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Detection of Cu2+ ion by Synthesis of Bio- mass-Silver Nanoparticle Nanocomposite Gadadhar Barman, Atanu Samanta, Swarnali Maiti and J. Konar Laha*

Department of Chemistry, Midnapore College (Vidyasagar University), Midnapore-721101, W.B, India

Abstract

Monitoring the levels of potentially toxic metal ion like Cu2+ in aquatic ecosystems is important because this ion can have severe effects on human health and the environment. In this work we concentrate on an optical assay of the Cu2+ ion using a nanocomposite of bio-mass (humic acid)-silver nanoparticle (HA-AgNP). A sensitive colorimetric method has been developed using this composite for detection of Cu2+ ion based on changes in absorbance resulting from metal ion-induced aggregation of silver nanoparticles. Thereby morphology of the nanoparticles changes from hexagonal or spherical to rod like structure. At the same time stable inner complex formation of the metal ion is evidenced from the appearance of a peak at around 720 nm in UV-Visible spectra. Various concentrations of Cu2+ ion were used to test the linearity and sensitivity limit of the nanocomposite. The complex formation between Cu2+ ion and humic acid present in HA-AgNP nanocomposite is further confirmed from the FTIR spectra. Values for Cu2+-complexes available in the literature are in conformity with our findings. XRD pattern confirms that Cu exists in the form of ions in the complex. The quenching properties of the fluorescence study of the nanocomposite have been utilized for detection of Cu2+ ion.

Index Term: HA-AgNP nanocomposite • Humic acid • Colorimetric sensor • Detection of Cu2+ ion • Estimation of Cu2+ ion • Cu-complex • Calibration curve

1. Introduction

——————————  ——————————

Most of the copper ion detection methods that have been reported so far include
Copper is a transition metal which plays
significant roles in environmental and biological
systems. Copper is widely used in the industrial and agricultural sectors but it is also highly toxic to organisms, such as certain algae, fungi and many bacteria and viruses [1]. The extensive and excessive use of copper in these areas has resulted in serious environment contamination [2]. High concentrations of copper in drinking water are hazardous to human health [3]. Some cases of liver damage of children have been proved to be associated with the excessive intake of copper [4]. Moreover, high concentrations of copper may destroy the biological reprocessing systems in water [5]. Therefore, the fabrication of sensitive and selective methods to monitor the presence of Cu2+ ions is of great significance.

• Gadadhar Barman is currently pursuing Ph. D degree program in

Midnapore College (Vidyasagar University), India, PH-+91

8900642399. E-mail:bgadadhar@rediffmail.com

• Atanu Samanta and Swarnali Maiti is currently pursuing Ph. D degree program in Midnapore College (Vidyasagar University), India, PH-+91 9932666591. E-mail: maiti.sonalie@gmail.com

• Dr. Jayasree Konar Laha is Faculty as Head and associate Professor in Midnapore College (Vidyasagar University), India, PH-+91 9434029182. E-mail: j.laha@yahoo.co.in, Fax: +91

3222275847

methods like organic fluorophore-based assays
[6,7], chromogenic sensors [8], quantum dots [9],
atomic absorption spectroscopy (AAS) [10], inductively coupled plasma mass spectroscopy (ICP-MS) [11], inductively coupled plasma atomic emission spectroscopy (ICP-AES) [12], electrochemical assays [13] and voltametry [14]. Peptide and oligopeptide modified electrodes have been widely developed in recent years for the detection of metal ions in environmental samples due to their high affinities to different metal ions [15, 16]. Self-assembled monolayers of L-cysteine on gold electrode have been reported for low concentration detection of metal ions [17]. Although the sensitivities of these methods for Cu2+ detection are generally high, most of them need expensive, and specialized instruments which limits their usefulness and make them unsuitable for field work.
Color changes associated with the aggregation of metal nanoparticles have led to the development of a number of assays for a variety of target species [18-23]. Gold nanoparticles (GNPs) seem to be becoming increasingly attractive in the fabrication of optical sensors and devices as well as in bio-

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diagnostics and medical therapeutics due to their excellent chemical and physical properties
[24,25]. They have strong surface plasmon resonance (SPR) absorption in the visible wavelength range with extremely high extinction coefficients. One attractive property of GNPs is that their SPR frequency changes dramatically with the distance between the GNPs. Therefore, the analytic-induced aggregation of GNPs offers a facile colorimetric approach for the simple visual detection of nucleic acids [26,27], proteins [28], small molecules [29,30], and metal ions [31,32]. Inspired by these findings, we attempted a colorimetric method using bio-mass (humic acid)-silver nanoparticle (HA-AgNP) nanocomposite based colorimetric assay for
detection of Cu2+ ion, the result of which are reported here.
Silver nanoparticles have been produced by using an organic macromolecule, humic acid (HA). Humic acid results from the microbiological decomposition of animals and plants and is capable of interacting with metallic cations. It contains several functional groups like phenolic -OH, quinines, hydroxyls, methoxyls, aldehydes, ketones and enolic -OH which could be responsible for the reduction of silver ions. The HA from different sources is found to be different. The probable structure [33,34] of HA and the most likely reaction pathway for interaction with Cu2+ ion are shown in Scheme 1.

