Research ArticleReduced Graphene Oxide/Gold Nanoparticles Modified Screen-Printed Electrode for the Determination of Palmitic Acid

Chin Boon Ching,1 Jaafar Abdullah ,1,2 and Nor Azah Yusof1,2

1Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia2Institute of Advanced Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Jaafar Abdullah; jafar@upm.edu.my

Received 9 October 2020; Revised 12 December 2020; Accepted 1 April 2021; Published 14 April 2021

Academic Editor: Domenico Caputo

Copyright © 2021 Chin Boon Ching et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Palm oil is one of the major oils and fats produced in the world today. The quality of palm oil is crucial to be investigated, and one ofthe quality indices is free fatty acid (FFA) content. Therefore, in this study, an electrochemical approach for the determination ofFFA has been explored as an alternative to replace the conventional method (titration method). The electrochemical method wasdeveloped based on electrochemically reduced graphene oxide (rGO) coupled with gold nanoparticles (AuNPs) deposited onto ascreen-printed carbon electrode (SPCE) via drop-casting technique. The voltammetric behaviour of 2-methyl-1,4-naphthoquinone (VK3) in the presence of palmitic acid at the modified electrode was investigated in an acetonitrile/watermixture containing lithium perchlorate (LiClO4). The electrochemical detection of palmitic acid was based on the voltammetricreduction of VK3 to form the corresponding hydroquinone which is proportional to the concentration of palmitic acid. Underoptimum conditions, the developed method showed a good linear relationship towards palmitic acid in the concentrationranging from 0.192mM to 0.833mM with the detection limit of 0.015mM. The exploration of the developed system is expectedto achieve high sensitivity and excellent selectivity towards the determination of FFA content in palm oil.

1. Introduction

The palm oil (Elaeis guineensis) originated in the tropical rainforest of Western Equatorial Africa and leads to an astonish-ing increase of plantation size throughout Southeast Asia [1].19.52 million tonnes of crude palm oil have been produced inMalaysia, making Malaysia the world second-largest pro-ducer of palm oil in the world [2]. Determination of free fattyacids (FFA) in palm oil is the main issue focused by manyresearchers nowadays because FFA content will affect thequality and marketing of palm oil products. The commonfactors which may cause an increase in FFA content aredamaged palm fruits during harvesting and poor storagemethods. Besides, crude palm oil itself naturally releases freefatty acids and the reaction can be increased by the microbiallipase enzyme. High FFA content will lead to many problemssuch as low product yield, unpleasant flavour, and complica-tions in sample processing procedures which subsequentlyinfluence the commercial value of palm oil in industry. Stan-

dard determination for the FFA content (as palmitic acid)has been set by the Malaysian Palm Oil Board (MPOB),which its composition in crude palm oil (CPO) and refinedbleached deodorized oil (RBDO) should be less than 5%and 0.1%, respectively [3].

Graphene, a two-dimensional material with a planarsheet of sp2-bonded carbon atoms in a hexagonal structure,has widely been utilized in many sensor fields due to itsunique and interesting properties, such as high surface area,good chemical stability, high mechanical strength, and excel-lent conductivity [4]. After undergoing an oxidation processto produce graphene oxide (GO), oxygen functional groupssuch as hydroxyl, epoxy, and carbonyl functional groupsattach on the GO surface [5]. The presence of these func-tional groups on the graphene framework aids in interactingwith an analyte such as acid for an effective detection process.However, the initially perfect π-conjugated system in graph-ite will be disrupted by these oxygen functional groups whichthen breaks the long-conjugated network of the graphitic

HindawiJournal of SensorsVolume 2021, Article ID 6684770, 14 pageshttps://doi.org/10.1155/2021/6684770

lattice, resulting in the decrease of electron transfer mobilityand electron scattering, thus lowers the conductivity of gra-phene oxide [6]. Some of the oxygen functional groups needto be removed to overcome the problem caused by the oxida-tion process. In this study, electrochemical reduction hasbeen proposed to reduce GO, because this method is simple,nontoxic, time-saving, less expensive, and green in naturecompared to other traditional methods such as thermalreduction and chemical reduction [7]. The deposited GOon SPCE was electrochemically reduced to reduced grapheneoxide (rGO) by cyclic voltammetry (CV) to eliminate oxygenfunctionality and hence restore the electrical properties ofgraphene [8]. In addition, the development of nanocompos-ite materials based on the integration of graphene with somesemiconductor or conductor nanomaterials such as goldnanoparticles was studied to further improve the sensitivityof the electrochemical sensors [9]. The electrocatalytic activ-ity of gold is proved to be further enhanced when supportedon graphene or its derivatives, owing to the synergic effectbetween these two components. In addition, gold nanoparti-cles (AuNPs) have shown magnificent properties such asgreat surface area, small dimensional size, and good elec-tronic properties, suitable to be used in sensor applications[10].

