the direct method (without NaCl). This method was successfully applied to detect Hg2+ ion in water samples. With the advantages of simplicity, selectivity and low sample volume, this method can potentially suitable for on-site monitoring of Hg2+ ion. Keywords: Gln-His-Ag NPs, Hg2+ ion, TEM, UV-visible spectrometry and water samples.
2
1. Introduction Mercury is a highly toxic and bioaccumulative heavy metal ion that causes a number of toxicological effects such as brain damage, kidney failure, and various cognitive and motion disorders even at a very low concentration [1]. Because of its high toxicity even at a trace concentration, a simple, specific and sensitive determination of Hg2+ is of particular interest in biological, toxicological, and environmental monitoring. Recent years, many analytical techniques have been used for the detection of Hg2+ ion in various matrices, including atomic absorption/emission spectroscopy [2], selective cold vapor atomic fluorescence spectrometry [3], X-ray fluorescence spectrometry [4], inductively coupled plasma mass spectrometry [5], anodic stripping voltammetry [6], atomic force microscopy [7], surface plasmon resonance [8], quartz crystal microbalance (QCMB) [9], magnetic resonance imaging [10], surface-enhanced Raman scattering [11], and electrochemical-based analytical methods [12-13], respectively. Even though these methods are successfully used to detect Hg2+ ion in complex samples, unfortunately these are either time-consuming or laboratory-based sophisticated instruments, which limit their application in routine monitoring of Hg2+ ion with reduced sample treatment. Thus, it is still of great challenge to develop a simple method for detection of Hg2+ environmental samples. In recent years, metal nanoparticles have proven to be as promising colorimetric probes for selective and sensitive detection of a wide variety of chemical species in environmental and biological samples [14-16]. Among the metal NPs, Ag NPs have attracted much research interests owing to many advantages over Au NPs including cheap precursor (silver nitrate), higher extinction coefficient (1010 M-1 cm-1), higher ratio of scattering to extinction and sharper extinction bands which makes them more prominent candidate than Au NPs for sensing of various chemical species [17]. As a result, several research groups have been developed Ag NPs-
3
based colorimetric methods for the detection of various heavy metals including Hg2+ ions. Briefly, Wang’s group described the use of unmodified Ag NPs as a probe for the colorimetric detection of Hg2+ ion in water samples [18]. Farhadi et al., developed a green synthetic approach for the preparation of Ag NPs and used as a probe for the highly selective detection of Hg2+ without modification [19]. Yuan and co-workers functionalized Ag NPs with poly(amidoamine) dendrimers and used as a colorimetric probe for the detection of Hg2+ ion [20]. Ren’s group incorporated Ag NPs in surface-enhanced Raman spectroscopic (SERS) technique for the detection of Hg2+ ion with improved sensitivity [21]. Gelatin stabilized Ag NPs were used as probes for the label free colorimetric detection of Hg2+ ion in waste water and blood samples [22]. Similarly, Chen et al., functionalized Ag NPs with 4-mercaptopyridine for the sensitive detection of Hg2+ ion using SERS technique [23]. Furthermore, Li’s group described a simple and selective method for ultra sensitive detection of Cu2+ and Hg2+ ions using cysteine-functionalized Ag NPs as a probe in SERS [24]. Liu and co-workers described the use of Ag NPs as a fluorescent probe for the detection of Cu2+ and Hg2+ ions in mineral water samples [25]. Annadhasan’s group developed green synthetic approaches for preparation of Au and Ag NPs and used as probes for simultaneous detection of Hg2+, Pb2+ and Mn2+ ions in water samples [26]. Anthony and co-workers prepared Ag NPs using plant extracts (neem leaf, mango leaf, green tea and pepper seed) as reducing agents and used as a probe for colorimetric detection of multiple metal ions (Hg2+, Zn2+, Pb2+) in water samples [27]. Similarly, the same group illustrated the use of N-(2-hydroxybenzyl)-valine and N(2-hydroxybenzyl)-isoleucine functionalized Ag NPs as a probe for simultaneous colorimetric sensing of Cd2+, Hg2+ and Pb2+ ions in aqueous solution at ppm level [28]. Apart from these Ag NPs-based
methods,
several
research
groups
4
have
been
developed
UV-visible
spectrophotometric methods for detection of Hg2+ ion in various samples using gold nanoparticles (Au NPs) as probes. In these methods, Au and Ag NPs were functionalized with various organic molecules including malonamide dithiocarbamate (MA–DTC) [29], papain [30], NH2OH [31], oligonucleotides [32], gold nanoflowers [33], deoxyribonucleic acid [34], and unmodified Ag-Au NPs [35] for colorimetric detection of Hg2+ ion by UV-visible spectrometry with improved sensitivity. These reports illustrated that the potentiality of Ag NPs as probes for colorimetric detection of metal ions in various environmental and biological samples. Inspired by these works, we decided to functionalize Ag NPs with Gln-His that can act as a distinct probe for simple and selective colorimetric detection of metal ions. In this paper, we report a size and shape controlled synthesis of Ag NPs using Gln and His as capping agents in aqueous medium. The Gln-His-Ag NPs were characterized by UVvisible, Fourier transform infrared spectroscopic (FT-IR) and transmission electron microscopic (TEM) technique. The Hg2+ ion was induced the aggregation of Gln-His-Ag NPs via coordination covalent bond between Hg2+ ion and –COOH and –NH2 group of Gln-His-Ag NPs (Scheme 1), resulting a red-shift and a color change from yellow to orange. This colorimetric method for the detection of Hg2+ showed good selectivity and sensitivity. It could be applied to detect Hg2+ in water samples.
5
2. Experimental section 2.1. Chemicals and materials Silver nitrate, sodium borohydride, tris(hydroxymethyl) aminomethane (Tris), glutamine, histidine, trisodium citrate dihydrate, metal salts
((Cd(NO3)2.4H2O, Co(NO3)2.6H2O,
Cu(NO3)2.3H2O, FeCI2. 4H2O, FeCl3.6H2O, Hg(NO3)2.H2O, Mg(NO3)2.6H2O, Mn(NO3)2.4H2O, NiSO4.6H2O, Pb(NO3)2, Zn(NO3)2.6H2O, KNO3 and NaNO3) were purchased from SigmaAldrich, USA. Hydrochloric acid was purchased from Merck Ltd., India. All chemicals are from analytical grade and used without further purification. Milli-Q-purified water was used for sample preparations.
2.2. Synthesis of Gln-His-Ag NPs Ag NPs were synthesized by sodium borohydride reduction of silver nitrate. Briefly, 15 mL of 2 mM NaBH4 with ice-chilled solution was mixed with 1.0 mL of tri-sodium acetate (1%) and then stirred for 5 min. Then, 5.0 mL of 1.0 mM AgNO3 aqueous solution was added drop wise into the above solution under vigorous stirring at room temperature, producing yellow colloidal Ag NPs. To bifunctionalize Ag NPs with Gln and His, 250 μL of Gln (1.0 mM) and His (0.25 mM) were added into the above solution under constant stirring for 2 h to molecular assembly of Gln and His on the surfaces of Ag NPs (Supporting Information of Figure S1-S2). All the experiments were performed at room temperature. The size of Gln-His-Ag NPs was verified by TEM and average size of Gln-His-Ag NPs was found to be ~10 nm. The concentration of Gln-His-Ag NPs was calculated to be ~26.80 nM by UV-visible spectroscopy based on an extinction coefficient of 4.18×109 M -1 cm-1 at λ = 407 nm.
6
The stability of Gln-His-Ag NPs over time (1 to 17 days) was monitored using UVvisible absorption spectroscopy at room temperature. The stability of Gln-His-Ag NPs over time (1 to 17 days) was monitored using UV-visible spectroscopy by measuring the maximum SPR peak at 402 nm (Supporting Information of Fig. S3). It should be noticed that the characteristic SPR peak of Gln-His-Ag NPs was observed at 402 nm with negligible decrease in the SPR peak intensity, suggesting that the synthesized Gln-His-Ag NPs are stable and dispersed in aqueous media, which confirms that Gln and His molecules preserves stability and hinders agglomeration of Ag NPs.
2.3. Detection of Hg2+ ion To develop Gln-His-Ag NPs as a probe for the colorimetric detection of Hg2+ ion, we have taken 700 µL of metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+, Cr3+ and Hg2+, 1.0 mM) solutions and added separately into 1.0 mL of Gln-His-Ag NPs solutions that contains 100 µL of Tris-HCl buffer, resulting a specific color change from yellow to orange only by the addition of Hg2+ ion. The colorimetric changes were observed by UV-visible spectrophotometer. The practical application of the probe for real samples water samples (drinking water, tap water and river water) was also described. Briefly, water samples (drinking water, tap water and river water) were collected from Tirupati in and around and Swarnamukhi River. The collected samples were spiked with known concentration of Hg2+ ion (150 and 200 µM) and other metal ions (400 µM), mixed thoroughly, filtered by using 0.45 µm membrane and then analyzed by the aforesaid procedure.
7
2.4. Instrumentation UV-visible absorption spectra were measured by using Maya pro 2000 spectrophotometer (Ocean Optics,USA) at room temperature. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). Transmission electron microscopy (TEM) images were taken on a JEOL 3010.