.

2. Materials and Methods

2.1. Chemicals

Chemicals used in the experiment were mostly analytical reagent ( A.R ) grade or of the highest purity available. Silver nitrate (AgNO3 ) with purity greater than 99.0 % from sigma Aldrich Pvt. Ltd. was used as received. Humic acid was purchased from Himedia Lab Pvt. Ltd. The used metal salt Cu(NO3 ) 2 was purchased from Beijing Chemical Reagent Company (Beijing, China). NaOH (>97% purity) was purchased from Merck Specialties Pvt. Ltd. Double distilled water was used in all experiments.

2.2. Synthesis of Silver nanoparticles (HA- AgNP) Nanocomposite

Typically, 2.5 mL of 0.003(M) AgNO3 solution was added to 10 ml of 100 mgL-1 humic acid solutions and then 0.01(M) NaOH solution was added drop wise to maintain the pH of the reaction mixture from 7.5 to 8.5. The mixture was cooled in ice cold water and then heated to 850C for times ranging from 20 to 40 minutes depending on the concentration of HA. The brown color of the solution gradually changes to yellow, indicating the reduction of Ag+ to Ag(0). The UV-VIS spectroscopic study further confirmed the formation of HA-AgNP

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nanocomposite. The same procedure was carried out by varying the concentrations of both AgNO3
solution and HA. The humic acid concentration was varied from 1-200mgL-1 and AgNO3 concentration was varied from 0.001 to 0.003 (M).

2.3. Detection of Cu2+ by sensing the colorimetric property of HA-AgNP nanocomposite

The colorimetric detection of aqueous Cu2+ ion was studied using 50 nM concentration of nanocomposite at room temperature. In order to demonstrate the effect of Cu2+ ions on the HA- AgNP nanocomposite, 500 µL portions of several concentrations of Cu2+ ions (0-160 µM) were added individually to 3 mL of HA-AgNP nanocomposite solution and the resulting mixture was then allowed to stand for 10 minutes at room temperature, during which the yellow color changed to a bluish here. The intensity of this bluish colour gradually increased with increase of Cu2+ ions concentration. Subsequently, the UV-VIS spectroscopic study confirmed the presence of Cu2+ ions and absorbance changes at 720 nm were monitored for complex formation.

2.4. Characterization

The absorbance spectra were analyzed by using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high resolution transmission electron microscope (HR-TEM). Samples for the TEM studies were prepared by placing a drop of the aqueous suspension of particles on a carbon-coated copper grid followed by solvent evaporation under vacuum. The crystalline nature of the HA-AgNP and copper complex of HA-AgNP nanocomposite was examined using X’ Pert Pro X-ray diffractometer operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation. The FTIR measurements of the HA-AgNP and copper complex of HA-AgNP nanocomposite were studied by using PerkinElmer Spectrum Version
02 spectrometer. Fluorescence spectra of all
samples were obtained using a Horiba Jobin
Yvon spectrofluorimeter (Fluoromax 4).

3. Results and discussion

3.1. UV-Visible spectral studies

Bio-synthesis of nanoparticles using microbiological decomposition of animals and
plants is an importent area of research. The development of biological processes for the
synthesis of nanoparticles is evolving as an important branch of nanotechnology. The present study deals with the synthesis of bio- mass (humic acid)-silver nanoparticle nanocomposite. Humic Acid can act as capping agent due to its chelating potential towards metal and metallic ions [35]. The optical properties of nanoparticles are mainly dominated by Surface Plasmon resonance (SPR) or localised Surface Plasmon resonance (LSPR) i.e. the collective oscillation of electrons at their surface in resonance with the incident electro- magnetic radiation. The SPR absorption wavelengths are dependent on their size, shape, and refractive index. So any changes in their surface structure, aggregation, or medium’s refractive index may lead to colorimetric changes of the sol.
The HA-AgNP nanocomposite
produced exhibits deep yellow color in water
(Fig. 1A and B). At 100 mgL-1 concentration of humic acid, a smooth and narrow absorption band for HA-AgNP nanocomposite is observed at 397 nm. The Plasmon band shifts to higher values with the increase of the concentration of humic acid (200mgL-1). This shift of the plasmon band to higher wavelengths with higher concentration may be explained by supposing a more dense coating of hydrophobic humic substance on the composite, which affects the value of the dielectric constant of the medium surrounding the nanoparticles [36]. It is well studied that the extinction spectrum of noble metals changes shape by the influence of the dielectric constant of the surrounding material and the type of the metal [37]. It is expected that humic acid capped silver nanoparticles have much better colloidal stability at higher concentrations (Fig. 1B) and hence, the concentration of humic acid used in this work was 200 mgL-1.
The extinction spectra of HA-AgNP
nanocomposite synthesized from different concentrations of AgNO3 solution are shown in Fig. 1A. With the increase of the concentration of AgNO3 in the nanocomposite, silver plasmon band shifted to higher wavelength (from 398 nm to 413 nm) and the spectrum was broad at higher concentrations. The broadening of the peak may be assigned to the changes in morphology as well as the developing of polydispersed nature of the sol.

