Palisoc 2017

Materials Research Express
ACCEPTED MANUSCRIPT
Determination of heavy metals in mussel and oyster samples with tris (2,
2’- bipyridyl) ruthenium (II)/graphene/Nafion® modified glassy carbon
electrodes
To cite this article before publication: Shirley Palisoc et al 2017 Mater. Res. Express in press https://doi.org/10.1088/2053-1591/aa9745
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Page 1 of 19 AUTHOR SUBMITTED MANUSCRIPT - MRX-104957.R1
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Determination of Heavy Metals in Mussel and Oyster Samples with Tris (2, 2’-bipyridyl)
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ruthenium (II)/Graphene/Nafion® Modified Glassy Carbon Electrodes
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7 Shirley T. Palisoc1,2, Donald Jans S. Uy1, Michelle T. Natividad1,2 and Toni Beth G. Lopez1,3
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Condensed Matter Physics Laboratory, De La Salle University, Manila 0922, Philippines
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Condensed Matter Research Unit, CENSER, De La Salle University, 2401 Taft Avenue, Manila
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0922, Philippines
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12 Technological University of the Philippines, Ayala Blvd, Ermita, Manila 1000, Philippines
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Abstract: Tris (2,2'-bipyridyl)ruthenium(II)/Graphene/Nafion® modified glassy carbon
16 electrodes (GCEs) were fabricated using the drop coating method. The modified electrode was
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17 used as the working electrode in differential pulse voltammetry (DPV) for the determination of
18 lead, cadmium, and copper in mussel and oyster samples. The concentration of Tris (2, 2’-
19 bipyridyl) ruthenium (II) and graphene were varied while those of Nafion®, methanol, and
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ethanol were held constant in the coating solution. The morphology and elemental composition
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22 of the fabricated electrodes were analyzed by scanning electron microscopy and energy-
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dispersive X-ray spectroscopy. Cyclic voltammetry (CV) was done to investigate the
reversibility and stability of the modified electrodes. The modified electrode with the best figures
of merit was utilized for the detection of copper (Cu2+), lead (Pb2+) and cadmium (Cd2+) via
2+
DPV. This was the electrode modified with 4 mg [Ru (bpy) 3] and 3mg graphene. The anodic
current and metal concentration showed linear relationship in the range of 48 ppb to 745ppb for
Pb2+, 49ppb to 613ppb for Cd2+, and 28 ppb to 472 ppb for Cu2+. The limits of detection for lead,
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30 cadmium, and copper were 48ppb, 49ppb, and 28ppb, respectively. Results from atomic
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32 absorption spectrometry (AAS) were compared with those measured with DPV. Lead, cadmium,
33 and copper were in mussels, oysters, and sea water. In addition, DPV was able to detect other
34 metals such as zinc, iron, tin and mercury in sea water samples and some samples of oysters.
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36 Keywords: Voltammetry, Ruthenium bipyridyl, heavy metals, glassy carbon electrode, mussels
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and oysters
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1. Introduction
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42 People have the misconception that the vastness of the sea allows it to absorb large
43 amount of waste without suffering observable damage making them dump their garbage and
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45 unwanted products into the sea. Manila Bay is facing various threats such as overpopulation,
46 land and sea pollution, over-fishing and habitat degradation. The poor water quality of manila
47 Bay can be attributed to the existing sewerage systems in the Metro Manila as well as the
48 untreated wastewater discharged from domestic, agricultural and commercial sources [1].
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Manila Bay today is considered as one of the most polluted bays in the world [2]. The
52 most common types of waste found in Manila Bay are sludge, human sewage, and industrial
53 waste. Some of these garbages decompose into chemicals that are harmful to the environment.
Degrading plastics, for instance, leach potentially toxic chemicals such as Bisphenol A which
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55 threatens ocean animals and humans [3].
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58 Even though Manila Bay is heavily polluted, it still contains marine life. Creatures such
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AUTHOR SUBMITTED MANUSCRIPT - MRX-104957.R1 Page 2 of 19
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as shrimp, fish, mussels, and other shellfish still survive the waters of Manila Bay despite its
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5 water pollution [4]. Fish caught in Manila Bay exceed the allowable concentration of lead in fish
6 at 0.05mg/kg [5]. In addition, mussels and oysters are good biological detectors for heavy metals
7 in sediments and metal pollution in water.
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9 Fang Zhang-qiang et al. [7] investigated the heavy metals found in mussels, oysters and
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clams in South China. Heavy metals such as cadmium, copper, zinc, lead, nickel, chromium,
12 antimony, and tin were found in aforementioned mollusks in South China [8]. Heavy metals such
13 as cadmium, lead, mercury, and arsenic have been extensively studied and reviewed by the
14 World Health Organization (WHO) [9]. Even though these metals are hazardous to human
15 health, they are used in industries such as in electronics [10].
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18 Lead, cadmium, and copper are some examples of trace heavy metals that are toxic in
19 high levels. Toxicity is defined as to when the internal intake of these metals exceeds a certain
20 threshold, only then would they show toxic effects; otherwise some metals, such as copper, zinc
21 and iron are called micronutrients that are essential to the biological systems, plants, human
22 body, and animals. Also, toxicity is not only dependent on the level of concentration of the metal
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but also on the source, acidity, and existence of that metal [11]. Some side effects of heavy
metals on human health include kidney and liver damage, decrease in the production of the
red and white blood cells, vomiting, among others. They also cause miscarriage for pregnant
women and even death [12]. Copper, on the other hand is a necessity in human health since it is
mixed in different enzyme component, and aids red blood cell formation. However, excessive
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intake of copper leads to depression, autism, tardive dyskinesia, memory loss, and Wilson’s
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31 disease [13].