3. Results and discussion 3.1. Characterization of Gln-His-Ag NPs Supporting Information of Figures S1-S2 show the UV-visible absorption spectra of Ag NPs using Gln and His as capping agents. To control the size and surface plasmon resonance (SPR) peak of Ag NPs, we studied the effect of Gln and His concentrations on the surfaces of Ag NPs. As shown in Supporting Information of Figure S1, the SPR peak of Ag NPs at 400 nm was slightly decreasing with increasing concentration of Gln from 0.5 to 5.0 mM. To control the SPR peak and color of Ag NPs, we selected 1.0 mM of Gln as the best concentration for the functionalization of Ag NPs. After optimization of Gln concentration, we also studied the effect of His concentration on the surfaces of Gln-Ag NPs (Supporting Information of Figure S2). It can be observed that 1.0 mM of Gln and 0.25 mM of His were found to be the best concentration for effective molecular assembly on the surfaces of Ag NPs, since the characteristic SPR peak is still remain < 410 nm, which suggests that the diameter of Gln-His-Ag NPs is less than 10 nm. Importantly, it can be noticed that very slight change in the SPR band of Ag NPs before and after modifications, which confirms the functionalization of Ag NPs with Gln and His. The both bare Ag NPs and Gln-His-Ag NPs have shown strong absorption peaks at 395 and 407 nm. Moreover,
8
before and after modifications it also observed that there is slight red – shift in SPR band of Ag NPs, which confirms that the functionalization of Ag NPs with Gln and His molecules. To confirm the attachment of Gln and His on the surfaces of Ag NPs, we studied the FTIR spectra of pure Gln, pure His and Gln-His-Ag NPs (Supporting Information of Figures S4S5). It can be observed that the broad band at 3410 cm-1 belongs to stretching of –OH group in Gln. The peaks at 1334, 1292 and 1257 cm-1 represent the bending, twisting and wagging of methylene groups in Gln. Similarly, the broad absorption bands intensities at 3353 and 3244 cm-1 are corresponded to the asymmetric and symmetric N-H stretching vibrations of - NH2 group. The pure N-H stretch of His appears at 3410 cm-1 and the broad band at 3078 cm-1 corresponds to N-H symmetric stretches of His and also protonated NH2 which are varyingly H-bonded to the environment. The band at 1639 cm-1 represents the anionic nature of carboxylate group in His, which is clearly evident from the C = O stretch. The skeletal vibrations of the imidazole ring were observed at 1581 cm-1. The peaks at 1336 and 1064 cm-1 are assigned to the OH plane deformation in COOH and NH symmetric stretch respectively and its shoulder at 1141 cm-1 is assigned to COO- stretch of the carboxylate anion in the ring of His. The bands at 821 and 1498 cm-1 correspond to the NH2 wagging vibrations and NH3+ symmetric deformation vibrations. The bands at 1287 and 550 cm-1 represent to the NH3+ rocking vibrations of the imidazole ring, CH2 rocking vibrations and the COO- rocking vibrations. The peak at 628 cm-1 is assigned to the C-OH bending vibrations and the C=O stretching vibration is observed at 1639 cm-1, respectively. However, the stretching and vibration bands of carboxylic and amino groups of Gln and His were shifted to lower and longer wave numbers, proving the interaction of Gln and His with the surfaces of Ag NPs, which actually stands as the evidence for the bonding interaction of Gln and His with Ag NPs. The morphology and size distribution of Gln-His-Ag NPs was characterized
9
by TEM. Figure 2a shows a typical TEM image of the as-synthesized Gln-His-Ag NPs. Figure 1b shows a related histogram of as-prepared Gln-His-Ag NPs. It can be noticed that the most of Gln-His-Ag NPs display well-distributed spherical nanoparticles with an average diameter of 5.5±1.0 nm.
3.2. Colorimetric sensing of Hg2+ ion To test their analytical application as a colorimetric sensor, we studied their SPR properties and tendency to agglomerate in the presence of analyte. The interaction of Gln-His-Ag NPs with various metal ions (1.0 mM) was monitored by UV-visible spectral change and also the color change was visualized with naked eye (Figure 2). Out of 16 metal ions tested (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+ and Hg2+), only Hg2+ ion exhibited significant change the UV-visible absorption spectra and the color of the solution, which can be easily observed with naked eye. This indicates that Gln-His-Ag NPs can be used as colorimetric sensor to detect Hg2+ ion in aqueous medium either without any modification or sample pretreatment. The selective interaction of Gln-His-Ag NPs with Hg2+ ions over the other environmentally relevant heavy metal ions could be due to the presence of amino (-NH2) and carboxylic (-COO-) groups on the surface of Gln-His-Ag NPs. It is noticed that only Hg2+ ion exhibited substantial change in the spectrum with a red-shift of the λmax from 407 to 480 nm and a sharp color change from yellow to orange, detectable by naked-eye. The shift in λmax of the plasmon band and the color change of the solution is due to aggregation of Gln-His-Ag NPs induced by Hg2+ ion through the coordination covalent bonding between the surface functional groups of Gln-His-Ag NPs and Hg2+ ion. The absorption ratio at A480/A407 is related to the quantities of dispersed and aggregated Gln-His-Ag NPs, respectively. Therefore,
10
the ratio of the values can be used to express the molar ratio of aggregated and dispersed forms of Gln-His-Ag NPs. In order to confirm the role of Gln and His molecules on the surface of Ag NPs, we studied the UV-visible absorption spectra and color of the bare Ag NPs (without capping of Gln and His) and Gln-His-Ag NPs with addition of Hg2+ ion. As shown in Figure 3, bare Ag NPs did not show any absorption spectral change or color change, indicating that Hg2+ ion did not interact with the bare Ag NPs. However, there is a drastic change in the absorption spectra and color of Gln-His-Ag NPs upon the addition of Hg2+ ion, confirming that the assembly of Gln and His molecules on the surfaces of Ag NPs play key role to interact with Hg2+ via coordinate covalent bond, resulting a red-shift and a color change.
3.3. Effect of pH To provide best condition for detection of Hg2+ ion with good selectivity and sensitivity, we have investigated the effect of various buffer media including phosphate-buffered saline (PBS), sodium acetate, and Tris-HCl pHs from 2.0 to 12.0 on the sensing ability fo Gln-His-Ag NPs towards Hg2+ ion. Initially, we studied the effect of PBS, sodium acetate and Tris-HCl buffer pHs ranging from 2 to 12 without addition of Hg2+ ion (Supporting Information of Figures S6-S8). It can be noticed that there is no change in the absorption spectra and color of Gln-HisAg NPs, indicating that the functionalized Gln-His-Ag NPs are stable in the above buffer media at pH from 2.0 to 12.0. Figure 4 and Supporting Information of Figures S9-S10 show the absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion in the presence of PBS, sodium acetate and Tris-HCl buffer media at pH 2.0-12. The absorption ratio was high at all the buffer media pH 2.0, confirming the self-aggregation of Gln-His-Ag NPs without induced by
11
Hg2+ ion. However, the absorption ratio at A480/A407 is very high at Tris-HCl pH 9.0, indicating that the high degree of aggregation of Gln-His-Ag NPs induced by Hg2+ ion. It can be noticed that the SPR peak is red-shifted in all buffer media at acidic media and neutral pH. The reason may be attributed to the fact that the functional groups (amino and carboxylic acid) at the surfaces of Gln-His-Ag NPs is protonated and is thus unable to coordinate with Hg2+, which leads to a red-shift with low intensity. However, at Tris-HCl pH 9.0, the absorption ratio at A480/A407 is very high, indicating that Hg2+ ion is highly induced the aggregation of Gln-His-Ag NPs via coordinate covalent bonds. Since, the coordinate covalent bonds will be facilitated between the –NH2 and -COOH groups of Gln-His-Ag NPs and Hg2+ ion, which facilitate to generate a new SPR peak around 480 nm. As a result, the characteristic SPR peak of Gln-His-Ag NPs was red-shifted from 407 to 480 nm, which yields a color change from yellow to orange that can be observed with naked-eye. These results indicate that Hg2+ ion shows strong ability to interact with –NH2 and -COOH groups of Gln-His-Ag NPs surfaces, resulting the aggregation of Gln-His-Ag NPs induced by Hg2+ ion through coordinate covalent bonds between Gln-His-Ag NPs and Hg2+ ion, which favors for electric dipole-dipole interactions, neutralization of surface charges and coupling between the plasmon of neighboring Ag NPs. Therefore, Tris-HCl pH 9.0 was selected as the best pH for the colorimetric detection of Hg2+ ion using Gln-His-Ag NPs as a probe. In order to confirm the aggregation of Gln-His-Ag NPs induced by Hg2+ ion, we studied the TEM image of Gln-His-Ag NPs before and after addition of Hg2+ ion. It can be noticed that Gln-His-Ag NPs showed highly dispersed nature (Figure 1a) with an average size of 10 nm, however after addition of Hg2+ ion Gln-His-Ag NPs came closer and induced the aggregation, resulting to from localized clusters (Figure 1b). This observation
12
indicates that the aggregation of Gln-His-Ag NPs induced by Hg2+ ion and in accordance with the Mie theory explains red-shift observed in SPR. Furthermore, we also studied the turbidance of the solutions with and without addition of Gln-His-Ag NPs in the presence of Hg2+ ion (100 and 500 µM) at pH 9.0 (Supporting Information of Figure S11). Since Hg2+ ion can be formed turbidity at higher pH (>8.0). It was observed that negligible turbidity was observed at pH 9.0, however it does not effects the measurement of absorption spectra of the solutions. To ensure selective sensing ability of GlnHis-Ag NPs towards Hg2+ ion, the sensing ability of Gln-His-Ag NPs was studied upon the addition of divalent metal ions (Cu2+, Cd2+, Ca2+, Co2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+, 1.0 mM) separately at Tris-HCl pH in the range of 2.0 – 12.0 (Supporting Information of Figures S12-S16). It can be noticed that the absorption spectra and color of Gln-His-Ag NPs did not show any obvious red-shift and color changes upon the addition of divalent metal ions (Cu2+, Cd2+, Ca2+, Co2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+, 1.0 mM) at Tris-HCl pH in the range of 2.0 – 12.0, suggesting the other divalent metal ions did not induce spectral and color changes of GlnHis-Ag NPs at Tris-HCl pH ranging from 2.0 – 12.0, which confirms that the Gln-His-Ag NPs acted as a selective probe for detection of Hg2+ ion.
3.4. Sensing mechanism As shown in Scheme 1, Gln-His-Ag NPs surfaces contain the amino (-NH2) and carboxylic acid (-COOH) which provides the binding sites for Hg2+ ion to form the complex. Since Hg2+ ion forms stable complexes due to the strong chemical bonding of Hg2+ ion to carboxylic acid and amino groups [36-38]. With this, we assume that the carboxylic acid and amino groups of Gln and His in Ag NPs play key role to bind with Hg2+ ion, favoring to decrease
13
interparticle distance through the formation of coordination covalent bonds, which results a redshift in the SPR band and a color change from yellow to orange. The surface functional groups (NH2 and –COOH) of Gln-His on Ag NPs dictate their specific interaction with Hg2+ ion, where Hg2+ ion acts as a driving force to induce the aggregation of Gln-His-Ag NPs [37-38]. Therefore, it was expected that the carboxylic acid and amino groups of both Gln-His-Ag NPs show high affinity to bind with Hg2+ ion, leading to the aggregation of Gln-His-Ag NPs induced by Hg2+ ion, which results drastic changes in the SPR peak and color . To confirm Hg2+ ion coordination bond with Gln-His-Ag NPs, we studied the FT-IR spectrum of Gln-His-Ag NPs with Hg2+ ion (Supporting Information of Figure S5b). The FT-IR spectrum of Gln-His-Ag NPs exhibited a broad band at 3450 cm−1 which is assigned to ν(OH) of Gln and His molecules. This band intensity is very less at the same position in the FT-IR spectrum of Gln-His-Ag NPs with Hg2+ ion, indicating the participation of these OH group in bonding. The intense peak at 1454 cm-1 is attributed to O-H bending (in-plane) and is shifted toward lower wave number at 1442 cm-1 which suggests that –OH group involves in the coordination bond with Hg2+ ion. The peaks at 1583 and 1641 cm-1 belong to NH2 group scissoring(1°-amines) and decreased peak intensities, proving the involvement of –NH2 groups in the coordination bonding with Hg2+ ion. The peak at 1795 cm-1 is attributed to stretching vibration of carbonyl groups (C=O) of Gln and His molecules, however the intensity of the peak was slightly decreased, which confirms that the involvement of the carboxylic group in covalent bonding with Hg2+ ion. These results indicate that carboxylic (-COOH) and NH2 groups of Gln and His molecules are involved in coordination covalent bonds with Hg2+ ion, resulting the neutralization of charges on the surfaces of Gln-His-Ag NPs, which yields a red-shift in SPR peak of Gln-His-Ag NPs and a color change.
