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Simultaneous electrochemical detection of Cd(II), Pb(II), As(III) and Hg(II) ions using ruthenium(II)-textured graphene oxide nanocomposite
Author’s Accepted Manuscript Simultaneous Electrochemical Detection of Cd(II), Pb(II), As(III) and Hg(II) Ions using Ruthenium(II)textured Graphene Oxide Nanocomposite Manju Bhargavi Gumpu, Murugan Veerapandian, Uma Maheswari Krishnan, John Bosco Balaguru Rayappan www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(16)30830-X http://dx.doi.org/10.1016/j.talanta.2016.10.076 TAL16996
To appear in: Talanta Received date: 22 August 2016 Revised date: 19 October 2016 Accepted date: 19 October 2016 Cite this article as: Manju Bhargavi Gumpu, Murugan Veerapandian, Uma Maheswari Krishnan and John Bosco Balaguru Rayappan, Simultaneous Electrochemical Detection of Cd(II), Pb(II), As(III) and Hg(II) Ions using Ruthenium(II)-textured Graphene Oxide Nanocomposite, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.076 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 galley proof before it is published in its final citable 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.
Simultaneous Electrochemical Detection of Cd(II), Pb(II), As(III) and Hg(II) Ions using Ruthenium(II)-textured Graphene Oxide Nanocomposite Manju Bhargavi Gumpu1,2, Murugan Veerapandian3, Uma Maheswari Krishnan4, John Bosco Balaguru Rayappan1,2* 1
Nano Sensors Lab @ Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), SASTRA University, Thanjavur - 613 401, Tamil Nadu, India 2 School of Electrical & Electronics Engineering, SASTRA University, Thanjavur - 613 401, Tamil Nadu, India 3 National Institute of Pharmaceutical Education & Research, Kolkata - 700 032, West Bengal, India 4 School of Chemical and Biotechnology, SASTRA University, Thanjavur - 613 401, Tamil Nadu, India
*Corresponding author at: Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) & School of Electrical & Electronics Engineering SASTRA University. Thanjavur – 613 401. India. T: +91 4362 264 101; Ext: 255; Fax: +91 4362 264120. Email: [email protected]
Abstract Simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions was carried out based on the synergistic effect of graphene oxide (GO) textured with redox active ruthenium(II) bipyridine complex ([Ru(bpy)3]2+). [Ru(bpy)3]2+-GO nanocomposite on the modified gold (Au) electrode acts as an electrocatalyst and favours the sensitive and selective detection of metal ions. Also, it exhibited an enhanced electron transfer rate with a low solution resistance examined by cyclic voltammetry and impedance analysis. The inherent electrochemical and electrocatalytic behaviours of [Ru(bpy)3]2+-GO on gold electrode were demonstrated for simultaneous detection of heavy metal ions in water matrix. The proposed sensor exhibited a higher sensitivity towards Cd(II), Pb(II), As(III) and Hg(II) metal ions with a lowest detection limit of 2.8, 1.41, 2.3 and 1.6 nM respectively. The observed detection limits were less than the World Health Organization standards and hence the developed sensor can be deployed for detecting heavy metal ions in water bodies. Simultaneous electrochemical detection of heavy metal ions in river and tap water was carried out using the developed sensor and the observed results were validated with atomic absorption spectroscopy.
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Keywords: Heavy metal ions, Simultaneous detection, Ruthenium (II) bipyridine, Graphene oxide, Redox-active nanocomposite
1. Introduction Environmental and water pollution due to heavy metal ions have been a major concern and even threat to human life [1,2]. Increase in industrial activities, usage of pesticides, insecticides, paints and modernized civilization have spread the heavy metal ions through air, water and soil [3,4]. As a result, heavy metal ions enter the food chain and contaminates the ecosystem. Many tragedies like Minamata poisoning, Bhopal disaster, arsenic poisoning, Sandoz disaster [5] are some of the historic evidences for the loss of human lives and economy due to the consumption of metal ion contaminated water. In view of these disasters, organizations such as Centre for Disease Control (CDC), International Agency for Research on Cancer (IARC), World Health Organization (WHO) have put forth limitations in permissible levels for various heavy metal ions [6]. Intake of heavy metal ions beyond these permissible levels lead to reproductive toxicity, respiratory disorders, impaired neurologic development, cardiovascular diseases, pulmonary fibrosis and behavioural issues [7–9]. Hence, rapid and reliable detection of these heavy metal ions is need of the hour
[10–12]. Various instrumental methods such as electrolyte cathode
atmospheric glow discharge (ELCAD) [13], microwave plasma torch (MPT)-atomic emission spectroscopy (AES), atomic absorption spectroscopy (AAS) [14], inductively coupled plasma atomic emission spectroscopy (ICP-AES)[15], laser-induced breakdown spectroscopy (LIBS) [16] are being used for detecting heavy metal ions. Aforementioned methods are restricted in terms of pre-sample preparation, time-consuming and complex instrumentation [13,17,18]. To overcome the drawbacks of conventional methods, electrochemical biosensors have emerged as an effective tool to quantify the heavy metal ion elements in water matrix with the advantages of rapidity, portability, simple sample preparation and easy handling [19,20]. Page 2 of 25
In this context, electrochemical sensors have been considered to detect heavy metal ions with additional advantages of sensitivity and specificity. Further, enhancement of sensitivity and selectivity in electrochemical sensors can be achieved through proper modification of electrode surface using various nanomaterials, organic and inorganic materials [21]. Among various nanomaterials, carbon based nanomaterials are highly compatible, offers high adsorption capacity, provides fast electron transfer rate and high surface coverage area [22]. Among them, graphene oxide (GO) is the most prominent nanomaterial with hydrophilic oxygenated functional groups [23]. Due to its biocompatible property, 2D thin layer structure and abundant oxygen functional groups it has been recently identified and employed as a novel interface layer in electrode/biosensor research. Compared to conductive graphene sheets bulk synthesis of GO is simple and cost-effective. Using chemisorption and physical treatment such as functional group activation and photoirradiation, the property of GO-based materials could be transformed for advanced electrochemical biosensing applications. Besides, GO provides high π-conjunction and doping of organic or inorganic complexes such as ruthenium (II) complexes based on bipyridine ligands [Ru(bpy)3]2+, further enhances the mass transport efficiency, charge transfer [24], sensitivity and selectivity [25]. [Ru(bpy)3]2+ complex also provides unique features such as octahedral geometry, tunable electrochemical properties and different stable oxidation states ranging from I to III [26]. Functionalization of GO with [Ru(bpy)3]2+ is well coordinated because of the electrostatic and π-π stacking interactions forming [Ru(bpy)3]2+-GO nanocomposite [27,28]. Even though [Ru(bpy)3]2+-GO nanocomposite was used for various applications like detecting cancer biomarkers, tripropylamine, and non-esterified fatty acids [29], to the best our knowledge it is the first time [Ru(bpy)3]2+-GO nanocomposite is being used for simultaneous detection of heavy metal ions in water. Herein, we integrated the redox active molecule (ruthenium (II) complex) on the surface
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of GO to improve the inherent electrical conductivity and demonstrate its use in detection of heavy metal ions. Earlier report demonstrated that the composite of SnO2/graphene modified on glassy carbon electrode could simultaneously detect the heavy metal ions, viz., Cd(II), Pb(II),Cu(II) and Hg(II) ions [30]. Though anodic stripping voltammetry helped in simultaneous detection of heavy metal ions, it was bounded in terms of detection limit, sensitivity and relative correlation co-efficient. This was due to the competition of Cd(II), Pb(II), Cu(II) and Hg(II) ions towards the limited number of active sites. Jena et al., reported the usage of gold nano electrode ensembles (GNEE) to detect As(III), Hg(II) and Cu(II) simultaneously [31]. But, this study suffered due to high over voltage, low stability and limited sensitivity [6]. Along with various metals, metal oxide nanointerfaces, polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) were also used for simultaneous detection of Cd(II) and Pb(II) [14]. Though this method has achieved the better detection limit, it has issues in terms of sensitivity due to slow diffusion rate and high polymer thickness [32]. By considering these limitations, [Ru(bpy)3]2+-GO nanocomposite was developed as an interface material on gold working electrode for the simultaneous detection of heavy metal ions Cd(II), Pb(II), As(III) and Hg(II) by employing differential pulse voltammetry (DPV). Herein, it is evident that [Ru(bpy)3]2+-GO nanocomposite acts as an efficient electrocatalyst for the simultaneous detection of heavy metal ions without any complexity in sample preparation and data analysis. 2. Experimental 2.1.1. Chemicals Graphite powder (<20 µm, synthetic), tris (2,2’-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)3Cl2 •6H2O) were procured from Sigma-Aldrich, Canada. Sodium citrate, citric acid, sodium chloride, potassium chloride, boric acid, phospohoric acid, acetic acid, sodium
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hydroxide, lead nitrate, cadmium acetate, cobalt acetate, zinc chloride, mercuric sulphate, arsenic (III) iodide and chitosan were purchased from Sigma Aldrich, India. All the studies were carried out at room temperature using deionized water. Citrate buffer of 0.1 M was prepared by dissolving 0.25 g of sodium citrate and 0.21 g of citric acid in 100 mL of deionized water.