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33 The common techniques in detecting heavy metals are atomic absorption spectrometry
34 (AAS), inductively-coupled plasma mass spectrometry (ICPMAS), high-performance liquid
35 chromatography (HPLC), and neutron activation analysis (NAA). Notwithstanding the favorable
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37 results given by these techniques, the length of preparation, prohibitive cost and number of
38 steps to conduct the experiment are the downside of these methods [14, 15]. An alternative
39 method in detecting heavy metals is the electrochemical technique. In comparison to other
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40 analytical methods, electrochemical techniques such as cyclic voltammetry (CV) and differential
41 pulse voltammetry (DPV) would be more than sufficient since they offer high sensitivity, low
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cost, relative simplicity, and good selectivity [14, 15].
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45 Chemically modified electrodes (CME) are currently being used in voltammetric
46 detection of heavy metals [16-26] instead of mercury based electrodes which are no longer used
47 because of its toxicity and the surface area of a drop of mercury is never constant. Furthermore,
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it has limited applications in the analysis of more positive potential range [27].
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51 In this study, [Ru (bpy)3]2+/Graphene/ Nafion® modified glassy carbon electrodes were
52 fabricated via the drop coating technique and were used to detect heavy metal ions in mussels
53 and oysters from Manila Bay. The redox mediator used was [Ru (bpy)3]2+), with Nafion® as the
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54 polymer for its anti-fouling properties. Graphene exhibits remarkable electronic and physical
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properties due to its high conductivity. Also, it is strong, stiff and very light and its electron
57 mobility is very high. These properties of graphene enhance the sensitivity and electron transport
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properties of the modified GCE.
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7 2. Methodology
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9 2.1 Glassware and equipment
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12 The potentiostat/galvanostat software BST8Cyclic was utilized for the cyclic
13 voltammetry (CV) and differential pulse voltammetry (DPV) measurements. Three-electrode
14 setup consisted of the modified electrode as the working electrode, a saturated calomel electrode
15 as the reference electrode and a platinum wire as the counter electrode. A BANDALIN
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SONOREX sonicator bath was used for glassware cleaning and for homogenization of solution.
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18 A BOSCH SAE200 electronic balance was used in all weight measurements. Atomic absorption
19 spectroscopy (AAS) was done with the AA-6300 Shimadzu Atomic Absorption
20 Spectrophotometer. Solutions were coated on the surface of the electrodes with a Transferpette®
21 micropipette.
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2.2 Chemicals and Reagents
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Graphene nanopowder multilayer was sourced from Graphene SUPERMARKET. Tris (2,
2’- bipyridyl) ruthenium (II), methanol, ethanol, 0.3microns and 0.05 microns alumina slurry,
sodium chloride, copper chloride, and cadmium chloride were all procured from Sigma Aldrich.
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Ten (10) wt% of Nafion obtained from Fuel Cell Store was diluted with methanol and ethanol
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31 in this study. Lead chloride was purchased from Techno Parchem. Other chemicals of analytical
32 grade and liquid solutions were prepared using deionized water from JCS Company.
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34 2.3 Preparation of the electrode modifier solution
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37 A 50ml beaker was cleaned using a sonicator bath for a minimum of 5 minutes. The
38 beaker was subsequently washed with tap water followed by distilled water and was air dried.
39 Twenty (20) ml methanol, 0.2ml of 10 wt% Nafion and 0.2 ml of ethanol was placed inside the
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40 50ml beaker and was cling-wrapped to avoid contamination and evaporation of the solution. The
41 [Ru (bpy)3]2+ and graphene powders were weighed using an electronic balance. The beaker was
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sonicated for a minimum of five minutes to thoroughly mix the solutions. The powders were then
44 mixed with the solution and sonicated again for one and a half hours to homogenize the solution.
45 Thirty (30) microlitters of the prepared modified solution were deposited to the surface of the
46 glassy carbon electrode (GCE) via drop coating using a transferpette micropipette. After drop
47 coating, the electrode was air dried for at least two hours.
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51 2.4 Cleaning and polishing of the electrode
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53 Cleaning was done before fabrication of the electrode and aftermeasurements. The
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54 electrode was subjected to a potential of 1.0 V to strip off metals from the surface of the
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56 electrode back to the analyte. For polishing, 0.3 microns was used followed by 0.05 microns of
57 alumina slurry. The GCEs were then placed in a petri dish with isopropanol which was
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consequently placed in an ultrasonic bath for five minutes. After sonication, the GCEs were
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5 rinsed with distilled water and were air-dried to attain a mirror-like surface.
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7 2.5 Cyclic Voltammetry
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9 Cyclic voltammetry (CV) using the three-electrode setup was performed to characterize
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the fabricated electrodes. Reversibility, stability, and anodic peak current were observed. The
12 analyte inside the voltammetric cell contained 100 mL of deionized water and 0.5844g of sodium
13 chloride to produce a 0.1M salt solution. Sparging with high purity nitrogen was employed to
14 remove oxygen that may interfere with the results.
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2.6 Differential Pulse Voltammetry
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19 Differential pulse voltammetry (DPV) consists of three steps: accumulation or
20 preconcentration, deposition or quiet time equilibration, and stripping. As in cyclic voltammetry,
21 the three-electrode setup was used and the solutions were subjected to sparging. The recipe used
22 for DPV consisted of a pulse amplitude of 0.0025mV, interval time of 0.50ms, pulse time of
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0.25ms, and step size of 0.002mV [28].
2.7 Sample preparation an