On the other hand, quinone-hydroquinone redox cou-ples are unique compounds and are studied intensely byresearchers due to the electrochemical behaviour of quinone,associated with its electron-proton transfer equilibrium andkinetics [11]. In detail, the electrochemical behaviour of qui-nones is sensitive to the presence of hydrogen ions which arethen involved in the proton transfer reaction. Protonsreleased from acid are accepted easily by quinone to fromhydroquinone and subsequently generate a peak which hasa good correlation with the concentration of acid; hence,the determination of the concentration of palmitic acidscan be determined [12]. In neutral aprotic solvents such asacetonitrile (AN), semiquinone (Q⋅−) and quinone dianion(Q2−) can be produced from quinones (Q) after two succes-sive one-electron reduction steps, generating two distinctcathodic peaks. The first electron transfer step, Q to Q⋅−,exhibits a clearly defined reversible voltammetric signal,whereas the second electron transfer, Q to Q2-, is generallyrelated to quasireversible signal. The behaviour of the secondelectron transfer is reported to be more sensitive to the inter-action between the materials deposited and the surface of theelectrode depending on the concentration of proton donorsand cationic species capable of ion pairing. When the acidis added, hydroquinone QH2 is formed and subsequently apeak formation will be observed. In short, the sequence ofthe reaction of quinones can be explained as two-electrontransfer coupled with two-proton acceptance process, suit-able to be used in the determination of acid [13].

Usually, the traditional way for the determination of freefatty acids in palm oil is through the acid-base titrationmethod by titration sample against potassium hydroxide inhot 2-propanol solution, and phenolphthalein is used as anindicator [14]. Although this method is direct and easy, it isencountered with some problems such as time-consuming,labour-intensive, and lack of accuracy [15]. Moreover, during

the neutralization process, some samples contain colouredsubstances, mainly carotenoids, which causes difficulty todetect a subtle colour change of the indicator in the transitionrange, thus leading to the inaccurate result [16]. In this study,an electrochemical method based on screen-printed carbonelectrode (SPCE) modified reduced graphene oxide coupledwith gold nanoparticles for the determination of FFA contentin palm oil has been proposed. The principle of the determi-nation is based on the electrochemical reduction of VK3 in anacetonitrile/water mixture containing lithium perchlorate(LiClO4) corresponding to concentrations of FFA.

2. Experimental

2.1. Chemicals and Instruments.All chemicals were of analyt-ical grade. Graphene oxide was purchased from GOAdvanced Solutions Sdn. Bhd (Malaysia). Gold chloroauricacid salt (HAuCl4·4H2O), potassium ferrocyanide (K3Fe(CN)6), potassium chloride (KCl), 2-methyl-1,4-naphtho-quinone (VK3), lithium perchlorate (LiClO4), phenolphtha-lein, and phosphate buffer saline solution with a pH of 7.4(PBS) were purchased from Sigma-Aldrich (USA). Palmiticacid (C16H32O2), 98%, was purchased from Acros Organics(USA). Potassium hydroxide (KOH), 2-propanol, and aceto-nitrile were purchased from R&M Chemicals (United King-dom). Deionized water (18.2 mΩ · cm at 25°C, Milli-Q) wasused throughout the experiments. The screen-printed carbonelectrode (SPCE) consists of carbon-based working andcounter electrodes, and a silver/silver chloride (Ag/AgCl) ref-erence electrode was purchased from Rapid Labs Sdn Bhd(Malaysia).

Electrochemical measurements were done using anAutolab Instrument Model Autolab Type III (Eco ChemieB. V., Netherlands). Cyclic voltammetry (CV), linear sweepvoltammetry (LSV), and electrochemical impedance spec-troscopy (EIS) data analyses were carried out using Nova1.11 software. Fourier transform infrared (FTIR) spectrawere analysed using a Thermo Nicolet 6700 FT-IR spectrom-eter (USA) in the range of 4000 to 400 cm−1. Field EmissionScanning Electron Microscopy (FESEM) coupled withEnergy Dispersive X-ray (EDX) images was obtained usinga Nova Nanosem 230, FEI (USA) instrument, and Ramanspectra were obtained using aWitec instrument model Alpha300R (Germany).

2.2. Preparation of Stock Solution

2.2.1. Preparation of Working Solution. For the preparationof working solution, 2-methyl-1,4-naphthoquinone (VK3)was dissolved in water/acetonitrile mixture solvent contain-ing lithium perchlorate (LiClO4). For VK3 concentration,the range of 2mM to 6mM of VK3 was prepared. For LiClO4concentration, the range of 1.5M to 4.0M of LiClO4 was pre-pared. Both VK3 and LiClO4 were dissolved in mixture sol-vent of water and acetonitrile. For the ratios of the mixturesolvent, 3 ratios were prepared, which were 1 : 1, 1 : 3, and3 : 1 of water to acetonitrile ratios. The optimization studywas carried out using cyclic voltammetry (CV).

2 Journal of Sensors

2.2.2. Preparation of Palmitic Acid Solution. 5mM of stocksolution was prepared by dissolving 12.8mg palmitic acidin 10mL acetonitrile. Different concentrations of palmiticacid ranged from 0.192mM to 0.833mM were studied byspiking a known amount of palmitic acid solution into theworking solution prepared above. The performance studyof the electrode towards palmitic acid was carried out usinglinear sweep voltammetry (LSV).

2.2.3. Preparation of Cooking Oil Stock Samples. Samplepreparation for correlation study was carried out based onthe reported methods [17], with slight alteration. Cookingoil (Buruh, Lam Soon Edible Oils Sdn. Bhd.) was used as areal sample in this study. A stock oil solution of 0.5 g/mLwas prepared by dissolving 5 g of cooking oil in 10mL 2-propanol. This solution was sonicated for 10 minutes untilhomogenous mixture was obtained. This sample was thenused to prepare stock solution and stock palmitic acidsolutions.