14
3.5. Sensitivity of the probe For quantitative detection of Hg2+ ion, the absorption spectra of Gln-His-Ag NPs were examined upon the addition of different concentrations of Hg2+ ranging from 100 to 1000 µM at optimal conditions. At the same time, the change of Hg2+ concentration could be observed by the naked-eye, as presented in Figure 5. Upon addition of increasing concentrations of Hg2+, the color of Gln-His-Ag NPs solution was changed gradually, initially from yellow to light brown, and finally to orange. Analogously, the characteristic SPR peak at 407 nm was gradually decreased and a new SPR peak was increased with increasing concentration of Hg2+ ion (Figure 5). The absorption ratio of A480/A407 was linear with a correlation coefficient of 0.993 within the range of 100 – 1000 μM of Hg2+ concentration (Supporting Information of Figure S17). The limit of detection (LOD) was calculated using the formula, LOD = 3 × sb/S (in which, sb = standard deviation of blank signal (n=3) and S = slope of the concentration peak intensity of calibration curve) and found to be 25.48 µM.
3.6. Improving the sensitivity of probe with the addition of NaCl as an ionic strength In order to improve the sensitivity of the probe, we studied the effect of NaCl (0.001 to 1.0 M) on the absorption spectra and color of Gln-His-Ag NPs without addition of Hg2+ ion. The UV-visible absorption spectra and color of Gln-His-Ag NPs show significant changes with increasing concentration of NaCl from 0.1 to 1.0 M, suggesting the aggregation of Gln-His-Ag NPs at high ionic strength. However, the absorption spectra and color of Gln-His-Ag NPs did not show any obvious effect at 0.2 M of NaCl, which confirms that the prevention of aggregate formation in NaCl as ionic strength (Supporting Information of Figure S18). In order to know the influence of NaCl on the aggregation of Gln-His-Ag NPs induced by Hg2+, we studied the UV-
15
visible absorption spectra and color changes of Gln-His-Ag NPs upon the addition of Hg2+ ion in the range of 1.0 – 500 µM separately in the presence of 0.2 M NaCl at Tris-HCl pH 9.0 (Figure 6). The SPR band shift relative to Gln-His-Ag NPs-Hg2+ ion, the absorption ratio at A500/A402 is used to quantify the concentration of Hg2+ ion. Since, the Gln-His-Ag NPs solution at 402 and 500 nm are related to the quantities of dispersed and aggregated Gln-His-Ag NPs in the presence of Hg2+ ion, respectively. Thus, we used the absorption ratio at A500/A402 to express the molar concentration of Hg2+ ion based on the aggregated and dispersed Gln-His-Ag NPs using 0.2 M of NaCl as a destabilizing agent. As shown in Figure 6, when the concentration of Hg2+ increased from 1.0 to 500 μM, the characteristic SPR band at 402 nm was gradually decreased and generated new SPR band at 500 nm, yielding a color change from yellow to orange. As a result, the absorption ratio (A500/A402) increases linearly with the increasing concentration of Hg2+ ion from 1.0 to 500 μM (R2 = 0.991) (Supporting Information of Figure S19). The limit of detection was found to be 0.90 µM for Hg2+ ion, respectively. It was observed that the LOD of the present method for Hg2+ ion is either lower than the reported methods [19] or close to other reported methods [27, 33] for colorimetric detection of Hg2+ ions using functionalized Au and Ag NPs. Table 1 shows the comparison of present method with the other reported NPs assisted UVVisible, fluorescence, SERS, and quartz crystal microbalance methods for the detection of Hg2+ ion. It was observed that the LOD of the present method is not comparable with the LOD value of NPs assisted surface enhanced Raman scattering, and fluorescence methods due to their high sensitivity than the NPs assisted UV-visible spectrometric methods. Furthermore, we simplified the synthetic procedure for molecular assembly of Gln-His-Ag NPs and allowed to detect the maximum allowable level of Hg2+ in drinking water with reduced sample preparations and volumes.
16
3.7. Interference study To evaluate the selectivity of the proposed method, the response of the sensor to other environmentally relevant metal ions, including Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+, was tested under the same experimental conditions. As shown in Figure 7, the addition of different metal ions to Gln-His-Ag NPs solution did not cause obvious change of the UV–vis absorption spectrum of Gln-His-Ag NPs except Hg2+ ion. The photographic image of Gln-His-Ag NPs solutions in the presence of 0.5 mM of various metal ions, the Gln-His-Ag NPs solution remained disperesed with yellow color except for Hg2+. After addition of 0.5 mM Hg2+, the color of Gln-His-Ag NPs solution changed from yellow to orange due to Hg2+-induced aggregation of Gln-His-Ag NPs. The results revealed that the other metal ions did not interfere with the determination of Hg2+, indicating the Gln-HisAg NPs probe had good selectivity for sensing of Hg2+ ion.
3.8. Detection of Hg2+ in water samples In order to apply this probe for detection of Hg2+ ion in real samples, the collected water samples were first filtered through 0.45 μm membrane to remove insoluble materials, if any, and maintained pH of 9.0 using Tris-HCl buffer. To make the calibration line for quantitative analysis, standard solutions of Hg2+ were prepared and spiked with collected water samples. To these, Gln-His-Ag NPs solutions were added over the concentration range of 100 – 1000 μM. The absorption spectra of the solutions were then recorded and A480/A407 was plotted against the concentration of Hg2+, a linear relationship was obtained throughout the concentration range used. As shown in Supporting Information of Table S1, the recovery values were satisfied
17
(95.00–98.66%), demonstrating this colorimetric method was suitable for Hg2+ detection in environmental samples.
4. Conclusions In conclusion, we have developed a facile colorimetric sensor for the detection of Hg2+ ion with good selectivity and sensitivity based on the aggregation of Gln-His-Ag NPs induced by Hg2+ ion. The concentration of Hg2+ is easily detected by simply mixing Gln-His-Ag NPs and free from any complicated modifying procedure. It exhibits sharp colour change with red-shift of the absorption band and the change in color from yellow to orange can be detected by naked eye. The Gln-His-Ag NPs-based UV–visible spectroscopic method is economy, simple, convenient, and shown potential application for the colorimetric detection of Hg2+ ion other heavy metal ions based on the aggregation Gln-His-Ag NPs.
Acknowledgements The authors thank the Department of science and technology, India for providing the financial support (DST Fast Track Scheme No.SR/ FTP/ES-16/2011).
18
References 1. H.H. Harris, I.J. Pickering and G.N.George, The chemical form of mercury in fish, Science., 301, (2003), 1203. 2. J.V. Cizdziel and S. Gerstenberger, Determination of total mercury in human hair and animal fur by combustion atomic absorption spectrometry Talanta., 64, (2004), 918–921. 3. Y. Yamini, N. Alizadeh and M. Shamsipur, Solid phase extraction and determination of ultra trace amounts of mercury(II) using octadecyl silica membrane disks modified by hexathia18-crown-6-tetraone and cold vapour atomic absorption spectrometry, Anal. Chim. Acta., 355, (1997), 69–74. 4. L. Bennun and J. Gomez, Determination of mercury by total-reflection X-ray fluorescence using amalgamation with gold Spectrochim. Acta Part B At. Spectrosc., 52, (1997), 1195 – 1200. 5. E. Kenduzler, M. Ates, Z. Arslan, M. McHenry and P.B. Tchounwou, Determination of mercury in fish otoliths by cold vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS), Talanta., 93, (2012), 404–410. 6. P. Ugo, L.M. Moretto and G.A. Mazzocchin, Voltammetric determination of trace mercury in chloride media at glassy carbon electrodes modified with polycationic ionomers, Anal. Chim. Acta., 305,(1995), 74–82. 7. T. Zhang, Z. G.Cheng, Y. B.Wang, Z. G.Li, C. X.Wang, Y. B.Li and Y. Fang, SelfAssembled 1-Octadecanethiol Monolayers on Graphene for Mercury Detection, Nano Lett., 10, (2010), 4738−4741. 8. H.Y.Zhang, L.Q. Yang, B.J. Zhou, W.M. Liu, J.E.C. Ge, J.S. Wu,Y. Wang and P.F. Wang, Ultrasensitive and selective gold film-based detection of mercury (II) in tap water using a
19
laser scanning confocal imaging-surface plasmon resonance system in real time, Biosens. Bioelectron., 47, (2013), 391−395. 9. M.H.Wang, S.L. Liu, Y.C. Zhang, Y.Q.Yang, Y. Shi, L.H. He, S.M. Fang and Z.H. Zhang, Graphene nanostructures with plasma polymerized allylamine biosensor for selective detection of mercury ions, Sensors and Actuators, B., 203, (2014), 497−503. 10. G.H. Liang, P. Zhang, H.X. Li, Z.Y. Zhang, H. Chen, S. Zhang and J.L. Kong An efficient strategy for unmodified nucleotide-mediated dispersion of magnetic nanoparticles, leading to a highly sensitive MRI-based mercury ion assay, Anal. Chim. Acta., 726, ( 2012), 73−78. 11. L.G. Xu, H.H. Yin, W.Ma, H. Kuang, L.B. Wang and C.L. Xu, Ultrasensitive SERS detection of mercury based on the assembled gold nanochains, Biosens. Bioelectron., 67, (2015), 472−476. 12. Y.Q.Yang, M.M.Kang, S.M. Fang,M.H. Wang, L.H. He, J.H. Zhao, H.Z. Zhang and Z.H. Zhang, Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for selective detection of mercury ions, Sensors and Actuators, B., 214, (2015), 63−69. 13. J.L.Wei, D.G. Yang, H.B. Chen, Y. Gao and H.M. Li, Stripping voltammetric determination of mercury(II) based on SWCNT-PhSH modified gold electrode , Sensors and Actuators, B., 190, (2014), 968−974. 14. B. S. B. Kasibabu, J. R. Bhamore, S. L. D'souza and S.K. Kailasa, Dicoumarol assisted synthesis of water dispersible gold nanoparticles for colorimetric sensing of cysteine and lysozyme in biofluids , RSC Advances., 5, (2015), 39182–39191.
20
15. G. M Patel, J. V. Rohit, R. K. Singhal and S.K. Kailasa, Recognition of carbendazim fungicide in environmental samples by using 4-aminobenzenethiol functionalized silver nanoparticles as a colorimetric sensor , Sensors and Actuators B., 206, (2015), 684-691. 16. V. N. Mehta, J. N. Solanki and S.K. Kailasa, Selective visual detection of Pb(II) ion via gold nanoparticles
coated
with
a
dithiocarbamate-modified
4′-aminobenzo-18-crown-6
Microchimica Acta., 181, (2014), 1905-1915. 17. T. C. Prathna, N. Chandrasekaran, A. M. Raichur and A. Mukherjee, Biomimetic synthesis of silver nanoparticles by Citrus limon (lemon) aqueous extract and theoretical prediction of particle size, Colloids Surf. B., 82, (2011), 152-159. 18. Y. Wang, F. Yang and X. Yang, Colorimetric Detection of Mercury(II) Ion Using Unmodified Silver Nanoparticles and Mercury-Specific Oligonucleotides, ACS Appl. Mater. Interfaces., 2, (2010), 339-342. 19. K. Farhadi, M. Forough, R. Molaei, S. Hajizadeh and A. Rafipour,
Highly selective
Hg2+ colorimetric sensor using green synthesized and unmodified silver nanoparticles, Sensors and Actuators, B., 161, (2012), 880-845. 20. X. Yuan, S. Wen, M. Shen and X. Shi, Dendrimer-stabilized silver nanoparticles enable efficient colorimetric sensing of
mercury ions in aqueous solution, Anal. Methods., 5,
(2013), 5486-5492. 21.