2.1.2. Synthesis of GO GO was synthesized using modified Hummers’ method [33]. Graphite powder and sulphuric acid (H2SO4) were mixed for 2 h. Potassium permanganate (KMnO4) of 6 g was added to this mixture by maintaining a temperature less than 293 K. Later the mixture was stirred at 308 K for 2 h and the resulting solution was treated with 10 mL of 30% of hydrogen peroxide and 150 mL of distilled water. The obtained graphite oxide solution was centrifuged multiple times until pH reaches neutral. The precipitate was ultra-sonicated for 1 h and GO was formed by exfoliating multi layered graphite oxide. 2.1.3. Synthesis of [Ru(bpy)3]2+-GO nanocomposite A mixture of 10 mL aqueous GO (1 mg/mL) and 10 mL ethanolic solution of Ru(bpy)3Cl2 (1 mg/mL) was magnetically stirred at 800 rpm for overnight under dark conditions. The [Ru(bpy)3]2+-GO nanocomposite mixture was centrifuged at 12,000 rpm for 45 min and unreacted [Ru(bpy)3]2+ was removed from the mixture by continuous washing in anhydrous ethanol and deionized water [29]. Isolated [Ru(bpy)3]2+-GO nanocomposite was used for further experimental investigations. 2.2. Characterization of [Ru(bpy)3]2+-GO nanocomposite Morphology of [Ru(bpy)3]2+-GO nanocomposite was investigated using Field Emission Scanning Electron Microscope (FE-SEM) (JEOL, 6701F, Japan) and Field Emission Transmission Electron Microscope (FE-TEM) (JSM2100, JEOL, Japan). Structural and functional group analysis of [Ru(bpy)3]2+-GO nanocomposite were carried out using Energy Page 5 of 25
Dispersive X-Ray Spectroscope (EDXS) (Thermo Fisher Scientific Inc., K Alpha, USA), X-ray Photoelectron Spectrometer (XPS, Omicron equipped with a hemispherical analyzer that utilizes the monochromated Al Kα radiation (hν = 1486.6 eV), operated at 12 kV and power of 300 W) and Fourier Transform Infrared Spectrometer (FT-IR) (Spectrum 100, Perkin Elmer, USA). Optical absorbance and photoluminescence properties were studied using Cary 5000 UV-VisNIR spectrophotometer (Agilent Technologies) and Varian Cary Eclipse Fluorescence Spectrometer respectively. Electrochemical analysis was carried out using CH electrochemical analyzer (CH600C, CH Instruments, USA). Gold working electrode (2 mm diameter, CH Instruments, USA), platinum (Pt) wire electrode (0.5 mm diameter, CHI115, CH Instruments, USA) as counter electrode and Ag/AgCl (0.5 mm diameter. CHI111, CH Instruments, USA) saturated with 0.1 M KCl as a reference electrode were used in three electrode cell. The concentration studies were validated using Atomic Absorption Spectrometer (AAS) (Model no. 2380, Perkin Elmer). 2.3. Fabrication of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode [Ru(bpy)3]2+-GO nanocomposite of 1 mg was dispersed in 0.05 (w/v) chitosan solution and sonicated for 30 min. From the well dispersed solution of [Ru(bpy)3]2+-GO nanocomposite, 3 µL was coated on the top of Au electrode and allowed to air dry. The nanocomposite film was crack free and hence used for electrochemical analysis. 3. Results and Discussion 3.1. Microstructural studies Structural Analysis Structural characteristics of GO and [Ru(bpy)3]2+-GO nanocomposite were studied from Raman spectrum (Fig. 1). GO exhibited two characteristic peaks at 1351 and 1594 cm-1 corresponding to the D and G bands. [Ru(bpy)3]2+-GO nanocomposite also exhibited D (1361cm-1) and G (1594 cm-1) bands with a slight shift. Besides, it also exhibited an additional peak at 1483 cm-1 Page 6 of 25
corresponding to the C=N vibrations of [Ru(bpy)3]2+ complex. A slight decrease in ID/IG ratio of [Ru(bpy)3]2+- GO nanocomposite (1.17) than GO (1.18) was observed due to the change in π-π stacking to π-conjugated structure of [Ru(bpy)3]2+-GO nanocomposite. Morphological characterization of [Ru(bpy)3]2+-GO nanocomposite The surface morphologies of GO and [Ru(bpy)3]2+-GO nanocomposite were analyzed using FESEM (Fig. 2a-b) and FE-TEM (Fig. 3a-c,e). GO showed a transparent wrinkled sheet like morpholgy with multiple layers. The EDS spectra showed peaks corresponding to carbon and oxygen for GO whereas an additional ruthenium peak was observed for [Ru(bpy)3]2+-GO nanocomposite. The transparent nature of GO was well evident from FE-TEM micrograph indicating that GO has less number of layers with large network of thin sheet topography. Selected Area Electron Diffraction (SAED) exhibited a hexagonal diffraction pattern for GO (Fig. 3d). In comparision to GO, [Ru(bpy)3]2+-GO nanocomposite showed a tightly packed sheet structures with lightly coorugated morphology.
Its SAED pattern confirmed the chemical
interaction between [Ru(bpy)3]2+ and GO (Fig.3f). Further, the EDS spectra of modified Au electrodes are shown in Fig. 3(g-h). It showed an additional Au peak for GO and [Ru(bpy)3]2+ modified Au electrode. This has confirmed that GO and [Ru(bpy)3]2+ were well immobilized on the surface of Au working electrode. Surface chemistry and optical properties of [Ru(bpy)3]2+-GO nanocomposites XPS analysis Vibrational changes in the surface elemental moieties of GO, before and after interaction with [Ru(bpy)3]2+ were observed from high-resolution XPS. Supplementary Fig. S1A shows the C1s peak of GO material with relevant deconvolution by Gaussian’s fit. This resulted in the five characteristic peaks related to the carbon atoms with different functional groups. In relation to previous reports [29,34] the C1s peaks were denoted as follows: non-oxygenated carbon (C-C, C=C and C-H, 284. 4 eV), hydroxyl (C-OH, 285 eV), epoxy (286.2 eV), carbonyl (287 eV) and
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carboxylic acid (288.5 eV) groups, respectively. Interaction of [Ru(bpy)3]2+ on the surface of GO significantly influenced the vibrational signals of inbuilt elemental binding groups. These include appearance of new peak at 281.1 eV, attributed to the Ru3d5/2 (Supplementary Fig. S1B). The structural vibrations of epoxy is shifted from 286.2 to 285.7 eV, and the hydroxyl groups are shifted from 285 to 285.2 eV. Further, disappearance of carboxylic acid and carbonyl group vibrations denote that the interaction of [Ru(bpy)3]2+ significantly modified the surface texture of GO [29]. Such chemically interacted electroactive molecules with nanosize distributions are expected to have advanced electron-charge transfer behaviours. FT-IR analysis FT-IR spectral analysis showed the characteristic peaks for GO (Supplementary Fig. S2A-i)) at 1039, 1183 and 1422 cm-1, which are attributed to the vibrations of carbonyl (C-O), epoxy (C-OC) and OH groups of oxidized graphitic domains. C-C stretching frequencies of graphitic lattice network is observed at 1620 cm-1. C=O stretching vibrations of carboxylic groups are located at 1712 cm-1 [33]. Upon interaction with [Ru(bpy)3]2+ complex the stretching and bending vibrations of GO are slightly modified (Supplementary Fig. S2A-ii). For instance, epoxyl group frequencies are notably diminished and hydroxyl group vibrations are disappeared. Functional groups of bipyridyl components from [Ru(bpy)3]2+ complex are well resolved at 728, 767, 1420, 1445 and 1467 cm-1 (highlighted in blue frame). In association with C-C skeletal vibrations of GO, the conjugated C=C signal from bipyridyl group is observed at 1600 cm -1. From the observed structural vibrations, it is evident that the [Ru(bpy)3]2+ is chemically interacted with the oxygenated functional groups of GO. Supplementary Fig. S2B represents the absorption bands of [Ru(bpy)3]Cl2 powder. The sharp and intense peaks located at 731 and 772 cm-1 are related to the C-H group stretch from the pyridine. Multiple peaks located in the region 897-1162 cm-1 correspond to the in-plane C-H bending vibrations. Other peaks located in the region 1220-1318, 1423, 1442, 1464 and 1600 cm-1 are the characteristics vibrations of bipyridyl groups [35].