2.2.4. Preparation of Stock Palmitic Acid Solution. 0.1 g/mLstock solution of palmitic acid was prepared by dissolving0.5 g palmitic acid in 5mL of the stock oil solution as pre-pared above. The solution was sonicated for 10 minutes atroom temperature until homogeneous solution was obtained.

For real sample analysis, the prepared stock oil solutionwas spiked with palmitic acid to the desired concentrationsin the range from 1 a.d. to 40 a.d.

2.3. Preparation of Reduced Graphene Oxide/GoldNanoparticle-Modified SPCE. 2.5mg/mL graphene oxide(GO) solution was diluted using 0.1M phosphate buffer solu-tion (pH7.4) and ultrasonicated for 2 hours to obtain ahomogeneous of 1.0mg/mL GO suspension. After that,5μL of the as-prepared solution was drop cast on the surfaceof SPCE and it was allowed to dry at room temperature. Sub-sequently, SPCE was electrochemically reduced using cyclicvoltammetry (CV) in the potential range of 0V to -1.5V witha scan rate of 100mV/s in a 0.1M KCl solution for 15 cycles.The rGO-modified SPCE was then washed carefully with dis-tilled water and dried at room temperature. After the rGO-modified SPCE was dried, 10μL of 1.5mM gold nanoparti-cles was drop cast on the surface of SPCE and it was let todry overnight.

The prepared rGO/AuNP-modified SPCE was character-ized using FESEM and EDX for morphological and elementalanalysis. FTIR was used to analyse the functional groups pres-ent on graphene oxide, reduced graphene oxide, and reducedgraphene oxide/gold nanoparticle composites. Raman spec-troscopy was used to reveal the detailed structural informationof graphene materials.

2.4. Electrochemical Study of Reduced Graphene Oxide/GoldNanoparticle-Modified SPCE. The electrochemical behaviourof rGO/AuNP-modified SPCE was investigated by cyclicvoltammetry (CV) in 0.005M K3Fe(CN)6/0.1M KCl solu-tion. Optimization parameter for the fabrication process ofthe modified electrode such as rGO concentration, rGO vol-ume, and AuNP concentration was also studied.

Scan rate study of the modified electrodes was performedby CV in 0.005M K3Fe(CN)6/0.1M KCl solution by varyingthe scan rate from 10mV/s to 100mV/s. Electrochemicalimpedance spectroscopy (EIS) of the modified electrodeswas carried out using CV with a frequency range from100 kHz to 0.1Hz and applied potential of 225mV with ACvoltage amplitude of 0.01V.

2.5. Analytical Performance of the Reduced GrapheneOxide/Gold Nanoparticle-Modified SPCE towards WorkingSolution. The electrochemical behaviour of rGO andrGO/AuNP-modified SPCE towards 2-methyl-1,4-naphtho-quinone (VK3) in acetonitrile/water mixture containing lith-ium perchlorate was evaluated using cyclic voltammetry(CV). In order to obtain the maximum response of the mod-ified electrode, the optimization of VK3 concentrations,LiClO4 concentrations, and the ratios of the solvent mixturewas studied.

Investigation of rGO and rGO/AuNP-modified SPCEtowards palmitic acid detection using the optimized workingsolution was also evaluated using linear sweep voltammetry(LSV) scanning from 1.5V to -1.5V at a scan rate of50mV/s. Different concentrations of palmitic acid including0.192mM, 0.370mM, 0.536mM, 0.690mM, and 0.833mMwere tested. The current response of rGO and rGO/AuNP-modified SPCE was obtained by injected different volumesof palmitic acid into the prepared working solution.

2.6. Applicability of the Reduced Graphene Oxide/GoldNanoparticle-Modified SPCE towards Spiked Real SampleAnalysis. Investigation of rGO/AuNP-modified SPCEtowards real sample spiked with known concentration of pal-mitic acid of 1 a.d., 10 a.d., 20 a.d., 30 a.d., and 40 a.d. wasevaluated using linear sweep voltammetry (LSV) at the scanrange of 1.5V to -1.5V with a scan rate of 50mV/s.

2.7. Validation of the Developed Method with the StandardTitrimetric Method Using Spiked Real Sample. In order toevaluate the potential application of the developed systemin real sample analysis, a validation study of the developedmethod towards the standard method (MPOB standard titri-metric method) for analysis of free fatty acids (palmitic acid)was investigated. Different palmitic acid concentrations of5 a.d., 15 a.d., and 35 a.d. were used.

For the standard method, the mixture of cooking oil(1 g), palmitic acid with known concentration (10mL), 2-propanol (50mL), and phenolphthalein (0.5mL) as indica-tor was put into an Erlenmeyer flask. The flask was thenplaced on a hot plate, and the temperature was adjusted toabout 40°C. The flask consisting of the mixture was shakengently and continuously titrated against standard KOH(0.1M) until the first permanent pink colour appears. Thefirst permanent pink colour change must persist for 30 s.The volume of KOH needed to neutralise the acid wasrecorded. The concentration of palmitic acid (% FFA) canbe calculated from

%FFA Palmitic acidð Þ = v − bð Þ ×N × 25:6w

, ð1Þ

3Journal of Sensors

where v is the volume in mL of titration solution, b is thevolume in mL of the blank, N is the normality of the stan-dard KOH solution, and w is the weight of the sample ofoil in grams.