Approximately 100 samples of oyster and mussels were used in this study. Thirty (30)
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samples were harvested and used during the months of April, June, and August. Prior to acid
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31 digestion, the samples were placed in a crucible and were dehydrated with a use of a hot plate.
32 Dehydration of the samples was necessary to avoid stains inside the furnace that may be caused
33 by the foam produced from the samples. All samples were subjected to white ashing with the use
34 of a furnace for at least 20hrs at a temperature of 600˚C. After ashing the samples, 10ml of nitric
35 acid were pipetted to the crucible and was left inside a fume hood for a minimum of 12 hours. The
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37 crucibles containing the same samples were transferred in a weighed beaker. The beaker was
38 heated up until all nitric acid have evaporated. The weight of the ash produced was determined
39 after the beaker was cooled. Approximately 1 to 1.5g of ash was produced for every batch of
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2.8 Real sampling
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45 To conduct real sampling analysis, 0.5844g of NaCl was added to the residue from the
46 acid digestion and was dissolved in 100ml deionized water. Solutions were sonicated for atleast
47 one and a half hours (1.5hr) and were swirled every 30 minutes for the homogenization of the
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sample. The sonicated solution was then analyzed with DPV using the optimum parameters.
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51 2.9 Atomic Absorption Spectroscopy
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53 Atomic absorption spectroscopy (AAS) was performed for validation of the results
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for DPV. A calibration curve was obtained for every metal.
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3 Results and Discussion
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6 3.1 Characterization of the fabricated electrodes
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8 3.1.1 SEM and EDX
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The morphology and elemental composition of the fabricated electrodes were analyzed by
12 scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX),
13 respectively. Scanning electron micrographs of the modified electrode surface revealed the
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15 presence of graphene in the form of flakes and [Ru (bpy)3] in the form of globules on the
16 graphene surfaces (Fig. 3.1). The presence of the modifiers on the electrode surface was also
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17 confirmed by EDX analysis (Fig. 3.2). The high wt% of carbon can be attributed to graphene and
18 the glassy carbon electrode. Fluorine is present due to Nafion and the other elements (Si, Al, Fe,
19 and Ca) are the impurities of the glassy carbon electrode.
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43 2+
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graphene (b) 2 mg [Ru (bpy)3] and 3 mg graphene, (c) 4 mg [Ru (bpy)3] and 3 mg graphene,
47 and (d) magnified image of (c).
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16 Fig. 3.2 EDX analysis of the fabricated electrode modified with 4 mg [Ru (bpy)3]2+ and 3 mg.
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21 3.1.2 Cyclic Voltammetry
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Figure 3.3 shows the voltammograms produced by the fabricated electrodes. The
voltammograms showed an anodic current peak at 1.0 V which is the reduction potential of [Ru
(bpy)3] . It can be observed that the highest peak was obtained with the combination of 4mg [Ru
2+
(bpy)3] and 3mg graphene.
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44 Figure 3.3 Cyclic voltammograms of the fabricated electrodes with different amounts of [Ru
45 (bpy)3]2+ and graphene.
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47 The reversibility of the fabricated electrodes was obtained by dividing the anodic peak
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current by the cathodic peak current. The most reversible electrodes are those with quotients
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Figure 3.4 Reversibility of the fabricated electrodes.
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16 Multiple scans were used to determine the stability of each modified electrode. Stability
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17 was taken from the relative standard deviation (RSD) from the average of the multiple scans. The
18 most stable electrodes are those with RSD values closest to zero. Figure 3.5 shows that the
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20 electrode with 4 mg [Ru (bpy)3] and 2 mg graphene was the most stable since it has the lowest
21 %RSD. A low %RSD indicates that the multiple scans of the electrode are relatively consistent
22 with each other which signifies better stability of the modified electrode and allows for more
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to time.
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precise and more reproducible results. A high %RSD indicates loss of [Ru (bpy)3] with respect
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41 Figure 3.5 Stability of different modified electrodes.
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43 3.2 Differential Pulse Voltammetry
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3.2.1 Determination of the best modified electrode
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48 To determine the best electrode, the fabricated electrodes were used in DPV to detect
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49 10ppm each of Cd2+ and Pb2+, simultaneously, and sequentially for Cu2+. The anodic current
50 peaked at potentials between -0.65 V to -0.75 V, -0.40 V to -0.50 V, and -0.10V to 0.00V, which
51 are the reduction potentials of cadmium, lead, and copper, respectively, as seen in Fig. 3.6. The
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histogram of the anodic peak current produced by the different fabricated electrodes is shown in
Figure 3.7. It can be seen that the peaks produced by the Cu2+, Pb2+ and Cd2+ increased linearly
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55 as the amounts of [Ru (bpy)3] and graphene increased. This indicates that increasing the
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amounts of the modifiers enhances the sensitivity of the electrode. This increase in the sensitivity
58 of the modified electrode can be attributed to the combined benefits of graphene and
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4 [Ru(bpy)3] . The high surface area-to-volume ratio which is a unique property of graphene
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5 increased the number of active sites within the electrode resulting to an increase in the amount of
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7 metal ions deposited on the electrode’s surface while [Ru(bpy)3] enhanced the conductivity of
8 the electrode by accelerating the electron transfer [23]. The histogram also shows that the
9 highest anodic peak currents for lead, cadmium and copper were obtained from the electrode
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11 with 4mg [Ru(bpy)3] and 3mg graphene since it has the highest amounts of [Ru(bpy)3] and
12 graphene. Thus, the combination of 4mg [Ru(bpy)3]2+and 3mg graphene was chosen as the best
13 concentration since the anodic current of Pb2+, Cd2+ and Cu2+ increased linearly for this said
14 combination. It was also observed that for a higher amount of graphene, the solution became
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more viscous making it hard to drop coat on the surface of the electrode. In addition, it can be
seen in some voltammograms for copper detection, that there were stripping peaks produced at
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18 0.1 V to 0.2 V. These peaks are caused by the oxidation of Cu2+ [29].
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19 Figure 3.7 Anodic peak currents of the fabricated electrodes for lead cadmium and copper via
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3.2.2 Optimization of the recipe for Differential Pulse Voltammetry
The accumulation or preconcentration potential, accumulation time, and deposition or