W. Ren, C. Zhu and E. Wang, Enhanced sensitivity of a direct SERS technique for Hg2+ detection based on the investigation of the interaction between silver nanoparticles and mercury ions, Nanoscale., 4, (2012), 5902-5909.
22. Z. Sun, N. Zhang, Y. Si, S. Li, J. Wen, X. Zhu and H. Wang, High-throughput colorimetric assays
for mercury(II) in blood and wastewater based on the mercury-stimulated catalytic
21
activity of small silver nanoparticles in a temperature-switchable gelatin matrix, Chem. Commun., 50, (2014), 9196-9199. 23. L. Chen, N. Qi, X. Wang, L. Chen, H. You and J. Li, Ultrasensitive surface- enhanced Raman scattering nanosensor for mercury ion detection based on functionalized silver nanoparticles, RSC Advances., 4, (2014), 15055-15060. 24. F. Li, J. Wang,Y. Lai,C. Wu, S. Sun,Y. He and H. Ma, Ultrasensitive and selective detection of copper (II) and mercury (II) ions by dye-coded silver nanoparticle-
based
SERS probes, Biosens. Bioelectron., 39, (2013), 82-87. 25. J. Liu, X. Ren, X. Meng, Z. Fang and F. Tang, Sensitive and selective detection of Hg2+ and Cu2+ ions by fluorescent Ag nanoclusters synthesized via a hydrothermal method, Nanoscale., 5, (2013), 10022-10028. 26. M. Annadhasan, T. Muthukumarasamyvel, V. R. S. Babu and N. Rajendiran, Green Synthesized Silver and Gold Nanoparticles for Colorimetric Detection of Hg2+, Pb2+ and Mn2+ in Aqueous Medium., ACS Sustainable Chem. Eng., 2, (2014), 887-896. 27. D. Karthiga and S. P. Anthony, Selective colorimetric sensing of toxic metal cations by green synthesized silver nanoparticles over a wide pH range, RSC Advances., 3, (2013), 1676516774. 28. V. Vinod Kumar and S. P. Anthony, Silver nanoparticles based selective colorimetric sensor for Cd2+, Hg2+ and Pb2+ ions: Tuning sensitivity and selectivity using co-stabilizing agents, Sensors and Actuators, B., 191, (2014), 31-36. 29. V. N. Mehta and S. K. Kailasa, Malonamide dithiocarbamate functionalized gold nanoparticles for colorimetric sensing of Cu2+and Hg2+ ions, RSC Adv., 5, (2015), 42454255.
22
30. Y. Guo, Z. Wang, W. Qu, H. Shao, X. Jiang, Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles, Biosensors and Bioelectronics., 26, (2011), 4064–4069. 31. A. Fan, Y. Ling, C. Lau, and J. Lu, Direct colorimetric visualization of mercury (Hg2+) based on the formation of gold nanoparticles, Talanta., 82, (2010), 687–692. 32. Z. Sheng, J. Han, J. Zhang, H. Zhao and L. Jiang, Method for detection of Hg2+ based on the specific thymine-Hg2+ thymine interaction in the DNA hybridization on the surface of quartz crystal microbalance, Colloids and Surfaces B: Biointerfaces,87, (2011),289–292. 33. P. Nalawade, and S. Kapoor, Gold nanoflowers based colorimetric detection of Hg2+ and Pb2+ ions, Spectrochimica Acta Part A., 116, (2013), 132–135. 34. J. Wu, L. Li, D. Zhu, P. He, Y. Fang, and G. Cheng, Gold nanoflowers based colorimetric detection of Hg2+ and Pb2+ ions, Anal. Chim. Acta., 694, (2011), 115-119. 35. L. Li, D. Feng, X. Fang, X. Han, and Y. Zhang, Visual sensing of Hg2+ using unmodified Au@Ag core–shell nanoparticles, J. Nanostruct. Chem., 4,(2014),117 -125. 36. P. Carney, S. Lopez, A. Mickley, K. Grinberg, W. Zhang, Z. Dai, Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine, Chirality., 10, (2011), 916-920. 37. Y. Chen, L. Yao, Y. Deng, D. Pan, E. Ogabiela, J. Cao, S. B. Adeloju, W. Chen, Rapid and ultrasensitive colorimetric detection of mercury(II) by chemically initiated aggregation of gold nanoparticles, Microchimica Acta., 182, (2015), 2147–2154. 38. C. Zong, K. Ai, G. Zhang, H. Li, L. Lu, Dual-Emission Fluorescent Silica NanoparticleBased Probe for Ultrasensitive Detection of Cu2+ , Anal. Chem., 83, (2011), 3126–3132.
23
Biographies: Prasad Buduru received his Ph.D degree in Chemistry from Sri Venkateswara University, Tirupati, India in 2006. He got two postdoc fellowships in Italy and South Korea. After completing his postdoc he joined as a Young Scientist-DST in Sri Venkateswara University. His research interest is functionalization of nanoparticles for colorimetric sensing of biomolecules and metal ions.
Sundhar Raja Reddy B.C. received his Ph.D degree in Geology from Sri Venkateswara University, Tirupati, India in 2007. He Joined as Research Associate in Geology after compleation of RA he joined as a Young Scientist-DST in Sri Venkateswara University. His research interest is Coastal pollution-ecology and environment.
Subba Naidu N.V. Professor , Dept of Chemistry, Sri Venkateswara University, Tirupati, India. He Received Young Scientist Award from the Government of Andhra Pradesh in the year 2006. His research interest in Analytical and Inorganic Chemistry.
24
Figure captions Figure 1. TEM images of (a) Gln-His-Ag NPs, (b) histogram of Gln-His-Ag NPs, and (b) GlnHis-Ag NPs with Hg2+ ion. Figure 2. (a) UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of various metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+1.0 mM). (b) Photograph of corresponding solutions. Figure 3. UV-visible absorption spectra of bare Ag and Gln-His-Ag NPs with and without Hg2+ ion. Figure 4. Effect of Tris-HCl pH from 2.0 to 12.0 on the UV-visible absorption spectra of GlnHis-Ag NPs with 1.0 mM of Hg2+ ion. In set shows the photographic image of the above solutions. Figure 5. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion concentration in the range of 100 to 1000 μM at Tris-HCl pH 9.0. Inset is the photographic image of Gln-His-Ag NPs solutions with Hg2+ ion concentration in the range of 100 to 1000 μM mM at Tris-HCl pH 9.0. Figure 6. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion concentration in the range of 000 to 000 μM in the presence of 0.2 M of NaCl at Tris-HCl pH 9.0. Inset is the photographic image corresponding solutions. Figure 7. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of various metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+, 1.0 mM) along without and with the addition of Hg2+ ion at Tris-HCl pH 9.0. In set shows the photographic image of the above solutions.
25
26
27
28
29
30
31
32
Scheme 1. Schematic illustration for the synthesis of Gln-His-Ag NPs and Hg2+ ion induced Gln-His-Ag NPs aggregation through coordination covalent bond.
33
Table 1. Performance of Gln-His-Ag NPs as a probe for colorimetric detection of Hg2+ with the other NPs-based reported methods. NPs
Linear range (M)
LOD (M)
Analytical technique
Reference
Unmodified Ag NPs
25×10-9 - 500 ×10-9
17×10-9
UV-visible
[18]
Unmodified Ag NPs
10 – 10 ×10-6
2.2 × 10−6
UV-visible
[19]
Dendrimer-stabilized Ag NPs
10 ppb to 10 ppm
-
UV-visible
[20]
-
90.9 ×10-
SERS
[21]
Citrate-Ag NPs
Small gel-Ag NPs
12
0.50 – 800 ×10-9
0.125×10-
[22]
9
4-mercaptopyridine-Ag NPs
1–100 ×10-9
0.34×10-9
SERS
[23]
Cysteine-Ag NPs
-
1×10-12
SERS
[24]
Polymethacrylic acid-Ag NPs
10×10-9 to 20×10-9
<10×10-9
Fluorescence
[25]
L-Tyrosine-Ag NPs
1 – 300×10-9
53×10-9
UV-visible
[26]
Neem leaf extracts -Ag NPs
0 – 140×10-6
<1×10-6
UV-visible
[27]
MA–DTC–Au NPs
0.01 to 10×10-6
45×10-9
UV-visible
[29]
Papain-Au NPs
1.0 – 40×10-6
200×10-9
UV-visible
[30]
NH2OH reduced Au NPs
10 – 1000 ×10-9
10×10-9
UV-visible
[31]
Oligonucleotides-Au NPs
6.0 – 100×10-9
5×10-9
QCMB
[32]
Gold nanoflowers
1.0 × 10−6
<1.0×10-6
UV-visible
[33]
Deoxyribonucleic acid-Au NPs
0.1–10×10-6
<0.1×10-6
UV-visible
[34]
Unmodified Au-Ag NPs
0.1–1.0×10-6
5.0×10-9
UV-visible
[35]
0.9×10-6
UV-visible
Gln-His-Ag NPs
1.0 – 500×10-6
34
Present work
S0925-4005(17)30048-5 http://dx.doi.org/doi:10.1016/j.snb.2017.01.041 SNB 21566
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-9-2016 2-1-2017 5-1-2017
Please cite this article as: Buduru Prasad, B.C.Sundher Raja Reddy, N.V.S.Naidu, Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functionalization of silver nanoparticles with glutamine and histidine for simple and selective detection of Hg2+ ion in water samples
Buduru Prasad*a, B.C. Sundher Raja Reddyb and N.V.S. Naidua
a
Department of Chemistry, Sri Venkateswara University,Tirupati-517502, Andra Pradesh, India Department of Geology, Sri Venkateswara University, Tirupati-517502, Andra Pradesh, India
b
*
Corresponding author, E-mail: [email protected]
1
Research Highlights
Gln-His-Ag NPs act as a probe for colorimetric sensing of Hg2+ ion.
The aggregation of Gln-His-Ag NPs induced by Hg2+ ion causes red-shift and color change.
The sensitivity of the probe was improved to 28-times in the presence of 0.2 M NaCl.
A good linear response for Hg2+ ion was found with a detection limit of 0.90 µM.
This probe showed good selectivity towards Hg2+ ion in the presence of other metal ions.