3. Results and Discussion

3.1. Characterization of the Modified SPCE. Raman spectros-copy is a nondestructive spectroscopic technique that isimmensely used to provide the detailed structural informa-tion of carbon materials, particularly graphene materials[18]. From Figure 1(a), Raman spectra of the GO, rGO, andrGO/AuNP samples exhibit two obvious signals with theirpeaks located at approximately 1320–1360 cm−1 and 1570–1600 cm-1, which corresponds to the D and G bands, respec-tively. All Raman spectra depicted the same pattern of thecurve, which implies that the structure of graphene was notaffected when the reduction process of GO to rGO and theaddition of AuNPs onto rGO sheets take place. The ratiobetween the D and G bands (ID/IG) can be calculated andused to estimate the extent of structural disorder [19]. ID/IGintensity for rGO ð1:16Þ > GO (1.04), indicating GO is suc-cessfully reduced to rGO. In another word, a decrease in theaverage size of in-plane sp2 domains due to the removal ofthe oxygen functional groups in the rGO has occurred,proving the successful reduction which restores the sp2

domains of graphene [20]. Meanwhile, for the Raman spec-trum of rGO/AuNP nanocomposite material, the calculatedID/IG intensity is 1.06, which is lower than rGO after thebinding with AuNPs. The decrease in the ID/IG intensitywas because of the aggregation of AuNPs on the rGOmatrix and subsequently caused an increase in the numberof graphitic domains. The interaction of rGO and AuNPsaffects the lattice constant and electronic properties of thematerials [21].

FTIR was used to detect the functional groups and char-acterize the molecule structure change of the materials of themodified electrode. From Figure 1(b), FTIR spectra of GOshowed absorption peaks at 1061 cm-1, 1628 cm-1, and3250 cm-1 which are attributed to C-O-C, C=C, and O-Hstretching vibrations, respectively [22]. After the reductionof GO, the C-O-C and O-H stretching vibrations of GOdisappeared, proving that the reduction of GO to rGOwas successful. Ring strains present in C-O-C bonding con-figurations of GO are more susceptible to ring-openingreactions, which lead to sp2 carbon (C=C) upon the reduc-tion process. In addition, a strong C=C stretching peak at1612 cm-1 and –CH3 bending peak at 1348 cm-1 wereobserved in FTIR spectra of rGO, suggesting the restorationof the carbon basal plane in rGO after reduction. Moreover,FTIR spectra for rGO/AuNPs depicted the characteristicbands of citrate at 3366, 1589, and 1391 cm-1. The presenceof the signal at 3366 cm-1 was indicative of the stretchingvibration of the –OH group, and the band at 1589 cm-1

was assigned to the C=O stretching of carboxylate ions ofcitrate, which implied that the hydroxyl group and the car-boxyl group contained in the sodium citrate formed stronghydrogen bonds with the residual oxygen functionalities onthe rGO surfaces. Moreover, the peak at 1391 cm-1 indi-cated the intensity of C–OH deformation vibration, whichlikely arose from surface-bound sodium citrate. In short,the results showed that the sodium citrate moieties areattached to the surface of rGO and act as a capping agentwhich stabilizes the rGO [23].

The surface morphology of GO, rGO, and rGO/AuNP-modified SPCE was characterized using FESEM with 100Kmagnification. As shown in Figures 2(a) and 2(b), it wasnoted that both GO and rGO film were successfully depos-ited onto the electrode surface with a typical crumpled andwrinkled multilayer sheet structure. A larger surface area

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Figure 1: (a) Raman spectra of GO, rGO, and rGO/AuNP-modified SPCE and (b) FTIR spectra of GO, rGO, and rGO/AuNP-modifiedSPCE.

4 Journal of Sensors

can be provided for the interaction with the analytes due tothese wrinkled and edge nanostructures of rGO sheets [24].Moreover, the presence of carbon (C) and oxygen (O) in bothGO and rGO films could also be proven by the EDX image.In GO, it consisted of 36.3% of C element, 32.1% of O ele-ment, and other trace elements which involved during thepreparation process of GO involving the use of PBS solution.In rGO, it consisted of 89.3% of C element and 8.8% of O ele-

ment. Thus, the signal of O is significantly decreased in rGOfrom 32.1% to 8.8%, indicating that GO is successfullyreduced to rGO. This is because mainly the oxygen groupssuch as epoxy and hydroxyl groups present in GO areremoved after the electrochemical reduction occurs. How-ever, the reduction process cannot remove all oxygen func-tional groups because these functional groups left behindwill be involved in the reaction with other elements such as

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5Journal of Sensors

AuNPs, as verified by the presence of 8.8% of O element inrGO from EDX spectra.

Based on Figure 2(c), a large amount of AuNPs is denselyand uniformly dispersed on the surface of rGO film with littlecoagulation. EDX spectrum of the rGO-AuNP nanocompos-ites also showed the presence of all C, O, and Au signals, con-sisting of 65.8% C element, 4.7% O elements, and 29.5% Auelement. These results distinctly confirmed the formation ofrGO/AuNP nanocomposites on SPCE. The oxygen func-tional groups left behind in rGO could interact with AuNPsalong the graphene surface through electrostatic interaction,forming rGO/AuNP nanocomposite materials [25]. There-fore, based on the proposed method, rGO/AuNP nanocom-posite was successfully prepared and it is proved to be areliable preparation method.