equilibrating or quiet time were optimized to achieve the highest possible anodic current of the
best electrode. The optimization of the accumulation potential was done simultaneously for Cd2+
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30 and Pb2+ and separately for Cu2+ with a concentration of 10ppm. The accumulation time and
31 deposition time were both held constant at 30 s. It can be seen in Fig 3.8 that the highest peak
32 current was obtained at the accumulation potential of -1.0V. Higher potential causes the
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35 which leads to a lower surface activity of the electrode [30].
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15 Figure 3.8 Peak currents of different accumulation potential for (a) lead and cadmium; (b) for
16 copper.
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19 For the accumulation time, the accumulation potential was held at -1.0 V and the
20 deposition time was held at 30 s. As seen in Fig. 3.10, the highest peak current for Pb2+ and Cd2+
21 was at 150 s. This accumulation time was also chosen for Cu2+ since the peak current starts to
22 decrease at 180s.
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Figure 3.10 Peak currents for different accumulation time for (a) lead and cadmium; (b) for
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56 For the deposition time, the accumulation potential was held at -1.0 V and the
57 accumulation time was held at 30 s. As seen in Fig. 3.11, the peak increases as the deposition
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time increases for Cd2+, Pb2+ and Cu2+. The deposition time of 30 s was chosen for time
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32 Figure 3.11 Peak current for different deposition time for (a) lead and cadmium; (b) for copper.
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36 3.3 Calibration Curve
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38 3.3.1 Differential Pulse Voltammetry
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41 The calibration curves for Pb2+, Cd2+ and Cu2+ were obtained from the voltammograms.
42 Simultaneous scans were done for Pb2+ and Cd2+ and sequentially for Cu2+. For Pb2+, the
43 concentration range starts at 48 ppb to 745 ppb and for Cd2+, the concentration range starts from
44 49 ppb to 612 ppb and 28 ppb to 472 ppb for Cu2+. The calibration curves are shown in Figs.
45 3.12, 3.13, and 3.14 for Pb2+, Cd2+ and Cu2+, respectively. All the graphs showed a linear
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47 relationship which indicates that as the concentration of the metals increase, so does the peak
48 current. The calibration curve for Pb2+, Cd2+, and Cu2+ produced a correlation value of 0.9811,
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Figure 3.12 (a) Voltammograms and (b) calibration curves for lead.
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Figure 3.13 (a) Voltammograms and (b) calibration curves for cadmium.
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31 Figure 3.14 (a) Voltammograms and (b) calibration curves for copper.
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33 3.4 Calculation for the best electrode’s sensitivity
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36 3.4.1 Limit of detection (LOD)
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38 The limits of detection of the best electrode for Pb2+, Cd2+ and Cu2+ were determined to find the
39 lowest heavy metal concentration that it can detect in a stock solution. The theoretical LOD is
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40 the lowest concentration that can be detected but not necessarily quantified. The limit of
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42 quantitation (LOQ) is the lowest concentration of the analyte that can still be determined with
43 acceptable results [31]. Table 3.1 shows the limit of quantitation and theoretical LOD of the
44 electrode for every metal.
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46 Table 3.1 Limit of quantitation, theoretical LOD and the experimental LOD.
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Heavy LOQ Theoretical Experimental
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49 Metal (ppb) LOD (ppb) LOD (ppb)