Abstract A simple, rapid and sensitive colorimetric method was developed for the detection of Hg2+ ion in water samples using glutamine (Gln) and histidine (His) functionalized silver nanoparticles (Gln-His-Ag NPs) as a probe. The functionalized Ag NPs were confirmed by UVvisible, FT-IR and transmission electron microscopic (TEM) techniques, respectively. The Hg2+ ion was induced quickly the aggregation of Gln-His-Ag NPs, yielding a color change from yellow to orange, which can be readily seen by naked eye. The recognition mechanism is attributed to unique supramolecular nanostructures of Gln-His-Ag NPs, which yields strong interaction (cooperative metal-ligand interaction) between Gln-His-Ag NPs and Hg2+ ion. A linear correlation was obtained in the range of 100 – 1000 μM with a detection limit of 25.48 µM. However, the sensitivity of the probe was greatly improved by the addition of 0.2 M NaCl as an ionic strength. The calibration graph was constructed between absorption ratio (A500/A402) and concentration of Hg2+ ion in the range of 1.0 – 500 µM with the detection limit of 0.90 µM, which is 28-times lower than the direct method (without NaCl). This method was successfully applied to detect Hg2+ ion in water samples. With the advantages of simplicity, selectivity and low sample volume, this method can potentially suitable for on-site monitoring of Hg2+ ion. Keywords: Gln-His-Ag NPs, Hg2+ ion, TEM, UV-visible spectrometry and water samples.
2
1. Introduction Mercury is a highly toxic and bioaccumulative heavy metal ion that causes a number of toxicological effects such as brain damage, kidney failure, and various cognitive and motion disorders even at a very low concentration [1]. Because of its high toxicity even at a trace concentration, a simple, specific and sensitive determination of Hg2+ is of particular interest in biological, toxicological, and environmental monitoring. Recent years, many analytical techniques have been used for the detection of Hg2+ ion in various matrices, including atomic absorption/emission spectroscopy [2], selective cold vapor atomic fluorescence spectrometry [3], X-ray fluorescence spectrometry [4], inductively coupled plasma mass spectrometry [5], anodic stripping voltammetry [6], atomic force microscopy [7], surface plasmon resonance [8], quartz crystal microbalance (QCMB) [9], magnetic resonance imaging [10], surface-enhanced Raman scattering [11], and electrochemical-based analytical methods [12-13], respectively. Even though these methods are successfully used to detect Hg2+ ion in complex samples, unfortunately these are either time-consuming or laboratory-based sophisticated instruments, which limit their application in routine monitoring of Hg2+ ion with reduced sample treatment. Thus, it is still of great challenge to develop a simple method for detection of Hg2+ environmental samples. In recent years, metal nanoparticles have proven to be as promising colorimetric probes for selective and sensitive detection of a wide variety of chemical species in environmental and biological samples [14-16]. Among the metal NPs, Ag NPs have attracted much research interests owing to many advantages over Au NPs including cheap precursor (silver nitrate), higher extinction coefficient (1010 M-1 cm-1), higher ratio of scattering to extinction and sharper extinction bands which makes them more prominent candidate than Au NPs for sensing of various chemical species [17]. As a result, several research groups have been developed Ag NPs-
3
based colorimetric methods for the detection of various heavy metals including Hg2+ ions. Briefly, Wang’s group described the use of unmodified Ag NPs as a probe for the colorimetric detection of Hg2+ ion in water samples [18]. Farhadi et al., developed a green synthetic approach for the preparation of Ag NPs and used as a probe for the highly selective detection of Hg2+ without modification [19]. Yuan and co-workers functionalized Ag NPs with poly(amidoamine) dendrimers and used as a colorimetric probe for the detection of Hg2+ ion [20]. Ren’s group incorporated Ag NPs in surface-enhanced Raman spectroscopic (SERS) technique for the detection of Hg2+ ion with improved sensitivity [21]. Gelatin stabilized Ag NPs were used as probes for the label free colorimetric detection of Hg2+ ion in waste water and blood samples [22]. Similarly, Chen et al., functionalized Ag NPs with 4-mercaptopyridine for the sensitive detection of Hg2+ ion using SERS technique [23]. Furthermore, Li’s group described a simple and selective method for ultra sensitive detection of Cu2+ and Hg2+ ions using cysteine-functionalized Ag NPs as a probe in SERS [24]. Liu and co-workers described the use of Ag NPs as a fluorescent probe for the detection of Cu2+ and Hg2+ ions in mineral water samples [25]. Annadhasan’s group developed green synthetic approaches for preparation of Au and Ag NPs and used as probes for simultaneous detection of Hg2+, Pb2+ and Mn2+ ions in water samples [26]. Anthony and co-workers prepared Ag NPs using plant extracts (neem leaf, mango leaf, green tea and pepper seed) as reducing agents and used as a probe for colorimetric detection of multiple metal ions (Hg2+, Zn2+, Pb2+) in water samples [27]. Similarly, the same group illustrated the use of N-(2-hydroxybenzyl)-valine and N(2-hydroxybenzyl)-isoleucine functionalized Ag NPs as a probe for simultaneous colorimetric sensing of Cd2+, Hg2+ and Pb2+ ions in aqueous solution at ppm level [28]. Apart from these Ag NPs-based
methods,
several
research
groups
4
have
been
developed
UV-visible
spectrophotometric methods for detection of Hg2+ ion in various samples using gold nanoparticles (Au NPs) as probes. In these methods, Au and Ag NPs were functionalized with various organic molecules including malonamide dithiocarbamate (MA–DTC) [29], papain [30], NH2OH [31], oligonucleotides [32], gold nanoflowers [33], deoxyribonucleic acid [34], and unmodified Ag-Au NPs [35] for colorimetric detection of Hg2+ ion by UV-visible spectrometry with improved sensitivity. These reports illustrated that the potentiality of Ag NPs as probes for colorimetric detection of metal ions in various environmental and biological samples. Inspired by these works, we decided to functionalize Ag NPs with Gln-His that can act as a distinct probe for simple and selective colorimetric detection of metal ions. In this paper, we report a size and shape controlled synthesis of Ag NPs using Gln and His as capping agents in aqueous medium. The Gln-His-Ag NPs were characterized by UVvisible, Fourier transform infrared spectroscopic (FT-IR) and transmission electron microscopic (TEM) technique. The Hg2+ ion was induced the aggregation of Gln-His-Ag NPs via coordination covalent bond between Hg2+ ion and –COOH and –NH2 group of Gln-His-Ag NPs (Scheme 1), resulting a red-shift and a color change from yellow to orange. This colorimetric method for the detection of Hg2+ showed good selectivity and sensitivity. It could be applied to detect Hg2+ in water samples.
5
2. Experimental section 2.1. Chemicals and materials Silver nitrate, sodium borohydride, tris(hydroxymethyl) aminomethane (Tris), glutamine, histidine, trisodium citrate dihydrate, metal salts
((Cd(NO3)2.4H2O, Co(NO3)2.6H2O,
Cu(NO3)2.3H2O, FeCI2. 4H2O, FeCl3.6H2O, Hg(NO3)2.H2O, Mg(NO3)2.6H2O, Mn(NO3)2.4H2O, NiSO4.6H2O, Pb(NO3)2, Zn(NO3)2.6H2O, KNO3 and NaNO3) were purchased from SigmaAldrich, USA. Hydrochloric acid was purchased from Merck Ltd., India. All chemicals are from analytical grade and used without further purification. Milli-Q-purified water was used for sample preparations.
2.2. Synthesis of Gln-His-Ag NPs Ag NPs were synthesized by sodium borohydride reduction of silver nitrate. Briefly, 15 mL of 2 mM NaBH4 with ice-chilled solution was mixed with 1.0 mL of tri-sodium acetate (1%) and then stirred for 5 min. Then, 5.0 mL of 1.0 mM AgNO3 aqueous solution was added drop wise into the above solution under vigorous stirring at room temperature, producing yellow colloidal Ag NPs. To bifunctionalize Ag NPs with Gln and His, 250 μL of Gln (1.0 mM) and His (0.25 mM) were added into the above solution under constant stirring for 2 h to molecular assembly of Gln and His on the surfaces of Ag NPs (Supporting Information of Figure S1-S2). All the experiments were performed at room temperature. The size of Gln-His-Ag NPs was verified by TEM and average size of Gln-His-Ag NPs was found to be ~10 nm. The concentration of Gln-His-Ag NPs was calculated to be ~26.80 nM by UV-visible spectroscopy based on an extinction coefficient of 4.18×109 M -1 cm-1 at λ = 407 nm.
6
The stability of Gln-His-Ag NPs over time (1 to 17 days) was monitored using UVvisible absorption spectroscopy at room temperature. The stability of Gln-His-Ag NPs over time (1 to 17 days) was monitored using UV-visible spectroscopy by measuring the maximum SPR peak at 402 nm (Supporting Information of Fig. S3). It should be noticed that the characteristic SPR peak of Gln-His-Ag NPs was observed at 402 nm with negligible decrease in the SPR peak intensity, suggesting that the synthesized Gln-His-Ag NPs are stable and dispersed in aqueous media, which confirms that Gln and His molecules preserves stability and hinders agglomeration of Ag NPs.
2.3. Detection of Hg2+ ion To develop Gln-His-Ag NPs as a probe for the colorimetric detection of Hg2+ ion, we have taken 700 µL of metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+, Cr3+ and Hg2+, 1.0 mM) solutions and added separately into 1.0 mL of Gln-His-Ag NPs solutions that contains 100 µL of Tris-HCl buffer, resulting a specific color change from yellow to orange only by the addition of Hg2+ ion. The colorimetric changes were observed by UV-visible spectrophotometer. The practical application of the probe for real samples water samples (drinking water, tap water and river water) was also described. Briefly, water samples (drinking water, tap water and river water) were collected from Tirupati in and around and Swarnamukhi River. The collected samples were spiked with known concentration of Hg2+ ion (150 and 200 µM) and other metal ions (400 µM), mixed thoroughly, filtered by using 0.45 µm membrane and then analyzed by the aforesaid procedure.
7
2.4. Instrumentation UV-visible absorption spectra were measured by using Maya pro 2000 spectrophotometer (Ocean Optics,USA) at room temperature. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). Transmission electron microscopy (TEM) images were taken on a JEOL 3010.