3.2. Electrochemical Characterization of the Modified SPCE

3.2.1. Electrochemical Study of the Modified SPCE. The elec-trochemical behaviours of bare GO, AuNPs, rGO, andrGO/AuNP-modified SPCE were characterized using cyclicvoltammetry (CV) in 0.005MK3[Fe (CN6)] containing0.1M KCl at a scan rate of 100mV/s in the potential rangeof -0.6V to 0.8V. Figure 3 depicts the voltammogram of bare,GO, AuNPs, rGO, and rGO/AuNP-modified SPCE, respec-tively. Bare SPCE gave a clearly defined reversible electro-chemical response. After modification with GO, a decreasein peak current has occurred, due to the poor conductivityof the GO which behaved as an electrical insulator becauseof the presence of polar and negatively charged functionalgroups such as –COOH, –COOR on the rGO surface. Thesenegatively charged functional groups can ionize in solutioneasily and subsequently generate a repulsive force over thenegatively charged [Fe (CN)6]

3− ions, which result in the dif-ficulty for the electron transfer between the surface of theelectrode and the electrolyte (ions) to occur [26].

In addition, the rGO-modified SPCE had the second-highest peak current with a smaller peak-to-peak separation(ΔEp) compared with bare and GO-modified electrodes,indicating that the excellent conductivity and great surfacearea of rGO could help to speed up the whole electrochemicalprocess. This is because of the removal of the negative chargefunctional groups in the GO sheet after the reduction pro-cess, leading to the absence of repulsive force which acceler-ates the electron transfer between the electrode surface andthe redox species. This phenomenon was also attributed tothe significant improvement of the electrical conductivity ofthe rGO film, presumably owing to the restoration of a gra-phitic network of sp2 bonds [26].

After the addition of AuNPs into the rGO sheet to formrGO/AuNP composites, the resulting modified SPCE washigher than the rGO-modified SPCE. The increase in thepeak current of rGO/AuNP-modified SPCE confirmed thecontribution from the nanocomposites. AuNP-modifiedSPCE only showed the third in conductivity among all elec-trodes. AuNPs need to be added and combined with rGOsheets to enhance the current response because AuNPs actas an electron transfer channel, which can further improvethe conductivity of the rGO film. As a consequence, thecharge transfer process between the modified layer and elec-trolyte (ions) was accelerated and occurred more efficiently[27]. On top of that, the electrochemical behaviour of thecomposite-modified electrode improved highest comparedwith a single ingredient modified electrode (rGO-modifiedSPCE or AuNP-modified SPCE) due to the synergic effectof both graphene and gold nanoparticles which showed agood electrical conductivity and large electroactive surfacearea [28]. It can be summarized that the redox signal of themodified electrode follows the order of rGO/AuNPs> rGO>AuNPs>bare>GO.

For the scan rate study of different electrodes, fromFigures 4(a)–4(c), the anodic peak shifted positively whilethe cathodic peak shifted negatively, generating slightlylarger ΔEp with increasing scan rates from 10mV/s to100mV/s. The linear graph of the anodic and cathodic peakcurrent (Ip) versus the square root of the scan rate (v1/2) wasplotted for all modified electrodes, and good linearity wasobtained, suggesting that all undergo diffusion-controlledprocess [29]. The kinetics of a diffusion-controlled systemis defined as the diffusion of ferrocyanide ions from the solu-tion into the interface of the electrode due to a concentrationgradient when ions are involved in redox reaction. By usingRandles-Sevcik equation:

Ipeak = 2:69 × 105� �

n2/3ACD1/2V1/2, ð2Þ

where Ipeak refers to the anodic peak current, n is the totalnumber of transferring electrons in redox reaction, A is themicroscopic surface area of the electrode, D is the diffusioncoefficient of ferrocyanate, and C is the concentration ofK3Fe(CN)6. Assuming n = 1 and D = 7:6 × 10−6 cm2s−1, theeffective surface areas for bare SPCE, rGO-modified SPCE,and rGO/AuNP-modified SPCE were 0.061 cm2, 0.127 cm2,and 0.130 cm2, respectively. The results indicate that rGO

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Figure 3: Cyclic voltammograms of different types of SPCE in 0.1MKCl solution containing 5.0mM [Fe (CN)6]

3−/4− solution at a scanrate of 100mV/s.

6 Journal of Sensors

and AuNPs were successfully deposited on the surface ofelectrode and subsequently increased the active surface areaof the electrode by approximately two times enhancement

compared to bare SPCE because both materials gave goodelectrical properties. Furthermore, the active surface areascalculated were proportional to the current response

y = 151.08x + 24.62R2 = 0.965

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Figure 4: (a) Cyclic voltammogram of (a) bare, (b) rGO, and (c) rGO/AuNP-modified SPCE in 0.1M KCl solution containing 5.0mM [Fe(CN)6]

3−/4− solution. The scan rate was varied from 100mV/s to 10mV/s (a to j). Inset is the linear graph of peak current (Ip/μA) against thesquare root of scan rate ½v1/2/ðV/sÞ1/2�.

7Journal of Sensors

obtained from CV measurements, which means the redoxsignal is expected to be higher when the larger surface areais ready to interact with the analytes.