50 Lead 129.7480 42.8169 48.4185
51 Cadmium 155.1459 51.1982 49.0312
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53 Copper 145.0597 47.8697 28.3385
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2
3
Table 3.2 Performance comparison of the modified electrode with previous works.
4
pt
5 Electrode Modifier Method Detection Limit Reference
3+
6 ITO [Ru(NH3)6] /Nafion ASV Pb and Cd -500 ppb [19]
7 ITO [Ru(bpy)3]2+/Nafion ASV Cu – 100 ppb [26]
8 GCE 3+
AuNP /[Ru(NH3)6] /Nafion ASV Pb –45 ppb [24]
9
Cd –200 ppb
cri
10
11 GCE chitosan/carbon nanotubes SWASV Pb -600 ppb [33]
12 Cd – 800 ppb
13 Cu – 100 ppb
14 2+
GCE [Ru(bpy)3] /Graphene/Nafion DPV Pb -48 ppb This work
15
Cd -49 ppb
16
Cu – 28 ppb
us
17
18
19
20 3.4.2 Sensitivity of the electrode
21
22
Sensitivity is the response of an instrument to changes in an analyte solution [32].
23
24
25
26
27
28
an
Calibration sensitivity was performed to determine on which heavy metal the electrode is the
most sensitive. The calibration sensitivity of the best electrode was determined by comparing the
slope of the calibration curve for each heavy metal. The higher the slope, the more sensitive the
electrode is. Table 3.3 shows the slopes of the calibration curves for lead, cadmium and copper.
The analytical sensitivity of the electrode for every metal is shown in Table 3.4.
dM
29
30
31 Table 3.3 Slope of the calibration curve for every metal.
32 Heavy Metal Slope(mA/ppb)
33 Lead 0.0268
34
35 Cadmium 0.0097
36 Copper 0.0408
37
38 Table 3.4 Analytical sensitivity of the three metals
39 Concentration slope (mA/ppb) Standard Analytical
pte
40 of Lead and Deviation (mA) Sensitivity