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Optical absorbance UV-Visible spectral analysis was employed to determine the optical properties of GO and [Ru(bpy)3]2+-GO nanocomposite. Supplementary Fig. S3 depicts the UV-Vis absorbance spectrum of GO (a) with a maximum absorbance at 235 nm. This corresponding to π-π* transition of electrons in the polyaromatic C-C bonds of GO. The inset in Supplementary Fig. S3 indicates the absorption spectra of [Ru(bpy)3]2+ complex showing peaks at 243, 290 and 420 nm, which are in good co-ordination with the previous work [29]. The prominent peaks at 243 and 420 nm of lower energy, signifies the oxidation of Ru(II) to Ru(III) in which dπ orbitals are stabilized leading to metal–to–ligand charge transfer (MLCT). The specific absorbance peak at 243 nm corresponds to the bpy π-π* intra-ligand transition and the peak at 290 nm corresponds to destabilization of bpy π* MOs (molecular orbitals) due to increased back bonding. This creates an energy gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Photoluminescence study Recent studies documented that aqueous dispersion of GO nanosheets possess finite electronic bandgap due to the disrupted π networks resulted from the oxygenation process [35]. Inherent broad PL property demonstrated from GO was believed to be originated from the sp3 carbon domains surrounded by the clusters of graphitic sp2 regions [33,36]. In this study, a complementary investigation on PL property was studied to understand the intrinsic changes in GO nanosheets their interaction with [Ru(bpy)3]2+. As observed from Supplementary Fig. S4A, at an excitation wavelength of 325 nm the GO nanosheets exhibited a near UV emission peak centered at 364 nm, in agreement with the earlier report [33]. Likewise, the [Ru(bpy)3]2+ modified GO nanomaterial also exhibited a PL emission peak with a notable broadness, which may be attributed to the localized transition between the adhered molecules. On adding [Ru(bpy)3]2+ to GO a slight decrease in wavelength was observed corresponding to the blue shift Page 9 of 25
signifying that the added [Ru(bpy)3]2+ has chemically interacted with COOH and C=C bonds of GO. This electrostatic interactions helped Ru metal of [Ru(bpy)3]2+ to bind with COO- and bpy to bind with C=C of GO to form a nanocomposite structure. To ensure a further electronic transition between the [Ru(bpy)3]2+ molecules and GO nanosheets, PL spectrum was recorded at an excitation wavelength of 450 nm (Supplementary Fig. S4B), which is the known standard for [Ru(bpy)3]2+. At this excitation, GO nanosheets only exhibited the inherent visible emission band centered at 529 nm, indicating the pristine nature of the GO layers. Earlier report studied that this PL behaviour of GO corresponds to the recombination of electron-hole pairs from conduction band and localized electronic state at the valence band [36]. In the case of [Ru(bpy)3]2+-GO nanocomposite, two characteristic broad visible emission bands were observed at 532 nm and 610 nm. The former one with a minor shift is attributed to the formation of new sp2/sp3 carbon domains associated from GO/[Ru(bpy)3]2+ and later is ascribed to the inherent triplet MLCT of [Ru(bpy)3]2+. The observed PL spectral changes provide a further complementary information on the surface modification of GO with [Ru(bpy)3]2+. 3.2. Electrochemical Studies 3.2.1 Electrocatalytic activitity of [Ru(bpy)3]2+- GO nanocomposite modified Au electrode Electrochemical behaviours of different modified Au electrodes were recorded in the presence of 0.5 mM K3[Fe(CN)6] in 0.1 M of KCl solution at a scan rate of 100 mVs-1 using cyclic voltammetry (CV). Fig. 4A depicts the anodic and cathodic peaks of bare Au, GO modified Au and [Ru(bpy)3]2+-GO modified Au electrodes with a potential difference (△E) of 189, 110 and 102 mV, respectively. Higher △E values of bare Au indicates the slow electron transfer rate and on modifying the Au electrode with GO and [Ru(bpy)3]2+-GO nanocomposite, the electron transfer rate was enhanced due to the electrocatalytic behavior of GO and [Ru(bpy)3]2+-GO nanointerface. The charge transport process of Au, GO modified Au and [Ru(bpy)3]2+-GO modified Au electrodes were studied using Electrical Impedance Spectroscopy (EIS) (Fig. 4B). The charge
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transfer resistance (Rct) of [Ru(bpy)3]2+-GO modified Au electrode was 281.1 Ω, which is almost 1.9 and 2.3 times lower than Au-GO and bare Au electrodes. This has signified the better electron transfer process between electrolyte and [Ru(bpy)3]2+-GO modified Au electrode. Further, to prove the electrocatalytic behaviour of bare Au, Au electrode modified with GO and [Ru(bpy)3]2+-GO, heterogeneous electron transfer rate constant (K0) of each was calculated (Eq. 1) based on
interfacial interaction.
K0 =
- (1)
where, R is the gas constant, T is the temperature, n is the electron transfer constant of the redox couple, F is the Faraday constant, Aec is the effective area of Au electrode, Rct is the charge transfer resistance and C is the concentration of redox couple in the bulk solution. The K0 value of [Ru(bpy)3]2+-GO modified Au electrode (15.3×10-6 s-1) was higher than GO modified Au electrode (6.74×10-6 s-1) and bare Au electrode (6.58×10-6 s-1) indicating a faster electron transfer exchange between the [Ru(bpy)3]2+-GO modified Au electrode interface and redox species, which was in good co-ordination with △E values. From CV and EIS studies, it is evident that GO modified Au electrode exhibited a higher conductivity than bare Au electrode due to the decreased oxygen content of GO. This can be estimated from the mass ratio of KMnO4 (6 g) to the graphite (2 g) concentration utilized for the synthesis of GO. Its ratio was observed to be 0.3, which corresponds to the decreased oxygen content of GO. Hence, it is conductive in nature and that is the reason why an enhanced peak current was observed. Further, [Ru(bpy)3]2+-GO modified Au electrode showed an enhanced peak current and electron transfer rates due to the further reduction of GO on accommodating [Ru(bpy)3]2+. Although [Ru(bpy)3]2+-GO nanocomposite can exhibit electrostatic and hydrophobic interactions, [Ru(bpy)3]2+-GO nanocomposite is dominated by the electrostatic interaction, which was evident from the irreversible nature of [Ru(bpy)3]2+-GO (△E > 60 mV). Page 11 of 25
3.2.2. Optimization of electrolyte and pH Electrolyte and buffer are the basic parameters that shows a major influence on potential. Optimization of electrolyte and pH helps in narrowing down the potential window and hydrolysis of metal ions [37]. Hence, voltammetric behaviour of different electrolyte solutions was tested at pH 6 in 0.1 M of phosphate buffer saline, citrate, acetate, Britton - Robinson and Tris-HCl buffer. In acetate and citrate buffer, quaternary mixture of Cd(II), Pb(II), As(III) and Hg(II) metal ions showed well-defined peaks (Supplementary Fig. S5A). But, citrate buffer exhibited a peak of maximum current which is 1 fold higher than the acetate buffer. In acetate buffer, lower concentrations of metal ions were not detected due to the complex formation behaviour of metal ions. In the case of Britton-Robinson buffer, peak overlap was observed. Tris-HCl buffer exhibited a single widened peak, which resulted in interference. PBS showed a well resolved peak for Pb(II) and As(III) with a lower current response than the citrate buffer due to binding of phosphate with metal ions [38]. Further, citrate buffer was optimized by varying pH in the range of 3.0 to 9.0 (Supplementary Fig. S5B). The maximum current was attained at the pH of 5.0. The current was increased in the pH range of 3.0 to 5.0 due to complexation mechanism and electrostatic attraction. Beyond pH 5, a decrease in current was observed due to the hydrolysis of metal ions. Thus 0.1 M citrate buffer of pH 5.0 was chosen as an optimal condition for further experiments. 3.2.3. Electrochemical determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions In order to achieve better sensitivity, DPV was employed as an analytical technique for effective detection of Cd(II), Pb(II), As(III) and Hg(II) quaternary metal ion mixture. Supplementary Fig. S5C shows the DPV response of bare Au electrode, GO modified Au electrode and [Ru(bpy)3]2+GO modified Au electrode in the presence of 0.1 M citrate buffer, pH 6. Bare Au electrode can detect only As(III) at 0.09 V vs Ag/AgCl from quaternary mixture of metal ions with a sensitivity of 0.886 µA µM-1. GO modified Au electrode exhibited two well resolved peaks at -0.698 and
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0.121 V vs Ag/AgCl for Cd(II) and As(III) with a sensitivity of 3.43 µA µM-1 and 2.11 µA µM-1. When compared to bare Au, GO modified Au electrode showed an increased sensitivity for As(III) due to the desorbing activity of adsorbed As(III) metal ions on GO. [Ru(bpy)3]2+-GO nanocomposite modified Au electrode exhibited four peaks at -0.638, -0.347, 0.127 and 0.404 V vs Ag/AgCl for Cd(II), Pb(II), As(III) and Hg(II) respectively (Supplementary Fig. S5C). Also, they showed a good separation of Cd(II) – Pb(II), Pb(II) – As(III) and As(III) – Hg(II) peaks that helps in avoiding the formation of inter-metallic compounds. Although, four metal peaks were observed on Au modified with [Ru(bpy)3]2+-GO, the intensity of peak currents was observed to be lower due to the coorugated morphology of [Ru(bpy)3]2+-GO. 3.2.4. Simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions Under the optimized conditions, Cd(II), Pb(II), As(III) and Hg(II) metal ions were detected individually as well as simultaneously using [Ru(bpy)3]2+-GO modified Au electrode. Supplementary Fig. S6 shows the DPV response towards Cd(II), Pb(II), As(III) and Hg(II) metal ions at -0.626, -0.375, 0.108 and 0.412 V respectively. The difference between each peak signifies the simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions is a feasible approach. Its analytical parameters were analyzed and the linear range of Cd(II), Pb(II), As(III) and Hg(II) metal ions were found to be 0.05-0.3, 0.05-0.25, 0.05-1.8 and 0.1-1.2 µM respectively covering the WHO limit. From Fig. 5, well resolved peaks at -0.638, -0.347, 0.127 and 0.404 V vs Ag/AgCl were observed for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. An increase in current with an increase in metal ion concentrations were also observed. Among these metal ions Cd(II) and Hg(II) showed weak peaks, which might be due to the intermetallic complex formation. The limit of detection of Cd(II), Pb(II), As(III) and Hg(II) metal ions were observed to be 0.013, 0.35, 0.06 and 0.34 µM respectively. The analytical parameters of Cd(II), Pb(II), As(III) and Hg(II) metal ions are presented in Table. 1.