Electrochemical impedance spectroscopy (EIS) is an elec-trochemical technique which is sensitive to the surface of theelectrode and used to investigate the interfacial charge trans-fer kinetics of the electrodes. A frequency range from100 kHz to 0.1Hz was used for the EIS measurement. Theapplied potential was set at 225mV with AC voltage ampli-tude of 0.01V. The calculated Rct values of the modified elec-trodes showed that the rate of charge transfer of therGO/AuNP-modified SPCE (Rct = 650Ω) is the fastest,followed by rGO-modified SPCE (Rct = 699Ω), bare SPCE(Rct = 5:84 kΩ), and the slowest at the GO-modified SPCE(Rct = 8:00 kΩ). As can be seen from Figure 5, when GOwas applied onto the electrode surface, the semicircleincreased significantly compared to the bare SPCE. GO actedas an insulating layer which increases the resistance of SPCEbecause of polar and negatively charged oxygen functionalgroups repelling the access of [Fe (CN)6]

3-/4- ions to the elec-trode surface which then make the interfacial charge transfertough between the electrode surface and ferrocyanide ions.After GO film was electrochemically reduced, the semicircledisappeared and gave almost straight lines, indicating thatrGO had facilitated the electron transfer between the electro-active species in the electrolyte and the electrode surfaceowing to the restoration of a graphitic network of sp2 bondswhich improved the conductivity of rGO. For rGO/AuNP-modified SPCE, at high frequencies, there was no semicircleobserved and the plot showed a more inclined straight linecompared with rGO, which meant that the internal resistanceof rGO/AuNP composites was smaller due to the good con-ductivity of AuNPs. AuNPs played a major role to ease andenhance the electron transfer rate at the solution and elec-trode interface and thus increased the overall conductivity.The outcomes from EIS experiments are consistent with theresults obtained from CV measurements.

3.3. Analytical Performance of rGO/AuNP-Modified SPCEtowards Palmitic Acid. In this study, 2-methyl-1,4-naphtho-quinone (Vitamin K3, VK3) was selected as a quinone reagentdue to its stability and solubility in acetonitrile solvent andacted as a redox couple where the reduced species of quinonewill interact with hydrogen ions to yield hydroquinone as theproduct. Lithium perchlorate (LiClO4) played a role as thesupporting electrolyte to decrease the electrochemical cellresistance. The working solution was prepared by dissolvingVK3 and LiClO4 in an acetonitrile/water mixture.

The presence of water in aprotic solvent is a very impor-tant factor that should be focused on because water repre-sents hydrogen-bond donor and acceptor. Thus, the use ofwater needed to be reduced to a minimal amount, yet themixture (acetonitrile and water) must be able to dissolve alltarget chemical species especially large amount of LiClO4which is difficult to dissolve in acetonitrile. Excess water mol-ecules may affect the detection of palmitic acid as water mol-ecules contain hydrogen ions that can interact with thereduced species of quinone in the system. Besides, water alsoplayed a role in providing a polar condition to dissolve theproton sources coming from the acids added; hence, the pro-tons would be able to move freely and react with dianion. Theexperiments were all performed under optimum conditionswhich the ratios of acetonitrile/water mixture, VK3 concen-tration, and LiClO4 concentration were 1 : 1, 4mM, and3.0M, respectively.

Under optimum conditions, the performance of thedeveloped sensor was first studied using bare electrode. How-ever, from Figure 6(a), only one reduction peak was obtainedat -0.958V which was due to reduction of quinone (Q) intosemiquinone radical anion (Q⋅−). The generation of the sec-ond reduction peak which was due to the reduction ofsemiquinone radical anion into dianion (Q2−) was unsuc-cessful. This is because the conductivity of bare electrodewas not strong enough to perform the second reductionprocess of semiquinone radical anion. The performance of

0

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6000

8000

10000

12000

0 5000 10000 15000 20000 25000

–Z″

(Ω)

Z′ (Ω)

BareGO

rGOrGO/AuNPs

Figure 5: Nyquist plots of bare SPCE, GO, rGO, and rGO/AuNP-modified SPCE in 0.1M KCl solution containing 5.0mM [Fe (CN)6]3−/4−

with frequency 100 kHz and amplitude 0.01 V.

8 Journal of Sensors

bare electrode towards palmitic acid was carried out. Theexperiment was done by injecting the palmitic acid withconcentration of 0.536mM into the working solution inthe scanning range from 1.5V to -1.5V at a scan rate of50mV/s using linear sweep voltammetry (LSV). FromFigure 6(b), no peak was obtained because the absence ofdianion owing to the fact that only dianion will interactwith hydrogen ion from acids to form hydroquinone.Moreover, the comparison between rGO-modified SPCEand rGO/AuNP-modified SPCE towards palmitic acid atconcentration of 0.536mM was also conducted. As shownin Figure 6(b), rGO/AuNP-modified SPCE showed a betterresponse for the detection of palmitic acid compared torGO-modified SPCE with a much higher reduction peakcurrent due to the presence of AuNPs which acceleratedthe electron transfer and further improved the electrochem-ical performance.