3. Results and discussion 3.1. Characterization of Gln-His-Ag NPs Supporting Information of Figures S1-S2 show the UV-visible absorption spectra of Ag NPs using Gln and His as capping agents. To control the size and surface plasmon resonance (SPR) peak of Ag NPs, we studied the effect of Gln and His concentrations on the surfaces of Ag NPs. As shown in Supporting Information of Figure S1, the SPR peak of Ag NPs at 400 nm was slightly decreasing with increasing concentration of Gln from 0.5 to 5.0 mM. To control the SPR peak and color of Ag NPs, we selected 1.0 mM of Gln as the best concentration for the functionalization of Ag NPs. After optimization of Gln concentration, we also studied the effect of His concentration on the surfaces of Gln-Ag NPs (Supporting Information of Figure S2). It can be observed that 1.0 mM of Gln and 0.25 mM of His were found to be the best concentration for effective molecular assembly on the surfaces of Ag NPs, since the characteristic SPR peak is still remain < 410 nm, which suggests that the diameter of Gln-His-Ag NPs is less than 10 nm. Importantly, it can be noticed that very slight change in the SPR band of Ag NPs before and after modifications, which confirms the functionalization of Ag NPs with Gln and His. The both bare Ag NPs and Gln-His-Ag NPs have shown strong absorption peaks at 395 and 407 nm. Moreover,
8
before and after modifications it also observed that there is slight red – shift in SPR band of Ag NPs, which confirms that the functionalization of Ag NPs with Gln and His molecules. To confirm the attachment of Gln and His on the surfaces of Ag NPs, we studied the FTIR spectra of pure Gln, pure His and Gln-His-Ag NPs (Supporting Information of Figures S4S5). It can be observed that the broad band at 3410 cm-1 belongs to stretching of –OH group in Gln. The peaks at 1334, 1292 and 1257 cm-1 represent the bending, twisting and wagging of methylene groups in Gln. Similarly, the broad absorption bands intensities at 3353 and 3244 cm-1 are corresponded to the asymmetric and symmetric N-H stretching vibrations of - NH2 group. The pure N-H stretch of His appears at 3410 cm-1 and the broad band at 3078 cm-1 corresponds to N-H symmetric stretches of His and also protonated NH2 which are varyingly H-bonded to the environment. The band at 1639 cm-1 represents the anionic nature of carboxylate group in His, which is clearly evident from the C = O stretch. The skeletal vibrations of the imidazole ring were observed at 1581 cm-1. The peaks at 1336 and 1064 cm-1 are assigned to the OH plane deformation in COOH and NH symmetric stretch respectively and its shoulder at 1141 cm-1 is assigned to COO- stretch of the carboxylate anion in the ring of His. The bands at 821 and 1498 cm-1 correspond to the NH2 wagging vibrations and NH3+ symmetric deformation vibrations. The bands at 1287 and 550 cm-1 represent to the NH3+ rocking vibrations of the imidazole ring, CH2 rocking vibrations and the COO- rocking vibrations. The peak at 628 cm-1 is assigned to the C-OH bending vibrations and the C=O stretching vibration is observed at 1639 cm-1, respectively. However, the stretching and vibration bands of carboxylic and amino groups of Gln and His were shifted to lower and longer wave numbers, proving the interaction of Gln and His with the surfaces of Ag NPs, which actually stands as the evidence for the bonding interaction of Gln and His with Ag NPs. The morphology and size distribution of Gln-His-Ag NPs was characterized
9
by TEM. Figure 2a shows a typical TEM image of the as-synthesized Gln-His-Ag NPs. Figure 1b shows a related histogram of as-prepared Gln-His-Ag NPs. It can be noticed that the most of Gln-His-Ag NPs display well-distributed spherical nanoparticles with an average diameter of 5.5±1.0 nm.
3.2. Colorimetric sensing of Hg2+ ion To test their analytical application as a colorimetric sensor, we studied their SPR properties and tendency to agglomerate in the presence of analyte. The interaction of Gln-His-Ag NPs with various metal ions (1.0 mM) was monitored by UV-visible spectral change and also the color change was visualized with naked eye (Figure 2). Out of 16 metal ions tested (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+ and Hg2+), only Hg2+ ion exhibited significant change the UV-visible absorption spectra and the color of the solution, which can be easily observed with naked eye. This indicates that Gln-His-Ag NPs can be used as colorimetric sensor to detect Hg2+ ion in aqueous medium either without any modification or sample pretreatment. The selective interaction of Gln-His-Ag NPs with Hg2+ ions over the other environmentally relevant heavy metal ions could be due to the presence of amino (-NH2) and carboxylic (-COO-) groups on the surface of Gln-His-Ag NPs. It is noticed that only Hg2+ ion exhibited substantial change in the spectrum with a red-shift of the λmax from 407 to 480 nm and a sharp color change from yellow to orange, detectable by naked-eye. The shift in λmax of the plasmon band and the color change of the solution is due to aggregation of Gln-His-Ag NPs induced by Hg2+ ion through the coordination covalent bonding between the surface functional groups of Gln-His-Ag NPs and Hg2+ ion. The absorption ratio at A480/A407 is related to the quantities of dispersed and aggregated Gln-His-Ag NPs, respectively. Therefore,
10
the ratio of the values can be used to express the molar ratio of aggregated and dispersed forms of Gln-His-Ag NPs. In order to confirm the role of Gln and His molecules on the surface of Ag NPs, we studied the UV-visible absorption spectra and color of the bare Ag NPs (without capping of Gln and His) and Gln-His-Ag NPs with addition of Hg2+ ion. As shown in Figure 3, bare Ag NPs did not show any absorption spectral change or color change, indicating that Hg2+ ion did not interact with the bare Ag NPs. However, there is a drastic change in the absorption spectra and color of Gln-His-Ag NPs upon the addition of Hg2+ ion, confirming that the assembly of Gln and His molecules on the surfaces of Ag NPs play key role to interact with Hg2+ via coordinate covalent bond, resulting a red-shift and a color change.
3.3. Effect of pH To provide best condition for detection of Hg2+ ion with good selectivity and sensitivity, we have investigated the effect of various buffer media including phosphate-buffered saline (PBS), sodium acetate, and Tris-HCl pHs from 2.0 to 12.0 on the sensing ability fo Gln-His-Ag NPs towards Hg2+ ion. Initially, we studied the effect of PBS, sodium acetate and Tris-HCl buffer pHs ranging from 2 to 12 without addition of Hg2+ ion (Supporting Information of Figures S6-S8). It can be noticed that there is no change in the absorption spectra and color of Gln-HisAg NPs, indicating that the functionalized Gln-His-Ag NPs are stable in the above buffer media at pH from 2.0 to 12.0. Figure 4 and Supporting Information of Figures S9-S10 show the absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion in the presence of PBS, sodium acetate and Tris-HCl buffer media at pH 2.0-12. The absorption ratio was high at all the buffer media pH 2.0, confirming the self-aggregation of Gln-His-Ag NPs without induced by
11
Hg2+ ion. However, the absorption ratio at A480/A407 is very high at Tris-HCl pH 9.0, indicating that the high degree of aggregation of Gln-His-Ag NPs induced by Hg2+ ion. It can be noticed that the SPR peak is red-shifted in all buffer media at acidic media and neutral pH. The reason may be attributed to the fact that the functional groups (amino and carboxylic acid) at the surfaces of Gln-His-Ag NPs is protonated and is thus unable to coordinate with Hg2+, which leads to a red-shift with low intensity. However, at Tris-HCl pH 9.0, the absorption ratio at A480/A407 is very high, indicating that Hg2+ ion is highly induced the aggregation of Gln-His-Ag NPs via coordinate covalent bonds. Since, the coordinate covalent bonds will be facilitated between the –NH2 and -COOH groups of Gln-His-Ag NPs and Hg2+ ion, which facilitate to generate a new SPR peak around 480 nm. As a result, the characteristic SPR peak of Gln-His-Ag NPs was red-shifted from 407 to 480 nm, which yields a color change from yellow to orange that can be observed with naked-eye. These results indicate that Hg2+ ion shows strong ability to interact with –NH2 and -COOH groups of Gln-His-Ag NPs surfaces, resulting the aggregation of Gln-His-Ag NPs induced by Hg2+ ion through coordinate covalent bonds between Gln-His-Ag NPs and Hg2+ ion, which favors for electric dipole-dipole interactions, neutralization of surface charges and coupling between the plasmon of neighboring Ag NPs. Therefore, Tris-HCl pH 9.0 was selected as the best pH for the colorimetric detection of Hg2+ ion using Gln-His-Ag NPs as a probe. In order to confirm the aggregation of Gln-His-Ag NPs induced by Hg2+ ion, we studied the TEM image of Gln-His-Ag NPs before and after addition of Hg2+ ion. It can be noticed that Gln-His-Ag NPs showed highly dispersed nature (Figure 1a) with an average size of 10 nm, however after addition of Hg2+ ion Gln-His-Ag NPs came closer and induced the aggregation, resulting to from localized clusters (Figure 1b). This observation
12
indicates that the aggregation of Gln-His-Ag NPs induced by Hg2+ ion and in accordance with the Mie theory explains red-shift observed in SPR. Furthermore, we also studied the turbidance of the solutions with and without addition of Gln-His-Ag NPs in the presence of Hg2+ ion (100 and 500 µM) at pH 9.0 (Supporting Information of Figure S11). Since Hg2+ ion can be formed turbidity at higher pH (>8.0). It was observed that negligible turbidity was observed at pH 9.0, however it does not effects the measurement of absorption spectra of the solutions. To ensure selective sensing ability of GlnHis-Ag NPs towards Hg2+ ion, the sensing ability of Gln-His-Ag NPs was studied upon the addition of divalent metal ions (Cu2+, Cd2+, Ca2+, Co2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+, 1.0 mM) separately at Tris-HCl pH in the range of 2.0 – 12.0 (Supporting Information of Figures S12-S16). It can be noticed that the absorption spectra and color of Gln-His-Ag NPs did not show any obvious red-shift and color changes upon the addition of divalent metal ions (Cu2+, Cd2+, Ca2+, Co2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+, 1.0 mM) at Tris-HCl pH in the range of 2.0 – 12.0, suggesting the other divalent metal ions did not induce spectral and color changes of GlnHis-Ag NPs at Tris-HCl pH ranging from 2.0 – 12.0, which confirms that the Gln-His-Ag NPs acted as a selective probe for detection of Hg2+ ion.
3.4. Sensing mechanism As shown in Scheme 1, Gln-His-Ag NPs surfaces contain the amino (-NH2) and carboxylic acid (-COOH) which provides the binding sites for Hg2+ ion to form the complex. Since Hg2+ ion forms stable complexes due to the strong chemical bonding of Hg2+ ion to carboxylic acid and amino groups [36-38]. With this, we assume that the carboxylic acid and amino groups of Gln and His in Ag NPs play key role to bind with Hg2+ ion, favoring to decrease
13
interparticle distance through the formation of coordination covalent bonds, which results a redshift in the SPR band and a color change from yellow to orange. The surface functional groups (NH2 and –COOH) of Gln-His on Ag NPs dictate their specific interaction with Hg2+ ion, where Hg2+ ion acts as a driving force to induce the aggregation of Gln-His-Ag NPs [37-38]. Therefore, it was expected that the carboxylic acid and amino groups of both Gln-His-Ag NPs show high affinity to bind with Hg2+ ion, leading to the aggregation of Gln-His-Ag NPs induced by Hg2+ ion, which results drastic changes in the SPR peak and color . To confirm Hg2+ ion coordination bond with Gln-His-Ag NPs, we studied the FT-IR spectrum of Gln-His-Ag NPs with Hg2+ ion (Supporting Information of Figure S5b). The FT-IR spectrum of Gln-His-Ag NPs exhibited a broad band at 3450 cm−1 which is assigned to ν(OH) of Gln and His molecules. This band intensity is very less at the same position in the FT-IR spectrum of Gln-His-Ag NPs with Hg2+ ion, indicating the participation of these OH group in bonding. The intense peak at 1454 cm-1 is attributed to O-H bending (in-plane) and is shifted toward lower wave number at 1442 cm-1 which suggests that –OH group involves in the coordination bond with Hg2+ ion. The peaks at 1583 and 1641 cm-1 belong to NH2 group scissoring(1°-amines) and decreased peak intensities, proving the involvement of –NH2 groups in the coordination bonding with Hg2+ ion. The peak at 1795 cm-1 is attributed to stretching vibration of carbonyl groups (C=O) of Gln and His molecules, however the intensity of the peak was slightly decreased, which confirms that the involvement of the carboxylic group in covalent bonding with Hg2+ ion. These results indicate that carboxylic (-COOH) and NH2 groups of Gln and His molecules are involved in coordination covalent bonds with Hg2+ ion, resulting the neutralization of charges on the surfaces of Gln-His-Ag NPs, which yields a red-shift in SPR peak of Gln-His-Ag NPs and a color change.