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3.2.5. Interference studies The selectivity and cross reactivity of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode in the presence of heavy metal ions were analyzed individually as well as simultaneously. As the concentration of As(III) and Hg(II) was varied from 0.02 to 2 µM, a slight increase in Pb(II) was observed while the current peaks of Cd(II) almost remained the same (Supplementary Fig. S7A). This may be due to the formation of As-Pb intermetallic compound. Similarly, the concentration of Cd(II) and Pb(II) was varied from 0.02 to 3 µM in the presence of 0.5 µM of As(III) and Hg(II) (Supplementary Fig. S7B). The current response of As(III) and Hg(II) showed no significant changes confirming the absence of inter-metallic complexation with Cd(II) and Pb(II) ions. Hence, the proposed sensor can be employed to detect Cd(II), Pb(II), As(III) and Hg(II) metal ions with no major interference among the four metal ions. 3.2.6. Precision and stability measurements The precision in terms of repeatability and reproducibility of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode was evaluated by taking repetitive measurements of DPV in the presence of 0.5 µM of Cd(II), Pb(II), As(III) and Hg(II) ions. For ten repetitive experiments, the relative standard deviation (RSD) was 2.86%, 3.02%, 1.87% and 2.44% for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. Reproducibility of the developed electrode was verified with three different electrodes and it showed an RSD of 3.2%, 2.88%. 3.3% and 3.69% for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. The operational stability of the developed electrode was estimated in the presence of 0.5 µM of Cd(II), Pb(II), As(III) and Hg(II) metal ions and retained a sensitivity of 89.2%, 91.1%, 90.7% and 73.8% respectively for a period of 60 days. 3.2.7 Application (Tap water) and validation with AAS The developed electrode showed a good reproducibility and repeatability with a better stability. Hence, it was used for testing Cauvery river water and tap water. The concentrations of Cd(II),
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Pb(II), As(III) and Hg(II) metal ions in Cauvery river water and tap water were estimated and validated using atomic absorption spectroscopy (AAS) (Table 2). It showed a better sensing characteristics of lower detection limit and higher sensitivity in detecting Cd(II), Pb(II), As(III) and Hg(II) metal ions in a simultaneous manner (Supplementary Table 1).
4. Conclusion In the present investigation, [Ru(bpy)3]2+-GO modified Au electrode was fabricated towards the simultaneous detection of Cd(II), Pb(II), As(III) and Hg(II)
metal ions. [Ru(bpy)3]2+-GO
modified Au electrode exhibited a good electrocatalytic activity with a large number of active sites, which resulted in an enhanced charge transfer rate. Improved electrocatalytic behaviour of [Ru(bpy)3]2+-GO helped in detecting the quaternary mixture with a good peak-to-peak separation and lower detection limit of 2.80, 1.41, 2.30 and 1.60 nM for Cd(II), Pb(II), As(III) and Hg(II) ions respectively. Also, the developed sensor exhibited a better sensitivity, repeatability and stability in presence of Cd(II), Pb(II), As(III) and Hg(II)
ions.
Further, [Ru(bpy)3]2+-GO
modified Au electrode was used to detect various concentrations of metal ions in river and tap water. The observed results were in good co-ordination with results of AAS. The developed [Ru(bpy)3]2+-GO modified Au electrode can be utilized for onsite qualitative and quantitative analysis of water bodies. Acknowledgements The authors are grateful to the Department of Science & Technology, New Delhi for their financial support (DST/TM/WTI/2K14/197(a)(G)), (DST/TSG/PT/2008/28), (SR/FST/ETI284/2011 (C)) and (SR/NM/PG-16/2007). We also acknowledge SASTRA University, Thanjavur for extending infrastructure support to carry out the study.
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List of Figures Fig.1: Raman spectral analysis of GO and [Ru(bpy)3]2+-GO nanocomposite. Fig. 2: FE-SEM image of GO and [Ru(bpy)3]2+-GO nanocomposite with relevant identified elemental mapping of uniformly distributed C-K, O-K and Ru-L. Fig. 3: FE-TEM micrographs of (a) GO (b) [Ru(bpy)3]2+-GO nanocomposite, (c) FE-TEM micrographs of GO mimcking the coating on the modifed electrode with an inset of (d) SAED patterns of GO, (e) FE-TEM micrographs of [Ru(bpy)3]2+- GO nanocomposite mimcking the Page 20 of 25
coating on the modifed electrode with an inset of (f) SAED patterns of GO and energy dispersive spectra of (g) GO and (h) [Ru(bpy)3]2+- GO nanocomposite mimcking the coating on the modifed electrode. Fig. 4: (A) Cyclic voltammetric analysis (B) EIS analysis of various modified electrodes in presence of 0.5 mM of K3[Fe(CN)6] in 0.1 M of KCl solution at a scan rate of 100 mVs-1. Fig. 5: (A) Simultaneous detection of Cd(II), Pb(II), As(III) and Hg(II) at different concentrations. (B) Calibration plots of As(III) and Pb(II), (C) calibration plots of Cd(II) and Hg(II) in 0.1 M citrate buffer at pH 5 using [Ru(bpy)3]2+-GO nanocomposite modified Au electrode.
List of Tables
Table 1: Analytical parameters of individual and simultaneous detection of heavy metal ions at [Ru(bpy)3]2+-GO nanocomposite modified Au electrode. Method of analysis
Analyte
Selective analysis
Cd(II) Pb(II) As(III) Hg(II) Cd(II) Pb(II) As(III) Hg(II)
Simultaneous analysis
LOD (nM) 132.28 350.60 293.52 350.20 2.80 1.41 2.30 1.60
Potential (V) -0.626 -0.375 0.108 0.412 -0.638 -0.347 0.127 0.404
Sensititvity (µA µM-1) 8.02 24.12 0.187 3.71 17.51 34.74 23.60 31.43
Table 2: Determination of Cd(II), Pb(II), As(III) and Hg(II) in water using [Ru(bpy)3]2+- GO nano-interface modified Au electrode and validation with AAS. Sample Analyte Added (µM) Found Recovery RSD AAS (µM) (%) Cauvery Cd(II) 0.00 1.07 95.4 2.39 ND river water 2.00 2.32 103.4 3.62 ND Pb(II) 0.00 19.63 102.68 0.19 19.21±0.32 2.00 22.72 99.79 3.53 23.17±0.53 As(III) 0.00 0.33 96.89 1.008 ND 2.00 5.05 102.42 0.019 5.15±0.81 Hg(II) 0.00 2.39 104.13 2.66 2.01±0.78 2.00 4.23 99.08 3.23 4.48±0.554 Tap river Cd(II) 0.00 3.9 94.62 4.21 ND water 2.00 5.32 97.72 2.11 5.15±0.812 Pb(II) 0.00 2.98 105.5 0.77 3.02±0.43 Page 21 of 25
As(III) Hg(II)
2.00 0.00 2.00 0.00 2.00
7.33 0.88 2.64 3.33 4.22
Fig.1:
Fig. 2:
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99.5 92.15 96.32 101.2 103.3
1.34 1.141 2.11 4.04 3.21
7.4±0.53 ND ND 3.67±0.21 4.4±0.48
Fig. 3:
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Fig. 4 :
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Fig. 5 : (B) -1
Sensitivity = 23.6 LOD = 2.3 n Linear range = 0.1 - 1.2
Current ()
4.4
As(III)
2
R = 0.96
4.0
-1
3.6
Sensitivity = 34.74 LOD = n Linear range = 0.05 - 1.5
3.2
2
R = 0.98
Pb(II)
2.8 0.0
0.3
0.6
0.9
Concentration ()
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1.2
1.5
Highlights Go and [Ru(bpy)3]2+-GO nanocomposite were synthesized and characterized for microstructural analysis [Ru(bpy)3]2+-GO modified Au electrode provides a better electron transfer process between electrolyte and electrode Electrocatalytic behaviour of [Ru(bpy)3]2+-GO nanocomposite was studied in detecting heavy metal ions Well resolved peaks with good peak-to-peak separation were observed for Cd(II), Pb(II), As(III) and Hg(II) ions simultaneously Obtained resulted were validated using AAS and the results are in good agreement with low RSD Graphical Abstract
PII: DOI: Reference:
S0039-9140(16)30830-X http://dx.doi.org/10.1016/j.talanta.2016.10.076 TAL16996
To appear in: Talanta Received date: 22 August 2016 Revised date: 19 October 2016 Accepted date: 19 October 2016 Cite this article as: Manju Bhargavi Gumpu, Murugan Veerapandian, Uma Maheswari Krishnan and John Bosco Balaguru Rayappan, Simultaneous Electrochemical Detection of Cd(II), Pb(II), As(III) and Hg(II) Ions using Ruthenium(II)-textured Graphene Oxide Nanocomposite, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.076 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 galley proof before it is published in its final citable 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.