In detail, by using rGO/AuNP-modified SPCE as shownin Figure 7(a), the first reduction peak was obtained at thepotential of approximately -0.897V due to the formation ofelectrochemically generated semiquinone radical anion fromquinone (E1) and the second reduction peak was obtainedapproximately at potential of -1.251V due to the formationof dianion from semiquinone radical anion (E2) [30]. Bothions generated were stable and could be successfully detectedby cyclic voltammetry where two distinct reduction peakswere observed. Active charged species such as quinones (Q,Q⋅−, and Q2-) could dissolve and move freely in the solutiondue to the high polarity of acetonitrile and ready for theinteraction with hydrogen ions released from acid. Electro-chemical reduction of quinones was carried out followingtwo consecutive one-electron transfer steps. Compared tosemiquinone radical anion, the electrochemical behaviourof dianion is more sensitive to hydrogen ions and can easilyform hydrogen bonding interaction and proton transfer.Thus, in the presence of acids, dianion is ready to interactwith hydrogen ions or protons from the palmitic acid to formhydroquinone and subsequently gave a reduction peak. The

height of the reduction peak obtained was proportional tothe concentration of acids added; hence, it could be utilizedto detect the palmitic acid content in palm oil [31]. The pos-sible mechanism of reduction of quinone in the solution issummarized as follows:

Q+e⇌Q⋅−

Q⋅− + e⇌Q2−

Q2− + 2H+ ⇌QH2

Q + 2H+2e⇌QH2

ð3Þ

The performance of the developed sensor was then stud-ied with different concentrations of palmitic acid including0.192mM, 0.370mM, 0.536mM, 0.690mM, and 0.833mM,respectively. The same procedure and measurement withthe experiment using bare electrode were used. It can beobserved from Figure 7(b) that the reduction peak currentresponses for both modified electrodes increase proportion-ally to increase concentration of palmitic acid. This observa-tion could be due to the higher concentration of palmiticacid, which could release more hydrogen ions, and ease theformation of hydrogen bonding, hence increasing the currentresponse [32]. Moreover, the LSV voltammogram showedthat the reduction potential shifted slightly towards a nega-tive potential when the concentration of palmitic acidincreased. This observation indicated that when quinonewas electrochemically reduced to form Q2-, it was ready tointeract with hydrogen ions from the acid source to formhydroquinone and the pH near the electrode surfaceincreases sharply in solution after the hydrogen ions wereconsumed [33]. When this happened, the reduction potentialwould decrease rapidly and hence a new peak located atslightly negative potential was formed [34].

From the calibration curve obtained for rGO/AuNP-modified SPCE as shown in Figure 7(c), it can be noted thatas the concentration of palmitic acid increased, the net

–300–250–200–150–100

–500

50100150200

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ent (𝜇

A)

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(a)

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BarerGOrGO/AuNPs

(b)

Figure 6: (a) Cyclic voltammogram of VK3 (4mM) in acetonitrile/water mixture in the presence of LiClO4 (3mM) using bare SPCE; (b) LSVvoltammograms of bare SPCE, rGO, and rGO/AuNP-modified SPCE in acetonitrile/water mixture in the presence of VK3, LiClO4, andpalmitic acid (0.536mM).

9Journal of Sensors

current produced also increased. A linear relationship wasestablished with a regression coefficient of y = 166:24x +285:62, R2 = 0:993. The limit of detection (LOD) is calculatedto be 0.015mM based on

LOD = 3σS, ð4Þ

where σ is the standard deviation of the blank and S is theslope of the calibration curve. Thus, rGO/AuNP-modifiedSPCE showed an excellent response in the detection of pal-mitic acid with low limit of detection and high sensitivitydue to the presence of rGO and AuNPs which aided theelectron transfer and further improved the electrochemicalperformance. The LOD of the developed method was alsocompared with previously reported work done by Yusofet al. [35] for the determination of palmitic acid using tita-nia nanotube electrode, and the LOD obtained was1.006mM. Thus, rGO/AuNP-modified SPCE had a better

performance towards the detection of palmitic acid with alower detection limit.

To evaluate the reproducibility study using rGO/AuNP-modified SPCE, five modified electrodes were fabricated bythe same procedure and tested in the presence of 0.536mMpalmitic acid using LSV. The current response also remainedalmost constant, and the obtained relative standard deviation(RSD) value was 0.98% as shown in Figure 8(a). Moreover,the repeatability was also evaluated under the same proce-dure and the RSD value of 1.67% was obtained as shown inFigure 8(b). The obtained results suggested that the proposedmethod for both modified electrodes considerably minimizedthe sensor-to-sensor deviation and excellent fabricationreproducibility was achieved.

3.4. Applicability of rGO/AuNP-Modified SPCE towardsSpiked Real Samples. To examine the applicability of the pro-posed sensor on real sample analysis, the experiment wasconducted by spiking a known concentration of stock palmi-tic acid solution into the stock oil solution (cooking oil). The

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E2E1

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–1.2 –1.1 –1 –0.9 –0.8 –0.7 –0.6 –0.5

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ent (𝜇

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0.192 mM0.370 mM0.536 mM

0.690 mM0.833 mM

(b)

y = 166.24x + 285.62R2 = 0.993

050

100150200250300350400450

0 0.2 0.4 0.6 0.8 1

Net

curr

ent (𝜇

A)

Concentration of palmitic acid (mM)

(c)

Figure 7: (a) Cyclic voltammogram of 4.0mM VK3 in acetonitrile: water (1 : 1, v/v) in the presence of 3.0M LiClO4 using rGO/AuNP-modified SPCE; (b) LSV voltammograms of rGO/AuNP-modified SPCE in 4.0mM VK3 consisting of 3.0M LiClO4 with variousconcentrations of palmitic acid: (i) 0.192mM, (ii) 0.370mM, (iii) 0.536mM, (iv) 0.690mM, and (v) 0.833mM; and (c) calibration curve ofLSV response of palmitic acid with a concentration range of 0.192-0.833mM scanning in a positive direction from 1.5V to -1.5V at ascan rate of 50mV/s, ðn = 3Þ.