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In this work, the detection of Cu2+ ion in aqueous solution has been carried out by chelating the metal ion and observing the color change.
Several concentrations of Cu(NO3 ) 2 solution, 0 to
160 µM were added to HA-AgNP
nanocomposite and corresponding changes of the absorption coefficients were noted. When Cu(NO3 )2 solution was added to the composite, a new peak at around 720 nm was found in addition to the peak at 413 nm. More interestingly, with the increase of concentration of Cu2+ ion solution the absorption coefficient of the 413 nm peak decreased whereas the absorption coefficient of the 720 nm peak increased gradually (Fig. 2A & 2B). The SPR of HA-AgNP nanocomposite at 413 nm and 720 nm
are related to the quantities of dispersed and aggregated versions of HA-AgNP in the composite respectively. Hence we used the ratio
of the absorbance values of 720/413 (E720/413 ) to express the molar ratio of aggregated and dispersed phase in the nanocomposite. The humic acid was used as a reducing as well as a capping agent in the synthesis of HA-AgNP nanocomposite and further as a receptor for the sensing of Cu2+ ion by forming a stable inner complex with the metal ion. Morphology changes or stable inner complex formation of the metal ion is evidenced from the color change (detectable by human eye) and appearance of a peak at around 720 nm [32, 38].

The changes of the UV-Visible absorbance ratios
(E720/413 ) was found to be linear with the Cu2+ ion
concentration within the concentration range 0 to
90 µM with the regression equation

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y=0.000178x+0.0693 (R2=0.99064). A slightly distorted linear curve was found within the
concentration range 100 to 160 µM with the regression equation y=0.00074x+0.2843 (R2=0.95124). Both results are shown in Figure 2C
&2D. These findings encourage us to suggest that
this method can be used for detecting Cu2+ ion in
solution with a minimum detectable limit of 10
µM. Same experiment using other metal ions like
Zn2+, Cd2+ and Mg2+ in place of Cu 2+ ion could not produce any significant color change and obviously no change in UV-Visible absorption (Fig.3) These findings tested the specificity of this assay for detection of Cu2+ ion.

3.2. HR-TEM studies

Figure 4 shows the TEM images of HA- AgNP nanocomposite produced from 200 mgL-1 humic acid and 0.003M AgNO3 . The particles were mostly spherical and their sizes varied from
20 to 50 nm, along with a hexagonal nanosheets structure. Being a good capping agent humic
acid induces rapid nucleation but it cannot restrict the growth of silver nanoparticles at higher concentration of humic caid. Hence polydispersed behaviour was observed in the TEM images. Selected area diffraction (SAED) pattern (Fig.4D) illustrates the crystalline nature of the nanocomposite.

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Figure 5 shows the TEM images of Cu-
complex of HA-AgNP nanocomposite. Due to the presence of both oxygen rich functional groups (such as -COOH, >C=O, -OH), and hydrophobic aliphatic and aromatic groups in humic acid, it is capable to act as a chelating ligand [39,40]. The strong binding of the Cu2+ metal ion with chelating ligand facilitates the aggregation of silver nanoparticles. The morphology of the nanocomposite thereby changes enormously which is shown in TEM micrograph (Fig.5). The explanation of strong binding of Cu2+ ion with the ligand is evidenced from FTIR data. Due to the complexation of the composite with Cu2+ ion, the shape of the
particles completely changes to rod like
structures instead of the hexagonal or spherical ones found in the nanocomposite (Fig.4). As explanation, it may be pointed out that the removal of humic acid from the composite due to the complex formation makes the surface of the composite charged. This charged surface facilitates the formation of aggregation in linear fashion but not in random fashion, to generate rod like structure (Fig. 5). The Cu-complex formed are found to be quite large in sizes (Fig.5) though it was not in a precipitated state but was well dispersed in the solution phase Fig. 2 B (inset).