Simultaneous Electrochemical Detection of Cd(II), Pb(II), As(III) and Hg(II) Ions using Ruthenium(II)-textured Graphene Oxide Nanocomposite Manju Bhargavi Gumpu1,2, Murugan Veerapandian3, Uma Maheswari Krishnan4, John Bosco Balaguru Rayappan1,2* 1
Nano Sensors Lab @ Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), SASTRA University, Thanjavur - 613 401, Tamil Nadu, India 2 School of Electrical & Electronics Engineering, SASTRA University, Thanjavur - 613 401, Tamil Nadu, India 3 National Institute of Pharmaceutical Education & Research, Kolkata - 700 032, West Bengal, India 4 School of Chemical and Biotechnology, SASTRA University, Thanjavur - 613 401, Tamil Nadu, India
*Corresponding author at: Centre for Nanotechnology & Advanced Biomaterials (CeNTAB) & School of Electrical & Electronics Engineering SASTRA University. Thanjavur – 613 401. India. T: +91 4362 264 101; Ext: 255; Fax: +91 4362 264120. Email: [email protected]
Abstract Simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions was carried out based on the synergistic effect of graphene oxide (GO) textured with redox active ruthenium(II) bipyridine complex ([Ru(bpy)3]2+). [Ru(bpy)3]2+-GO nanocomposite on the modified gold (Au) electrode acts as an electrocatalyst and favours the sensitive and selective detection of metal ions. Also, it exhibited an enhanced electron transfer rate with a low solution resistance examined by cyclic voltammetry and impedance analysis. The inherent electrochemical and electrocatalytic behaviours of [Ru(bpy)3]2+-GO on gold electrode were demonstrated for simultaneous detection of heavy metal ions in water matrix. The proposed sensor exhibited a higher sensitivity towards Cd(II), Pb(II), As(III) and Hg(II) metal ions with a lowest detection limit of 2.8, 1.41, 2.3 and 1.6 nM respectively. The observed detection limits were less than the World Health Organization standards and hence the developed sensor can be deployed for detecting heavy metal ions in water bodies. Simultaneous electrochemical detection of heavy metal ions in river and tap water was carried out using the developed sensor and the observed results were validated with atomic absorption spectroscopy.
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Keywords: Heavy metal ions, Simultaneous detection, Ruthenium (II) bipyridine, Graphene oxide, Redox-active nanocomposite
1. Introduction Environmental and water pollution due to heavy metal ions have been a major concern and even threat to human life [1,2]. Increase in industrial activities, usage of pesticides, insecticides, paints and modernized civilization have spread the heavy metal ions through air, water and soil [3,4]. As a result, heavy metal ions enter the food chain and contaminates the ecosystem. Many tragedies like Minamata poisoning, Bhopal disaster, arsenic poisoning, Sandoz disaster [5] are some of the historic evidences for the loss of human lives and economy due to the consumption of metal ion contaminated water. In view of these disasters, organizations such as Centre for Disease Control (CDC), International Agency for Research on Cancer (IARC), World Health Organization (WHO) have put forth limitations in permissible levels for various heavy metal ions [6]. Intake of heavy metal ions beyond these permissible levels lead to reproductive toxicity, respiratory disorders, impaired neurologic development, cardiovascular diseases, pulmonary fibrosis and behavioural issues [7–9]. Hence, rapid and reliable detection of these heavy metal ions is need of the hour
[10–12]. Various instrumental methods such as electrolyte cathode
atmospheric glow discharge (ELCAD) [13], microwave plasma torch (MPT)-atomic emission spectroscopy (AES), atomic absorption spectroscopy (AAS) [14], inductively coupled plasma atomic emission spectroscopy (ICP-AES)[15], laser-induced breakdown spectroscopy (LIBS) [16] are being used for detecting heavy metal ions. Aforementioned methods are restricted in terms of pre-sample preparation, time-consuming and complex instrumentation [13,17,18]. To overcome the drawbacks of conventional methods, electrochemical biosensors have emerged as an effective tool to quantify the heavy metal ion elements in water matrix with the advantages of rapidity, portability, simple sample preparation and easy handling [19,20]. Page 2 of 25
In this context, electrochemical sensors have been considered to detect heavy metal ions with additional advantages of sensitivity and specificity. Further, enhancement of sensitivity and selectivity in electrochemical sensors can be achieved through proper modification of electrode surface using various nanomaterials, organic and inorganic materials [21]. Among various nanomaterials, carbon based nanomaterials are highly compatible, offers high adsorption capacity, provides fast electron transfer rate and high surface coverage area [22]. Among them, graphene oxide (GO) is the most prominent nanomaterial with hydrophilic oxygenated functional groups [23]. Due to its biocompatible property, 2D thin layer structure and abundant oxygen functional groups it has been recently identified and employed as a novel interface layer in electrode/biosensor research. Compared to conductive graphene sheets bulk synthesis of GO is simple and cost-effective. Using chemisorption and physical treatment such as functional group activation and photoirradiation, the property of GO-based materials could be transformed for advanced electrochemical biosensing applications. Besides, GO provides high π-conjunction and doping of organic or inorganic complexes such as ruthenium (II) complexes based on bipyridine ligands [Ru(bpy)3]2+, further enhances the mass transport efficiency, charge transfer [24], sensitivity and selectivity [25]. [Ru(bpy)3]2+ complex also provides unique features such as octahedral geometry, tunable electrochemical properties and different stable oxidation states ranging from I to III [26]. Functionalization of GO with [Ru(bpy)3]2+ is well coordinated because of the electrostatic and π-π stacking interactions forming [Ru(bpy)3]2+-GO nanocomposite [27,28]. Even though [Ru(bpy)3]2+-GO nanocomposite was used for various applications like detecting cancer biomarkers, tripropylamine, and non-esterified fatty acids [29], to the best our knowledge it is the first time [Ru(bpy)3]2+-GO nanocomposite is being used for simultaneous detection of heavy metal ions in water. Herein, we integrated the redox active molecule (ruthenium (II) complex) on the surface
Page 3 of 25
of GO to improve the inherent electrical conductivity and demonstrate its use in detection of heavy metal ions. Earlier report demonstrated that the composite of SnO2/graphene modified on glassy carbon electrode could simultaneously detect the heavy metal ions, viz., Cd(II), Pb(II),Cu(II) and Hg(II) ions [30]. Though anodic stripping voltammetry helped in simultaneous detection of heavy metal ions, it was bounded in terms of detection limit, sensitivity and relative correlation co-efficient. This was due to the competition of Cd(II), Pb(II), Cu(II) and Hg(II) ions towards the limited number of active sites. Jena et al., reported the usage of gold nano electrode ensembles (GNEE) to detect As(III), Hg(II) and Cu(II) simultaneously [31]. But, this study suffered due to high over voltage, low stability and limited sensitivity [6]. Along with various metals, metal oxide nanointerfaces, polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) were also used for simultaneous detection of Cd(II) and Pb(II) [14]. Though this method has achieved the better detection limit, it has issues in terms of sensitivity due to slow diffusion rate and high polymer thickness [32]. By considering these limitations, [Ru(bpy)3]2+-GO nanocomposite was developed as an interface material on gold working electrode for the simultaneous detection of heavy metal ions Cd(II), Pb(II), As(III) and Hg(II) by employing differential pulse voltammetry (DPV). Herein, it is evident that [Ru(bpy)3]2+-GO nanocomposite acts as an efficient electrocatalyst for the simultaneous detection of heavy metal ions without any complexity in sample preparation and data analysis. 2. Experimental 2.1.1. Chemicals Graphite powder (<20 µm, synthetic), tris (2,2’-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)3Cl2 •6H2O) were procured from Sigma-Aldrich, Canada. Sodium citrate, citric acid, sodium chloride, potassium chloride, boric acid, phospohoric acid, acetic acid, sodium
Page 4 of 25
hydroxide, lead nitrate, cadmium acetate, cobalt acetate, zinc chloride, mercuric sulphate, arsenic (III) iodide and chitosan were purchased from Sigma Aldrich, India. All the studies were carried out at room temperature using deionized water. Citrate buffer of 0.1 M was prepared by dissolving 0.25 g of sodium citrate and 0.21 g of citric acid in 100 mL of deionized water.