41 Cadmium (1/ppb)
42 (pbb) Pb Cd Pb Cd Pb Cd
43
44 80 0.0268 0.0097 0.0315 0.0245 0.8498 0.3067
45
150 0.0268 0.0097 0.0200 0.0345 1.3384 0.4831
46
200 0.0268 0.0097 0.0700 0.0430 0.3824 0.1380
47
450 0.0268 0.0097 0.0400 0.0400 0.6692 0.2415
48
ce
49 600 0.0268 0.0097 0.4000 0.0150 0.0669 0.0242

50 800 0.0268 0.0097 0.2000 0.0465 0.1338 0.0483
51 1000 0.0268 0.0097 0.2000 0.0320 0.1338 0.0483
52
53 Concentration slope Standard Analytical
of Copper (mA/ppb) Deviation Sensitivity
Ac
54
55 (pbb) (mA) (1/ppb)
56 80 0.0408 0.0100 4.0783
57 150 0.0408 0.0770 0.5296
58
59
60 14
1
2
3 250 0.0408 0.0315 1.2947
4
pt
400 0.0408 0.0950 0.4293
5
6 500 0.0408 0.4050 0.1007
7 700 0.0408 0.2600 0.1569
8 1000 0.0408 0.0962 0.4237
9
cri
10
11 3.5 Real Sampling
12
13
The real samples and the sea water where the mussels and oysters were obtained were
14
15 analyzed using DPV and AAS. Both methods were able to detect lead, cadmium, and copper in
16 some samples. Also, DPV was able to detect other metals such as mercury, zinc, and tin in some
samples of the oysters. For the sea water, metals such as lead, cadmium, tin, iron, zinc, Cu2+,
us
17
18 Cu1+ and mercury were detected. The voltammograms for the real samples are shown in
19 Figs.3.15-3.17. Table 3.4 shows the comparison of the results from AAS and DPV.
20
21
22
23
24
25 100.00
an
Sea water
Cu1
Hg
μA )
26 Cu2
Cell Current (μ
50.00 +
27 Sn2 +
28 Pb2 +
0.00 Cd2 +
Fe2
dM
29
30 -50.00 Zn2 + +
31 -100.00
32 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
33 Cell potential (V)
34
35 Fig. 3.15 Voltammogram for Sea Water (Manila Bay).
36
37
38 Mussels (April 2016)
39
70.00
pte
40
μA)
41 50.00
Cell Current (μ
Cu2+
42
43 30.00
44 Pb2+
10.00 Cd2+
45
46
-10.00
47
48 -1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40
ce
49 Cell Potential (V)