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3.3. FTIR spectral studies

FTIR analysis was performed to identify the bio-molecules localized on the surface which are responsible for the reduction of silver ion and for Cu-complex formation. Representative FT-IR spectra of humic acid powder, synthesized HA- AgNP nanocomposite and Cu-complex of HA- AgNP nanocomposite are shown in Figure 5. Bands attributable to עRassym R(COO-) and עRsymR (COO-) modes of carboxylate groups present in humic acid appeared at 1585 and 1383 cm–l with comparable intensities (Fig. 6A). When HA- AgNP nanocomposite was prepared in presence
of humic acid, the respective peaks shift to 1618 and 1385 cm -1 due to reduction of silver ion, the former being less intense than the latter (Fig. 6B). Finally when Cu2+ ion was added to the above composite, the correspondin עRsym R(COO-) modes of carboxylate group further shifted to the 1632 cm–l (Fig. 6C). The dramatic change of frequencies of carboxylate band can only be explained if it is considered that the stable inner complex formation occurred between Cu2+ ion and humic acid present in HA-AgNP nanocomposite[41-44].

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3.4. XRD studies

The XRD analysis was performed to confirm the crystalline nature of the biologically synthesized HA-AgNP. Various Braggs’ diffraction patterns of the synthesized HA-AgNP nanocomposite and copper complex of the composite are shown in Figure 7. HA-AgNP nanocomposite shows sharp peaks at 32.32,
38.20o, 44.42o, 64.64o and 77.78o corresponding to
(111), (200), (220), (311) and (222) planes, respectively (Fig. 7A) which indicated that HA- AgNP nanocomposite has fcc structure and essentially crystalline in nature. XRD of the copper complex of the composite shows peaks at
12.45o, 24.95o, 28.61 o, 29.71o, 41.61o, 46.87o, 55.58o,
58.58o, 67.19o, 81.40o, and 86.90o in addition to
HA-AgNP nanocomposite peaks at 32.71o, 38.07o,
44.42o , 64.59o, 77.80o. Values for Cu2+-complexes available in the literature are in conformity with our findings [45,46]. This observation confirmed that Cu existed in the form of ions in the complex of the composite (Fig. 7B & C).

3.5. Fluorescence studies

A turn on approach of fluorescent sensor has been developed for sensing Cu2+ ions in aqueous solution, based on Cu2+ ions displacing humic acid unit on the surfaces of HA-AgNP nanocomposite [43,47]. Humic acid molecules shows fluorescence in bulk solution, but its intensity diminishes by more than four times when adsorbed onto nanocomposite surfaces (Fig. 8).

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In the presence of Cu2+ ions, due to the strong binding between metal ions and humic acid
molecules, the adsorbed HA molecules on the nanocomposite surface are released from it (Scheme 2) and thereby the restoration of the florescence is observed from the enhancement of the intensity of the 528 nm peak. From the structure of humic acid it is expected that it can make a complex with Cu2+ ion through the functional groups like -COOH and -OH. In an attempt to detect and estimate Cu2+ ions present in water, various solutions having concentrations between 0 to 50 µM were added in the composite and the corresponding changes in the fluorescence spectra were observed. It is noted that this probe is quite capable of detecting Cu2+ ions present at concentration as low as 25 µM upon excitation at 475 nm. With the increase of Cu2+ ion concentration (50 µM) an enhancement in the intensity was observed. It is also to be noted that this probe is unable to detect metal ion concentration lower than 25 µM.

4. Conclusion

We report on an optical assay for Cu2+ ion using HA-AgNP nanocomposite. A sensitive colorimetric method has been developed using HA-AgNP nanocomposite for detection of Cu2+ ion. The changes resulted might be due to the metal ion-induced aggregation in the nanocomposite or direct deposition of metal ion (Cu2+) onto the nanocomposite through inner copper complex formation. The change in frequencies of carboxylate band in FT-IR spectra confirmed the stable inner complex formation between Cu2+ ion and humic acid present in HA- AgNP nanocomposite. The strong binding of the Cu2+ metal ion with chelating ligand facilitates the aggregation of silver nanoparticles and thereby morphology of the nanoparticles changes from hexagonal or spherical to rod like structure. The observation from XRD pattern also confirmed that Cu existed in the form of ions in the complex. In the fluorescence study the
quenching properties of the nanocomposite have been employed to study the turn on fluorescence
approaches for detection of Cu2+ ion.

Acknowledgements

We are thankful to Central Research Facility at IIT Kharagpur, India for HR-TEM, XRD, and Fluorescence studies.

Conflict of Interest

The authors declare that there is no conflict of interest.

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