2.1.2. Synthesis of GO GO was synthesized using modified Hummers’ method [33]. Graphite powder and sulphuric acid (H2SO4) were mixed for 2 h. Potassium permanganate (KMnO4) of 6 g was added to this mixture by maintaining a temperature less than 293 K. Later the mixture was stirred at 308 K for 2 h and the resulting solution was treated with 10 mL of 30% of hydrogen peroxide and 150 mL of distilled water. The obtained graphite oxide solution was centrifuged multiple times until pH reaches neutral. The precipitate was ultra-sonicated for 1 h and GO was formed by exfoliating multi layered graphite oxide. 2.1.3. Synthesis of [Ru(bpy)3]2+-GO nanocomposite A mixture of 10 mL aqueous GO (1 mg/mL) and 10 mL ethanolic solution of Ru(bpy)3Cl2 (1 mg/mL) was magnetically stirred at 800 rpm for overnight under dark conditions. The [Ru(bpy)3]2+-GO nanocomposite mixture was centrifuged at 12,000 rpm for 45 min and unreacted [Ru(bpy)3]2+ was removed from the mixture by continuous washing in anhydrous ethanol and deionized water [29]. Isolated [Ru(bpy)3]2+-GO nanocomposite was used for further experimental investigations. 2.2. Characterization of [Ru(bpy)3]2+-GO nanocomposite Morphology of [Ru(bpy)3]2+-GO nanocomposite was investigated using Field Emission Scanning Electron Microscope (FE-SEM) (JEOL, 6701F, Japan) and Field Emission Transmission Electron Microscope (FE-TEM) (JSM2100, JEOL, Japan). Structural and functional group analysis of [Ru(bpy)3]2+-GO nanocomposite were carried out using Energy Page 5 of 25
Dispersive X-Ray Spectroscope (EDXS) (Thermo Fisher Scientific Inc., K Alpha, USA), X-ray Photoelectron Spectrometer (XPS, Omicron equipped with a hemispherical analyzer that utilizes the monochromated Al Kα radiation (hν = 1486.6 eV), operated at 12 kV and power of 300 W) and Fourier Transform Infrared Spectrometer (FT-IR) (Spectrum 100, Perkin Elmer, USA). Optical absorbance and photoluminescence properties were studied using Cary 5000 UV-VisNIR spectrophotometer (Agilent Technologies) and Varian Cary Eclipse Fluorescence Spectrometer respectively. Electrochemical analysis was carried out using CH electrochemical analyzer (CH600C, CH Instruments, USA). Gold working electrode (2 mm diameter, CH Instruments, USA), platinum (Pt) wire electrode (0.5 mm diameter, CHI115, CH Instruments, USA) as counter electrode and Ag/AgCl (0.5 mm diameter. CHI111, CH Instruments, USA) saturated with 0.1 M KCl as a reference electrode were used in three electrode cell. The concentration studies were validated using Atomic Absorption Spectrometer (AAS) (Model no. 2380, Perkin Elmer). 2.3. Fabrication of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode [Ru(bpy)3]2+-GO nanocomposite of 1 mg was dispersed in 0.05 (w/v) chitosan solution and sonicated for 30 min. From the well dispersed solution of [Ru(bpy)3]2+-GO nanocomposite, 3 µL was coated on the top of Au electrode and allowed to air dry. The nanocomposite film was crack free and hence used for electrochemical analysis. 3. Results and Discussion 3.1. Microstructural studies Structural Analysis Structural characteristics of GO and [Ru(bpy)3]2+-GO nanocomposite were studied from Raman spectrum (Fig. 1). GO exhibited two characteristic peaks at 1351 and 1594 cm-1 corresponding to the D and G bands. [Ru(bpy)3]2+-GO nanocomposite also exhibited D (1361cm-1) and G (1594 cm-1) bands with a slight shift. Besides, it also exhibited an additional peak at 1483 cm-1 Page 6 of 25
corresponding to the C=N vibrations of [Ru(bpy)3]2+ complex. A slight decrease in ID/IG ratio of [Ru(bpy)3]2+- GO nanocomposite (1.17) than GO (1.18) was observed due to the change in π-π stacking to π-conjugated structure of [Ru(bpy)3]2+-GO nanocomposite. Morphological characterization of [Ru(bpy)3]2+-GO nanocomposite The surface morphologies of GO and [Ru(bpy)3]2+-GO nanocomposite were analyzed using FESEM (Fig. 2a-b) and FE-TEM (Fig. 3a-c,e). GO showed a transparent wrinkled sheet like morpholgy with multiple layers. The EDS spectra showed peaks corresponding to carbon and oxygen for GO whereas an additional ruthenium peak was observed for [Ru(bpy)3]2+-GO nanocomposite. The transparent nature of GO was well evident from FE-TEM micrograph indicating that GO has less number of layers with large network of thin sheet topography. Selected Area Electron Diffraction (SAED) exhibited a hexagonal diffraction pattern for GO (Fig. 3d). In comparision to GO, [Ru(bpy)3]2+-GO nanocomposite showed a tightly packed sheet structures with lightly coorugated morphology.
Its SAED pattern confirmed the chemical
interaction between [Ru(bpy)3]2+ and GO (Fig.3f). Further, the EDS spectra of modified Au electrodes are shown in Fig. 3(g-h). It showed an additional Au peak for GO and [Ru(bpy)3]2+ modified Au electrode. This has confirmed that GO and [Ru(bpy)3]2+ were well immobilized on the surface of Au working electrode. Surface chemistry and optical properties of [Ru(bpy)3]2+-GO nanocomposites XPS analysis Vibrational changes in the surface elemental moieties of GO, before and after interaction with [Ru(bpy)3]2+ were observed from high-resolution XPS. Supplementary Fig. S1A shows the C1s peak of GO material with relevant deconvolution by Gaussian’s fit. This resulted in the five characteristic peaks related to the carbon atoms with different functional groups. In relation to previous reports [29,34] the C1s peaks were denoted as follows: non-oxygenated carbon (C-C, C=C and C-H, 284. 4 eV), hydroxyl (C-OH, 285 eV), epoxy (286.2 eV), carbonyl (287 eV) and
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carboxylic acid (288.5 eV) groups, respectively. Interaction of [Ru(bpy)3]2+ on the surface of GO significantly influenced the vibrational signals of inbuilt elemental binding groups. These include appearance of new peak at 281.1 eV, attributed to the Ru3d5/2 (Supplementary Fig. S1B). The structural vibrations of epoxy is shifted from 286.2 to 285.7 eV, and the hydroxyl groups are shifted from 285 to 285.2 eV. Further, disappearance of carboxylic acid and carbonyl group vibrations denote that the interaction of [Ru(bpy)3]2+ significantly modified the surface texture of GO [29]. Such chemically interacted electroactive molecules with nanosize distributions are expected to have advanced electron-charge transfer behaviours. FT-IR analysis FT-IR spectral analysis showed the characteristic peaks for GO (Supplementary Fig. S2A-i)) at 1039, 1183 and 1422 cm-1, which are attributed to the vibrations of carbonyl (C-O), epoxy (C-OC) and OH groups of oxidized graphitic domains. C-C stretching frequencies of graphitic lattice network is observed at 1620 cm-1. C=O stretching vibrations of carboxylic groups are located at 1712 cm-1 [33]. Upon interaction with [Ru(bpy)3]2+ complex the stretching and bending vibrations of GO are slightly modified (Supplementary Fig. S2A-ii). For instance, epoxyl group frequencies are notably diminished and hydroxyl group vibrations are disappeared. Functional groups of bipyridyl components from [Ru(bpy)3]2+ complex are well resolved at 728, 767, 1420, 1445 and 1467 cm-1 (highlighted in blue frame). In association with C-C skeletal vibrations of GO, the conjugated C=C signal from bipyridyl group is observed at 1600 cm -1. From the observed structural vibrations, it is evident that the [Ru(bpy)3]2+ is chemically interacted with the oxygenated functional groups of GO. Supplementary Fig. S2B represents the absorption bands of [Ru(bpy)3]Cl2 powder. The sharp and intense peaks located at 731 and 772 cm-1 are related to the C-H group stretch from the pyridine. Multiple peaks located in the region 897-1162 cm-1 correspond to the in-plane C-H bending vibrations. Other peaks located in the region 1220-1318, 1423, 1442, 1464 and 1600 cm-1 are the characteristics vibrations of bipyridyl groups [35].