14
3.5. Sensitivity of the probe For quantitative detection of Hg2+ ion, the absorption spectra of Gln-His-Ag NPs were examined upon the addition of different concentrations of Hg2+ ranging from 100 to 1000 µM at optimal conditions. At the same time, the change of Hg2+ concentration could be observed by the naked-eye, as presented in Figure 5. Upon addition of increasing concentrations of Hg2+, the color of Gln-His-Ag NPs solution was changed gradually, initially from yellow to light brown, and finally to orange. Analogously, the characteristic SPR peak at 407 nm was gradually decreased and a new SPR peak was increased with increasing concentration of Hg2+ ion (Figure 5). The absorption ratio of A480/A407 was linear with a correlation coefficient of 0.993 within the range of 100 – 1000 μM of Hg2+ concentration (Supporting Information of Figure S17). The limit of detection (LOD) was calculated using the formula, LOD = 3 × sb/S (in which, sb = standard deviation of blank signal (n=3) and S = slope of the concentration peak intensity of calibration curve) and found to be 25.48 µM.
3.6. Improving the sensitivity of probe with the addition of NaCl as an ionic strength In order to improve the sensitivity of the probe, we studied the effect of NaCl (0.001 to 1.0 M) on the absorption spectra and color of Gln-His-Ag NPs without addition of Hg2+ ion. The UV-visible absorption spectra and color of Gln-His-Ag NPs show significant changes with increasing concentration of NaCl from 0.1 to 1.0 M, suggesting the aggregation of Gln-His-Ag NPs at high ionic strength. However, the absorption spectra and color of Gln-His-Ag NPs did not show any obvious effect at 0.2 M of NaCl, which confirms that the prevention of aggregate formation in NaCl as ionic strength (Supporting Information of Figure S18). In order to know the influence of NaCl on the aggregation of Gln-His-Ag NPs induced by Hg2+, we studied the UV-
15
visible absorption spectra and color changes of Gln-His-Ag NPs upon the addition of Hg2+ ion in the range of 1.0 – 500 µM separately in the presence of 0.2 M NaCl at Tris-HCl pH 9.0 (Figure 6). The SPR band shift relative to Gln-His-Ag NPs-Hg2+ ion, the absorption ratio at A500/A402 is used to quantify the concentration of Hg2+ ion. Since, the Gln-His-Ag NPs solution at 402 and 500 nm are related to the quantities of dispersed and aggregated Gln-His-Ag NPs in the presence of Hg2+ ion, respectively. Thus, we used the absorption ratio at A500/A402 to express the molar concentration of Hg2+ ion based on the aggregated and dispersed Gln-His-Ag NPs using 0.2 M of NaCl as a destabilizing agent. As shown in Figure 6, when the concentration of Hg2+ increased from 1.0 to 500 μM, the characteristic SPR band at 402 nm was gradually decreased and generated new SPR band at 500 nm, yielding a color change from yellow to orange. As a result, the absorption ratio (A500/A402) increases linearly with the increasing concentration of Hg2+ ion from 1.0 to 500 μM (R2 = 0.991) (Supporting Information of Figure S19). The limit of detection was found to be 0.90 µM for Hg2+ ion, respectively. It was observed that the LOD of the present method for Hg2+ ion is either lower than the reported methods [19] or close to other reported methods [27, 33] for colorimetric detection of Hg2+ ions using functionalized Au and Ag NPs. Table 1 shows the comparison of present method with the other reported NPs assisted UVVisible, fluorescence, SERS, and quartz crystal microbalance methods for the detection of Hg2+ ion. It was observed that the LOD of the present method is not comparable with the LOD value of NPs assisted surface enhanced Raman scattering, and fluorescence methods due to their high sensitivity than the NPs assisted UV-visible spectrometric methods. Furthermore, we simplified the synthetic procedure for molecular assembly of Gln-His-Ag NPs and allowed to detect the maximum allowable level of Hg2+ in drinking water with reduced sample preparations and volumes.
16
3.7. Interference study To evaluate the selectivity of the proposed method, the response of the sensor to other environmentally relevant metal ions, including Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+, was tested under the same experimental conditions. As shown in Figure 7, the addition of different metal ions to Gln-His-Ag NPs solution did not cause obvious change of the UV–vis absorption spectrum of Gln-His-Ag NPs except Hg2+ ion. The photographic image of Gln-His-Ag NPs solutions in the presence of 0.5 mM of various metal ions, the Gln-His-Ag NPs solution remained disperesed with yellow color except for Hg2+. After addition of 0.5 mM Hg2+, the color of Gln-His-Ag NPs solution changed from yellow to orange due to Hg2+-induced aggregation of Gln-His-Ag NPs. The results revealed that the other metal ions did not interfere with the determination of Hg2+, indicating the Gln-HisAg NPs probe had good selectivity for sensing of Hg2+ ion.
3.8. Detection of Hg2+ in water samples In order to apply this probe for detection of Hg2+ ion in real samples, the collected water samples were first filtered through 0.45 μm membrane to remove insoluble materials, if any, and maintained pH of 9.0 using Tris-HCl buffer. To make the calibration line for quantitative analysis, standard solutions of Hg2+ were prepared and spiked with collected water samples. To these, Gln-His-Ag NPs solutions were added over the concentration range of 100 – 1000 μM. The absorption spectra of the solutions were then recorded and A480/A407 was plotted against the concentration of Hg2+, a linear relationship was obtained throughout the concentration range used. As shown in Supporting Information of Table S1, the recovery values were satisfied
17
(95.00–98.66%), demonstrating this colorimetric method was suitable for Hg2+ detection in environmental samples.
4. Conclusions In conclusion, we have developed a facile colorimetric sensor for the detection of Hg2+ ion with good selectivity and sensitivity based on the aggregation of Gln-His-Ag NPs induced by Hg2+ ion. The concentration of Hg2+ is easily detected by simply mixing Gln-His-Ag NPs and free from any complicated modifying procedure. It exhibits sharp colour change with red-shift of the absorption band and the change in color from yellow to orange can be detected by naked eye. The Gln-His-Ag NPs-based UV–visible spectroscopic method is economy, simple, convenient, and shown potential application for the colorimetric detection of Hg2+ ion other heavy metal ions based on the aggregation Gln-His-Ag NPs.
Acknowledgements The authors thank the Department of science and technology, India for providing the financial support (DST Fast Track Scheme No.SR/ FTP/ES-16/2011).
18
References 1. H.H. Harris, I.J. Pickering and G.N.George, The chemical form of mercury in fish, Science., 301, (2003), 1203. 2. J.V. Cizdziel and S. Gerstenberger, Determination of total mercury in human hair and animal fur by combustion atomic absorption spectrometry Talanta., 64, (2004), 918–921. 3. Y. Yamini, N. Alizadeh and M. Shamsipur, Solid phase extraction and determination of ultra trace amounts of mercury(II) using octadecyl silica membrane disks modified by hexathia18-crown-6-tetraone and cold vapour atomic absorption spectrometry, Anal. Chim. Acta., 355, (1997), 69–74. 4. L. Bennun and J. Gomez, Determination of mercury by total-reflection X-ray fluorescence using amalgamation with gold Spectrochim. Acta Part B At. Spectrosc., 52, (1997), 1195 – 1200. 5. E. Kenduzler, M. Ates, Z. Arslan, M. McHenry and P.B. Tchounwou, Determination of mercury in fish otoliths by cold vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS), Talanta., 93, (2012), 404–410. 6. P. Ugo, L.M. Moretto and G.A. Mazzocchin, Voltammetric determination of trace mercury in chloride media at glassy carbon electrodes modified with polycationic ionomers, Anal. Chim. Acta., 305,(1995), 74–82. 7. T. Zhang, Z. G.Cheng, Y. B.Wang, Z. G.Li, C. X.Wang, Y. B.Li and Y. Fang, SelfAssembled 1-Octadecanethiol Monolayers on Graphene for Mercury Detection, Nano Lett., 10, (2010), 4738−4741. 8. H.Y.Zhang, L.Q. Yang, B.J. Zhou, W.M. Liu, J.E.C. Ge, J.S. Wu,Y. Wang and P.F. Wang, Ultrasensitive and selective gold film-based detection of mercury (II) in tap water using a
19
laser scanning confocal imaging-surface plasmon resonance system in real time, Biosens. Bioelectron., 47, (2013), 391−395. 9. M.H.Wang, S.L. Liu, Y.C. Zhang, Y.Q.Yang, Y. Shi, L.H. He, S.M. Fang and Z.H. Zhang, Graphene nanostructures with plasma polymerized allylamine biosensor for selective detection of mercury ions, Sensors and Actuators, B., 203, (2014), 497−503. 10. G.H. Liang, P. Zhang, H.X. Li, Z.Y. Zhang, H. Chen, S. Zhang and J.L. Kong An efficient strategy for unmodified nucleotide-mediated dispersion of magnetic nanoparticles, leading to a highly sensitive MRI-based mercury ion assay, Anal. Chim. Acta., 726, ( 2012), 73−78. 11. L.G. Xu, H.H. Yin, W.Ma, H. Kuang, L.B. Wang and C.L. Xu, Ultrasensitive SERS detection of mercury based on the assembled gold nanochains, Biosens. Bioelectron., 67, (2015), 472−476. 12. Y.Q.Yang, M.M.Kang, S.M. Fang,M.H. Wang, L.H. He, J.H. Zhao, H.Z. Zhang and Z.H. Zhang, Electrochemical biosensor based on three-dimensional reduced graphene oxide and polyaniline nanocomposite for selective detection of mercury ions, Sensors and Actuators, B., 214, (2015), 63−69. 13. J.L.Wei, D.G. Yang, H.B. Chen, Y. Gao and H.M. Li, Stripping voltammetric determination of mercury(II) based on SWCNT-PhSH modified gold electrode , Sensors and Actuators, B., 190, (2014), 968−974. 14. B. S. B. Kasibabu, J. R. Bhamore, S. L. D'souza and S.K. Kailasa, Dicoumarol assisted synthesis of water dispersible gold nanoparticles for colorimetric sensing of cysteine and lysozyme in biofluids , RSC Advances., 5, (2015), 39182–39191.