50
51
Fig. 3.16 Voltammogram for Mussels (April 2016).
52
53
Ac
54
55
56
57
58
59
60 15
1
2
3
4 Oyster (April 2016)
pt
5
6 Cu2+ Cu1+ Hg
60.00
μ A)
7
Cell Current (μ
45.00
8 30.00 Pb2+
9 15.00 Cd2+
0.00 Zn2+
cri
10
11 -15.00
-30.00
12
13 -1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40
14 Cell Potential (V)
15
16 Fig. 3.17 Voltammogram for Oysters (April 2016).
us
17
18
19
20
21 Table 3.4 Comparison of DPV and AAS results.
22 Lead (ppb)
23
24
25
26
27
28
Real Samples
Mussels (April 2016)

Mussels (June 2016)
Mussels (August 2016)
Oysters (April 2016)
DPV
an
111.8294
164.6177
N/A
154.1572
AAS
N/A
347.1765
N/A
N/A
dM
29 Oysters (June 2016) 201.3416 166.7318
30 Oysters (August 2016) N/A N/A
31 Sea Water 415.1473 379.6439
32
Cadmium (ppb)
33 Real Samples
34 DPV AAS
35 Mussels (April 2016) 296.2467 657.9601
36 Mussels (June 2016) 567.1160 674.0306
37 Mussels (August 2016) N/A N/A
38 Oysters (April 2016) 603.1353 656.4992
39 Oysters (June 2016) 158.1727 397.6279
pte
40
Oysters (August 2016) N/A N/A
41
42 Sea Water 1715.2831 1035.6068
43 Copper (ppb)
Real Samples
44 DPV AAS
45 Mussels (April 2016) 110.6627 367.5046
46 Mussels (June 2016) 630.2744 1528.0456
47 Mussels (August 2016) 914.6574 1086.4605
48
ce
Oysters (April 2016) 134.5117 N/A

49
50 Oysters (June 2016) 237.4689 385.3191
51 Oysters (August 2016) 197.6851 375.1958
52 Sea Water 369.1302 602.1352
53
Ac
54
55 According to WHO, the allowable concentration of lead and cadmium in the human body
56
are 50ppb and 5ppb, respectively. In comparison with our DPV results, all samples, except those
57
58 procured on August 2016, exceeded the limit for consumption of lead and cadmium. On the
59
60 16
1
2
3
other hand, the limit of consumption for copper is 1.3 ppm which none of the samples exceeded.
4
pt
5
6 4. Conclusions
7
8
9 Tris(2,2’-bipyridyl) ruthenium (II)/Graphene/Nafion solution was successfully deposited
cri
10
11
on the surface of glassy carbon electrodes. The modified electrodes were characterized using
12 cyclic voltammetry and differential pulse voltammetry to obtain the best concentrations of
13 [Ru(bpy)3]2+and grapheme that would yield the best modified electrodes in terms of their figures
14 of merit. The electrode with 4 mg [Ru(bpy)3]2+and 3mg graphene was chosen to be the best
15 electrode and was used in obtaining the calibration curve for the detection of Pb2+, Cd2+ and
16
Cu2+. The electrode produced calibration curves with high linearity from 48 ppb to 745 ppb for
us
17
18 Pb2+, 49 ppb to 618 ppb for Cd2+, and 28ppb to 472 ppb for Cu2+. The limits of detection of the
19 best electrode for lead, cadmium, and copper were 48ppb, 49ppb, and 28ppb, respectively. The
20 electrode was successful in detecting heavy metals in the real samples which were sea water,
21 mussels and oysters. The presence of lead, cadmium and copper was detected in almost all
22 samples using differential pulse voltammetry (DPV). The results were validated using atomic
23
24
25
26
27
28
Acknowledgement
an
absorption spectroscopy (AAS). Other metals such as zinc, tin, mercury, and iron were also
detected in DPV.
dM
29
This work was supported in part by the University Research Coordination Office (URCO) of De
30
31 La Salle University, Manila, Philippines.
32
33 References:
34
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2.7 Sample preparation an

The accumulation or preconcentration potential, accumulation time, and deposition or

49 0.9769 and 0.9823, respectively.

49 Metal (ppb) LOD (ppb) LOD (ppb)

40 of Lead and Deviation (mA) Sensitivity

49 600 0.0268 0.0097 0.4000 0.0150 0.0669 0.0242

49 Cell Potential (V)

Mussels (April 2016)

Oysters (April 2016) 134.5117 N/A

49 Biological Sciences. 20. 353-355

New Materials for Electrochemical Systems, 19(4), 223-228.

54 [24] Palisoc S. Causing N., Natividad M. (2017). Gold

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