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Optical absorbance UV-Visible spectral analysis was employed to determine the optical properties of GO and [Ru(bpy)3]2+-GO nanocomposite. Supplementary Fig. S3 depicts the UV-Vis absorbance spectrum of GO (a) with a maximum absorbance at 235 nm. This corresponding to π-π* transition of electrons in the polyaromatic C-C bonds of GO. The inset in Supplementary Fig. S3 indicates the absorption spectra of [Ru(bpy)3]2+ complex showing peaks at 243, 290 and 420 nm, which are in good co-ordination with the previous work [29]. The prominent peaks at 243 and 420 nm of lower energy, signifies the oxidation of Ru(II) to Ru(III) in which dπ orbitals are stabilized leading to metal–to–ligand charge transfer (MLCT). The specific absorbance peak at 243 nm corresponds to the bpy π-π* intra-ligand transition and the peak at 290 nm corresponds to destabilization of bpy π* MOs (molecular orbitals) due to increased back bonding. This creates an energy gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Photoluminescence study Recent studies documented that aqueous dispersion of GO nanosheets possess finite electronic bandgap due to the disrupted π networks resulted from the oxygenation process [35]. Inherent broad PL property demonstrated from GO was believed to be originated from the sp3 carbon domains surrounded by the clusters of graphitic sp2 regions [33,36]. In this study, a complementary investigation on PL property was studied to understand the intrinsic changes in GO nanosheets their interaction with [Ru(bpy)3]2+. As observed from Supplementary Fig. S4A, at an excitation wavelength of 325 nm the GO nanosheets exhibited a near UV emission peak centered at 364 nm, in agreement with the earlier report [33]. Likewise, the [Ru(bpy)3]2+ modified GO nanomaterial also exhibited a PL emission peak with a notable broadness, which may be attributed to the localized transition between the adhered molecules. On adding [Ru(bpy)3]2+ to GO a slight decrease in wavelength was observed corresponding to the blue shift Page 9 of 25
signifying that the added [Ru(bpy)3]2+ has chemically interacted with COOH and C=C bonds of GO. This electrostatic interactions helped Ru metal of [Ru(bpy)3]2+ to bind with COO- and bpy to bind with C=C of GO to form a nanocomposite structure. To ensure a further electronic transition between the [Ru(bpy)3]2+ molecules and GO nanosheets, PL spectrum was recorded at an excitation wavelength of 450 nm (Supplementary Fig. S4B), which is the known standard for [Ru(bpy)3]2+. At this excitation, GO nanosheets only exhibited the inherent visible emission band centered at 529 nm, indicating the pristine nature of the GO layers. Earlier report studied that this PL behaviour of GO corresponds to the recombination of electron-hole pairs from conduction band and localized electronic state at the valence band [36]. In the case of [Ru(bpy)3]2+-GO nanocomposite, two characteristic broad visible emission bands were observed at 532 nm and 610 nm. The former one with a minor shift is attributed to the formation of new sp2/sp3 carbon domains associated from GO/[Ru(bpy)3]2+ and later is ascribed to the inherent triplet MLCT of [Ru(bpy)3]2+. The observed PL spectral changes provide a further complementary information on the surface modification of GO with [Ru(bpy)3]2+. 3.2. Electrochemical Studies 3.2.1 Electrocatalytic activitity of [Ru(bpy)3]2+- GO nanocomposite modified Au electrode Electrochemical behaviours of different modified Au electrodes were recorded in the presence of 0.5 mM K3[Fe(CN)6] in 0.1 M of KCl solution at a scan rate of 100 mVs-1 using cyclic voltammetry (CV). Fig. 4A depicts the anodic and cathodic peaks of bare Au, GO modified Au and [Ru(bpy)3]2+-GO modified Au electrodes with a potential difference (△E) of 189, 110 and 102 mV, respectively. Higher △E values of bare Au indicates the slow electron transfer rate and on modifying the Au electrode with GO and [Ru(bpy)3]2+-GO nanocomposite, the electron transfer rate was enhanced due to the electrocatalytic behavior of GO and [Ru(bpy)3]2+-GO nanointerface. The charge transport process of Au, GO modified Au and [Ru(bpy)3]2+-GO modified Au electrodes were studied using Electrical Impedance Spectroscopy (EIS) (Fig. 4B). The charge
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transfer resistance (Rct) of [Ru(bpy)3]2+-GO modified Au electrode was 281.1 Ω, which is almost 1.9 and 2.3 times lower than Au-GO and bare Au electrodes. This has signified the better electron transfer process between electrolyte and [Ru(bpy)3]2+-GO modified Au electrode. Further, to prove the electrocatalytic behaviour of bare Au, Au electrode modified with GO and [Ru(bpy)3]2+-GO, heterogeneous electron transfer rate constant (K0) of each was calculated (Eq. 1) based on
interfacial interaction.
K0 =
- (1)
where, R is the gas constant, T is the temperature, n is the electron transfer constant of the redox couple, F is the Faraday constant, Aec is the effective area of Au electrode, Rct is the charge transfer resistance and C is the concentration of redox couple in the bulk solution. The K0 value of [Ru(bpy)3]2+-GO modified Au electrode (15.3×10-6 s-1) was higher than GO modified Au electrode (6.74×10-6 s-1) and bare Au electrode (6.58×10-6 s-1) indicating a faster electron transfer exchange between the [Ru(bpy)3]2+-GO modified Au electrode interface and redox species, which was in good co-ordination with △E values. From CV and EIS studies, it is evident that GO modified Au electrode exhibited a higher conductivity than bare Au electrode due to the decreased oxygen content of GO. This can be estimated from the mass ratio of KMnO4 (6 g) to the graphite (2 g) concentration utilized for the synthesis of GO. Its ratio was observed to be 0.3, which corresponds to the decreased oxygen content of GO. Hence, it is conductive in nature and that is the reason why an enhanced peak current was observed. Further, [Ru(bpy)3]2+-GO modified Au electrode showed an enhanced peak current and electron transfer rates due to the further reduction of GO on accommodating [Ru(bpy)3]2+. Although [Ru(bpy)3]2+-GO nanocomposite can exhibit electrostatic and hydrophobic interactions, [Ru(bpy)3]2+-GO nanocomposite is dominated by the electrostatic interaction, which was evident from the irreversible nature of [Ru(bpy)3]2+-GO (△E > 60 mV). Page 11 of 25
3.2.2. Optimization of electrolyte and pH Electrolyte and buffer are the basic parameters that shows a major influence on potential. Optimization of electrolyte and pH helps in narrowing down the potential window and hydrolysis of metal ions [37]. Hence, voltammetric behaviour of different electrolyte solutions was tested at pH 6 in 0.1 M of phosphate buffer saline, citrate, acetate, Britton - Robinson and Tris-HCl buffer. In acetate and citrate buffer, quaternary mixture of Cd(II), Pb(II), As(III) and Hg(II) metal ions showed well-defined peaks (Supplementary Fig. S5A). But, citrate buffer exhibited a peak of maximum current which is 1 fold higher than the acetate buffer. In acetate buffer, lower concentrations of metal ions were not detected due to the complex formation behaviour of metal ions. In the case of Britton-Robinson buffer, peak overlap was observed. Tris-HCl buffer exhibited a single widened peak, which resulted in interference. PBS showed a well resolved peak for Pb(II) and As(III) with a lower current response than the citrate buffer due to binding of phosphate with metal ions [38]. Further, citrate buffer was optimized by varying pH in the range of 3.0 to 9.0 (Supplementary Fig. S5B). The maximum current was attained at the pH of 5.0. The current was increased in the pH range of 3.0 to 5.0 due to complexation mechanism and electrostatic attraction. Beyond pH 5, a decrease in current was observed due to the hydrolysis of metal ions. Thus 0.1 M citrate buffer of pH 5.0 was chosen as an optimal condition for further experiments. 3.2.3. Electrochemical determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions In order to achieve better sensitivity, DPV was employed as an analytical technique for effective detection of Cd(II), Pb(II), As(III) and Hg(II) quaternary metal ion mixture. Supplementary Fig. S5C shows the DPV response of bare Au electrode, GO modified Au electrode and [Ru(bpy)3]2+GO modified Au electrode in the presence of 0.1 M citrate buffer, pH 6. Bare Au electrode can detect only As(III) at 0.09 V vs Ag/AgCl from quaternary mixture of metal ions with a sensitivity of 0.886 µA µM-1. GO modified Au electrode exhibited two well resolved peaks at -0.698 and
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0.121 V vs Ag/AgCl for Cd(II) and As(III) with a sensitivity of 3.43 µA µM-1 and 2.11 µA µM-1. When compared to bare Au, GO modified Au electrode showed an increased sensitivity for As(III) due to the desorbing activity of adsorbed As(III) metal ions on GO. [Ru(bpy)3]2+-GO nanocomposite modified Au electrode exhibited four peaks at -0.638, -0.347, 0.127 and 0.404 V vs Ag/AgCl for Cd(II), Pb(II), As(III) and Hg(II) respectively (Supplementary Fig. S5C). Also, they showed a good separation of Cd(II) – Pb(II), Pb(II) – As(III) and As(III) – Hg(II) peaks that helps in avoiding the formation of inter-metallic compounds. Although, four metal peaks were observed on Au modified with [Ru(bpy)3]2+-GO, the intensity of peak currents was observed to be lower due to the coorugated morphology of [Ru(bpy)3]2+-GO. 3.2.4. Simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions Under the optimized conditions, Cd(II), Pb(II), As(III) and Hg(II) metal ions were detected individually as well as simultaneously using [Ru(bpy)3]2+-GO modified Au electrode. Supplementary Fig. S6 shows the DPV response towards Cd(II), Pb(II), As(III) and Hg(II) metal ions at -0.626, -0.375, 0.108 and 0.412 V respectively. The difference between each peak signifies the simultaneous determination of Cd(II), Pb(II), As(III) and Hg(II) metal ions is a feasible approach. Its analytical parameters were analyzed and the linear range of Cd(II), Pb(II), As(III) and Hg(II) metal ions were found to be 0.05-0.3, 0.05-0.25, 0.05-1.8 and 0.1-1.2 µM respectively covering the WHO limit. From Fig. 5, well resolved peaks at -0.638, -0.347, 0.127 and 0.404 V vs Ag/AgCl were observed for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. An increase in current with an increase in metal ion concentrations were also observed. Among these metal ions Cd(II) and Hg(II) showed weak peaks, which might be due to the intermetallic complex formation. The limit of detection of Cd(II), Pb(II), As(III) and Hg(II) metal ions were observed to be 0.013, 0.35, 0.06 and 0.34 µM respectively. The analytical parameters of Cd(II), Pb(II), As(III) and Hg(II) metal ions are presented in Table. 1.