20
15. G. M Patel, J. V. Rohit, R. K. Singhal and S.K. Kailasa, Recognition of carbendazim fungicide in environmental samples by using 4-aminobenzenethiol functionalized silver nanoparticles as a colorimetric sensor , Sensors and Actuators B., 206, (2015), 684-691. 16. V. N. Mehta, J. N. Solanki and S.K. Kailasa, Selective visual detection of Pb(II) ion via gold nanoparticles
coated
with
a
dithiocarbamate-modified
4′-aminobenzo-18-crown-6
Microchimica Acta., 181, (2014), 1905-1915. 17. T. C. Prathna, N. Chandrasekaran, A. M. Raichur and A. Mukherjee, Biomimetic synthesis of silver nanoparticles by Citrus limon (lemon) aqueous extract and theoretical prediction of particle size, Colloids Surf. B., 82, (2011), 152-159. 18. Y. Wang, F. Yang and X. Yang, Colorimetric Detection of Mercury(II) Ion Using Unmodified Silver Nanoparticles and Mercury-Specific Oligonucleotides, ACS Appl. Mater. Interfaces., 2, (2010), 339-342. 19. K. Farhadi, M. Forough, R. Molaei, S. Hajizadeh and A. Rafipour,
Highly selective
Hg2+ colorimetric sensor using green synthesized and unmodified silver nanoparticles, Sensors and Actuators, B., 161, (2012), 880-845. 20. X. Yuan, S. Wen, M. Shen and X. Shi, Dendrimer-stabilized silver nanoparticles enable efficient colorimetric sensing of
mercury ions in aqueous solution, Anal. Methods., 5,
(2013), 5486-5492. 21.
W. Ren, C. Zhu and E. Wang, Enhanced sensitivity of a direct SERS technique for Hg2+ detection based on the investigation of the interaction between silver nanoparticles and mercury ions, Nanoscale., 4, (2012), 5902-5909.
22. Z. Sun, N. Zhang, Y. Si, S. Li, J. Wen, X. Zhu and H. Wang, High-throughput colorimetric assays
for mercury(II) in blood and wastewater based on the mercury-stimulated catalytic
21
activity of small silver nanoparticles in a temperature-switchable gelatin matrix, Chem. Commun., 50, (2014), 9196-9199. 23. L. Chen, N. Qi, X. Wang, L. Chen, H. You and J. Li, Ultrasensitive surface- enhanced Raman scattering nanosensor for mercury ion detection based on functionalized silver nanoparticles, RSC Advances., 4, (2014), 15055-15060. 24. F. Li, J. Wang,Y. Lai,C. Wu, S. Sun,Y. He and H. Ma, Ultrasensitive and selective detection of copper (II) and mercury (II) ions by dye-coded silver nanoparticle-
based
SERS probes, Biosens. Bioelectron., 39, (2013), 82-87. 25. J. Liu, X. Ren, X. Meng, Z. Fang and F. Tang, Sensitive and selective detection of Hg2+ and Cu2+ ions by fluorescent Ag nanoclusters synthesized via a hydrothermal method, Nanoscale., 5, (2013), 10022-10028. 26. M. Annadhasan, T. Muthukumarasamyvel, V. R. S. Babu and N. Rajendiran, Green Synthesized Silver and Gold Nanoparticles for Colorimetric Detection of Hg2+, Pb2+ and Mn2+ in Aqueous Medium., ACS Sustainable Chem. Eng., 2, (2014), 887-896. 27. D. Karthiga and S. P. Anthony, Selective colorimetric sensing of toxic metal cations by green synthesized silver nanoparticles over a wide pH range, RSC Advances., 3, (2013), 1676516774. 28. V. Vinod Kumar and S. P. Anthony, Silver nanoparticles based selective colorimetric sensor for Cd2+, Hg2+ and Pb2+ ions: Tuning sensitivity and selectivity using co-stabilizing agents, Sensors and Actuators, B., 191, (2014), 31-36. 29. V. N. Mehta and S. K. Kailasa, Malonamide dithiocarbamate functionalized gold nanoparticles for colorimetric sensing of Cu2+and Hg2+ ions, RSC Adv., 5, (2015), 42454255.
22
30. Y. Guo, Z. Wang, W. Qu, H. Shao, X. Jiang, Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles, Biosensors and Bioelectronics., 26, (2011), 4064–4069. 31. A. Fan, Y. Ling, C. Lau, and J. Lu, Direct colorimetric visualization of mercury (Hg2+) based on the formation of gold nanoparticles, Talanta., 82, (2010), 687–692. 32. Z. Sheng, J. Han, J. Zhang, H. Zhao and L. Jiang, Method for detection of Hg2+ based on the specific thymine-Hg2+ thymine interaction in the DNA hybridization on the surface of quartz crystal microbalance, Colloids and Surfaces B: Biointerfaces,87, (2011),289–292. 33. P. Nalawade, and S. Kapoor, Gold nanoflowers based colorimetric detection of Hg2+ and Pb2+ ions, Spectrochimica Acta Part A., 116, (2013), 132–135. 34. J. Wu, L. Li, D. Zhu, P. He, Y. Fang, and G. Cheng, Gold nanoflowers based colorimetric detection of Hg2+ and Pb2+ ions, Anal. Chim. Acta., 694, (2011), 115-119. 35. L. Li, D. Feng, X. Fang, X. Han, and Y. Zhang, Visual sensing of Hg2+ using unmodified Au@Ag core–shell nanoparticles, J. Nanostruct. Chem., 4,(2014),117 -125. 36. P. Carney, S. Lopez, A. Mickley, K. Grinberg, W. Zhang, Z. Dai, Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine, Chirality., 10, (2011), 916-920. 37. Y. Chen, L. Yao, Y. Deng, D. Pan, E. Ogabiela, J. Cao, S. B. Adeloju, W. Chen, Rapid and ultrasensitive colorimetric detection of mercury(II) by chemically initiated aggregation of gold nanoparticles, Microchimica Acta., 182, (2015), 2147–2154. 38. C. Zong, K. Ai, G. Zhang, H. Li, L. Lu, Dual-Emission Fluorescent Silica NanoparticleBased Probe for Ultrasensitive Detection of Cu2+ , Anal. Chem., 83, (2011), 3126–3132.
23
Biographies: Prasad Buduru received his Ph.D degree in Chemistry from Sri Venkateswara University, Tirupati, India in 2006. He got two postdoc fellowships in Italy and South Korea. After completing his postdoc he joined as a Young Scientist-DST in Sri Venkateswara University. His research interest is functionalization of nanoparticles for colorimetric sensing of biomolecules and metal ions.
Sundhar Raja Reddy B.C. received his Ph.D degree in Geology from Sri Venkateswara University, Tirupati, India in 2007. He Joined as Research Associate in Geology after compleation of RA he joined as a Young Scientist-DST in Sri Venkateswara University. His research interest is Coastal pollution-ecology and environment.
Subba Naidu N.V. Professor , Dept of Chemistry, Sri Venkateswara University, Tirupati, India. He Received Young Scientist Award from the Government of Andhra Pradesh in the year 2006. His research interest in Analytical and Inorganic Chemistry.
24
Figure captions Figure 1. TEM images of (a) Gln-His-Ag NPs, (b) histogram of Gln-His-Ag NPs, and (b) GlnHis-Ag NPs with Hg2+ ion. Figure 2. (a) UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of various metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+1.0 mM). (b) Photograph of corresponding solutions. Figure 3. UV-visible absorption spectra of bare Ag and Gln-His-Ag NPs with and without Hg2+ ion. Figure 4. Effect of Tris-HCl pH from 2.0 to 12.0 on the UV-visible absorption spectra of GlnHis-Ag NPs with 1.0 mM of Hg2+ ion. In set shows the photographic image of the above solutions. Figure 5. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion concentration in the range of 100 to 1000 μM at Tris-HCl pH 9.0. Inset is the photographic image of Gln-His-Ag NPs solutions with Hg2+ ion concentration in the range of 100 to 1000 μM mM at Tris-HCl pH 9.0. Figure 6. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of Hg2+ ion concentration in the range of 000 to 000 μM in the presence of 0.2 M of NaCl at Tris-HCl pH 9.0. Inset is the photographic image corresponding solutions. Figure 7. UV-visible absorption spectra of Gln-His-Ag NPs upon the addition of various metal ions (Na+, K+, Cu2+, Zn2+, Cd2+, Fe2+, Mn2+, Mg2+, Co2+, Pb2+, Ni2+, Ca2+, Hg2+, Al3+, Fe3+ and Cr3+, 1.0 mM) along without and with the addition of Hg2+ ion at Tris-HCl pH 9.0. In set shows the photographic image of the above solutions.
25
26
27
28
29
30
31
32
Scheme 1. Schematic illustration for the synthesis of Gln-His-Ag NPs and Hg2+ ion induced Gln-His-Ag NPs aggregation through coordination covalent bond.
33
Table 1. Performance of Gln-His-Ag NPs as a probe for colorimetric detection of Hg2+ with the other NPs-based reported methods. NPs
Linear range (M)
LOD (M)
Analytical technique
Reference
Unmodified Ag NPs
25×10-9 - 500 ×10-9
17×10-9
UV-visible
[18]
Unmodified Ag NPs
10 – 10 ×10-6
2.2 × 10−6
UV-visible
[19]
Dendrimer-stabilized Ag NPs
10 ppb to 10 ppm
-
UV-visible
[20]
-
90.9 ×10-
SERS
[21]
Citrate-Ag NPs
Small gel-Ag NPs
12
0.50 – 800 ×10-9
0.125×10-
[22]
9
4-mercaptopyridine-Ag NPs
1–100 ×10-9
0.34×10-9
SERS
[23]
Cysteine-Ag NPs
-
1×10-12
SERS
[24]
Polymethacrylic acid-Ag NPs
10×10-9 to 20×10-9
<10×10-9
Fluorescence
[25]
L-Tyrosine-Ag NPs
1 – 300×10-9
53×10-9
UV-visible
[26]
Neem leaf extracts -Ag NPs
0 – 140×10-6
<1×10-6
UV-visible
[27]
MA–DTC–Au NPs
0.01 to 10×10-6
45×10-9
UV-visible
[29]
Papain-Au NPs
1.0 – 40×10-6
200×10-9
UV-visible
[30]
NH2OH reduced Au NPs
10 – 1000 ×10-9
10×10-9
UV-visible
[31]
Oligonucleotides-Au NPs
6.0 – 100×10-9
5×10-9
QCMB
[32]
Gold nanoflowers
1.0 × 10−6
<1.0×10-6
UV-visible
[33]
Deoxyribonucleic acid-Au NPs
0.1–10×10-6
<0.1×10-6
UV-visible
[34]
Unmodified Au-Ag NPs
0.1–1.0×10-6
5.0×10-9
UV-visible
[35]
0.9×10-6
UV-visible
Gln-His-Ag NPs
1.0 – 500×10-6
34
Present work