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3.2.5. Interference studies The selectivity and cross reactivity of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode in the presence of heavy metal ions were analyzed individually as well as simultaneously. As the concentration of As(III) and Hg(II) was varied from 0.02 to 2 µM, a slight increase in Pb(II) was observed while the current peaks of Cd(II) almost remained the same (Supplementary Fig. S7A). This may be due to the formation of As-Pb intermetallic compound. Similarly, the concentration of Cd(II) and Pb(II) was varied from 0.02 to 3 µM in the presence of 0.5 µM of As(III) and Hg(II) (Supplementary Fig. S7B). The current response of As(III) and Hg(II) showed no significant changes confirming the absence of inter-metallic complexation with Cd(II) and Pb(II) ions. Hence, the proposed sensor can be employed to detect Cd(II), Pb(II), As(III) and Hg(II) metal ions with no major interference among the four metal ions. 3.2.6. Precision and stability measurements The precision in terms of repeatability and reproducibility of [Ru(bpy)3]2+-GO nanocomposite modified Au electrode was evaluated by taking repetitive measurements of DPV in the presence of 0.5 µM of Cd(II), Pb(II), As(III) and Hg(II) ions. For ten repetitive experiments, the relative standard deviation (RSD) was 2.86%, 3.02%, 1.87% and 2.44% for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. Reproducibility of the developed electrode was verified with three different electrodes and it showed an RSD of 3.2%, 2.88%. 3.3% and 3.69% for Cd(II), Pb(II), As(III) and Hg(II) metal ions respectively. The operational stability of the developed electrode was estimated in the presence of 0.5 µM of Cd(II), Pb(II), As(III) and Hg(II) metal ions and retained a sensitivity of 89.2%, 91.1%, 90.7% and 73.8% respectively for a period of 60 days. 3.2.7 Application (Tap water) and validation with AAS The developed electrode showed a good reproducibility and repeatability with a better stability. Hence, it was used for testing Cauvery river water and tap water. The concentrations of Cd(II),
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Pb(II), As(III) and Hg(II) metal ions in Cauvery river water and tap water were estimated and validated using atomic absorption spectroscopy (AAS) (Table 2). It showed a better sensing characteristics of lower detection limit and higher sensitivity in detecting Cd(II), Pb(II), As(III) and Hg(II) metal ions in a simultaneous manner (Supplementary Table 1).
4. Conclusion In the present investigation, [Ru(bpy)3]2+-GO modified Au electrode was fabricated towards the simultaneous detection of Cd(II), Pb(II), As(III) and Hg(II)
metal ions. [Ru(bpy)3]2+-GO
modified Au electrode exhibited a good electrocatalytic activity with a large number of active sites, which resulted in an enhanced charge transfer rate. Improved electrocatalytic behaviour of [Ru(bpy)3]2+-GO helped in detecting the quaternary mixture with a good peak-to-peak separation and lower detection limit of 2.80, 1.41, 2.30 and 1.60 nM for Cd(II), Pb(II), As(III) and Hg(II) ions respectively. Also, the developed sensor exhibited a better sensitivity, repeatability and stability in presence of Cd(II), Pb(II), As(III) and Hg(II)
ions.
Further, [Ru(bpy)3]2+-GO
modified Au electrode was used to detect various concentrations of metal ions in river and tap water. The observed results were in good co-ordination with results of AAS. The developed [Ru(bpy)3]2+-GO modified Au electrode can be utilized for onsite qualitative and quantitative analysis of water bodies. Acknowledgements The authors are grateful to the Department of Science & Technology, New Delhi for their financial support (DST/TM/WTI/2K14/197(a)(G)), (DST/TSG/PT/2008/28), (SR/FST/ETI284/2011 (C)) and (SR/NM/PG-16/2007). We also acknowledge SASTRA University, Thanjavur for extending infrastructure support to carry out the study.
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List of Figures Fig.1: Raman spectral analysis of GO and [Ru(bpy)3]2+-GO nanocomposite. Fig. 2: FE-SEM image of GO and [Ru(bpy)3]2+-GO nanocomposite with relevant identified elemental mapping of uniformly distributed C-K, O-K and Ru-L. Fig. 3: FE-TEM micrographs of (a) GO (b) [Ru(bpy)3]2+-GO nanocomposite, (c) FE-TEM micrographs of GO mimcking the coating on the modifed electrode with an inset of (d) SAED patterns of GO, (e) FE-TEM micrographs of [Ru(bpy)3]2+- GO nanocomposite mimcking the Page 20 of 25
coating on the modifed electrode with an inset of (f) SAED patterns of GO and energy dispersive spectra of (g) GO and (h) [Ru(bpy)3]2+- GO nanocomposite mimcking the coating on the modifed electrode. Fig. 4: (A) Cyclic voltammetric analysis (B) EIS analysis of various modified electrodes in presence of 0.5 mM of K3[Fe(CN)6] in 0.1 M of KCl solution at a scan rate of 100 mVs-1. Fig. 5: (A) Simultaneous detection of Cd(II), Pb(II), As(III) and Hg(II) at different concentrations. (B) Calibration plots of As(III) and Pb(II), (C) calibration plots of Cd(II) and Hg(II) in 0.1 M citrate buffer at pH 5 using [Ru(bpy)3]2+-GO nanocomposite modified Au electrode.
List of Tables
Table 1: Analytical parameters of individual and simultaneous detection of heavy metal ions at [Ru(bpy)3]2+-GO nanocomposite modified Au electrode. Method of analysis
Analyte
Selective analysis
Cd(II) Pb(II) As(III) Hg(II) Cd(II) Pb(II) As(III) Hg(II)
Simultaneous analysis
LOD (nM) 132.28 350.60 293.52 350.20 2.80 1.41 2.30 1.60
Potential (V) -0.626 -0.375 0.108 0.412 -0.638 -0.347 0.127 0.404
Sensititvity (µA µM-1) 8.02 24.12 0.187 3.71 17.51 34.74 23.60 31.43
Table 2: Determination of Cd(II), Pb(II), As(III) and Hg(II) in water using [Ru(bpy)3]2+- GO nano-interface modified Au electrode and validation with AAS. Sample Analyte Added (µM) Found Recovery RSD AAS (µM) (%) Cauvery Cd(II) 0.00 1.07 95.4 2.39 ND river water 2.00 2.32 103.4 3.62 ND Pb(II) 0.00 19.63 102.68 0.19 19.21±0.32 2.00 22.72 99.79 3.53 23.17±0.53 As(III) 0.00 0.33 96.89 1.008 ND 2.00 5.05 102.42 0.019 5.15±0.81 Hg(II) 0.00 2.39 104.13 2.66 2.01±0.78 2.00 4.23 99.08 3.23 4.48±0.554 Tap river Cd(II) 0.00 3.9 94.62 4.21 ND water 2.00 5.32 97.72 2.11 5.15±0.812 Pb(II) 0.00 2.98 105.5 0.77 3.02±0.43 Page 21 of 25
As(III) Hg(II)
2.00 0.00 2.00 0.00 2.00
7.33 0.88 2.64 3.33 4.22
Fig.1:
Fig. 2:
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99.5 92.15 96.32 101.2 103.3
1.34 1.141 2.11 4.04 3.21
7.4±0.53 ND ND 3.67±0.21 4.4±0.48
Fig. 3:
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Fig. 4 :
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Fig. 5 : (B) -1
Sensitivity = 23.6 LOD = 2.3 n Linear range = 0.1 - 1.2
Current ()
4.4
As(III)
2
R = 0.96
4.0
-1
3.6
Sensitivity = 34.74 LOD = n Linear range = 0.05 - 1.5
3.2
2
R = 0.98
Pb(II)
2.8 0.0
0.3
0.6
0.9
Concentration ()
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1.2
1.5
Highlights Go and [Ru(bpy)3]2+-GO nanocomposite were synthesized and characterized for microstructural analysis [Ru(bpy)3]2+-GO modified Au electrode provides a better electron transfer process between electrolyte and electrode Electrocatalytic behaviour of [Ru(bpy)3]2+-GO nanocomposite was studied in detecting heavy metal ions Well resolved peaks with good peak-to-peak separation were observed for Cd(II), Pb(II), As(III) and Hg(II) ions simultaneously Obtained resulted were validated using AAS and the results are in good agreement with low RSD Graphical Abstract