10 Journal of Sensors

viscosity of oil may have a slight influence on the electricalconductivity. Theoretically, a decrease in viscosity of oil willresult in an increase of mobility of polar molecules and sub-sequently increase the electrical conductivity [36]. However,

in this work, small volume of cooking oil was used; thus,the effect is not significant.

The performance of rGO/AuNP-modified SPCE towardsvarious concentrations of palmitic acid of 1 a.d. (aciditydegree), 10 a.d., 20 a.d., 30 a.d., and 40 a.d. spiked in the stockoil solution was evaluated using the LSV technique.Figure 9(a) illustrates similar observation as previous results(without using the real sample) has been obtained, wherethe reduction peak current increased linearly with theincreasing concentration of palmitic acid. A calibration curvewas then plotted as shown in Figure 9(b), and a linear regres-sion coefficient of 1:48x + 15:95, R2 = 0:997 for the concen-tration ranging from 1 to 40 a.d. was obtained.

3.5. Detection of Palmitic Acid with the Standard TitrationMethod. Figure 10 depicts a linear calibration curve of thestandard titration method towards palmitic acid concentra-tions of 1 a.d., 10 a.d., 20 a.d., 30 a.d., and 40 a.d. spiked inthe stock oil solution. A linear relationship between the per-centage of free fatty acids (%FFA) and the acidity degree

200220240260280300320340360380400

1 2 3 4 5

Net

curr

ent (𝜇

A)

Reproducibility/number of measurement

(a)

200220240260280300320340360380400

1 2 3 4 5

Net

curr

ent (𝜇

A)

Repeatability/number of measurement

(b)

Figure 8: (a) Reproducibility study and (b) repeatability study of rGO/AuNP-modified SPCE in the presence of 0.536mM palmitic acid inacetonitrile: water (1 : 1, v/v) consisting of 4.0mM VK3 and 3.0M LiClO4, scanning from 1.5V to -1.5V at a scan rate of 50mV/s, ðn = 3Þ.

–100

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1 a.d.10 a.d.20 a.d.

30 a.d.40 a.d.

(a)

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Figure 9: (a) LSV voltammograms of rGO/AuNP-modified SPCE in 4.0mM VK3 consisting of 3.0M LiClO4 with various concentrations ofpalmitic acid: (i) 1 a.d., (ii) 10 a.d., (iii) 20 a.d., (iv) 30 a.d., and (v) 40 a.d.; (b) the calibration curve of LSV response of palmitic acid spiked incooking oil in the concentration range of 1-40 a.d. and scanning in a positive direction from 1.5V to -1.5 V at a scan rate of 50mV/s, ðn = 3Þ.

y = 0.15x + 1.11R2 = 0.979

012345678

0 10 20 30 40 50

% F

FA (p

alm

itic a

cid)

Acidity degree of oil (a.d.)

Figure 10: Calibration curve of percentage value of palmitic acid bythe standard titration method, ðn = 3Þ.

11Journal of Sensors

(a.d.) of palmitic acid with a linear regression coefficient of0:15x + 1:11, R2 = 0:979 was obtained. The volume of KOHneeded to neutralize the acid was then converted into%FFA using Equation (1). To conclude, the higher the con-centration of palmitic acid, the higher the volume of KOHneeded to neutralize the acid, and the higher the percentageof free fatty acids were obtained.

From the developed method (Figure 9(b)), the currentresponse obtained from rGO/AuNP-modified SPCE wasplotted against acidity degree (a.d.). On the other hand, fromthe standard method (Figure 10), the %FFA was plottedagainst acidity degree. Since both of the graphs shared thesame unit which is acidity degree, the relationship of the cur-rent response from the developed method was then plottedagainst %FFA from the standard titration method as shownin Figure 11(a). From the calibration curve obtained, the lin-ear regression coefficient of 9:75x + 5:61, R2 = 0:985 wasobtained, indicating that both methods were correlated andcomparable.

Another three concentrations of palmitic acid that con-sisted of 5 a.d., 15 a.d., and 35 a.d. were performed for the val-idation study using both the developed method and thestandard method. A validation curve was plotted by convert-ing the current response obtained from LSV into the percent-age of FFA using the linear equation from Figure 11(a).Figure 11(b) presents the results of the validation study (bothx- and y-axis with the same unit which is %FFA) obtained

from the analysis of palmitic acid performed using the devel-oped method by comparing to the result obtained with thestandard titration method for concentration of 5 a.d, 15 a.d.,and 35 a.d. A good linear correlation was found betweenthese two methods that the fit to the graph had a slope of1.14 and R2 = 0:998 which indicates good agreement andcomparable.

In addition, statistical analysis was also carried out bycomparing the two means of the developed sensor and thestandard titration method as shown in Table 1. The calcu-lated t-values were found to be less than the tabulated value;hence, the difference between the two methods used is insig-nificant at the 95% confidence level and the null hypothesis isaccepted. The palmitic acid concentrations measured by therGO/AuNP-modified SPCE sensor were in good agreementand comparable with results from the standard titrationmethod. This highly precise result proved that the proposedsensor can be implemented in the analysis of palmitic acidcontent in real samples.

4. Conclusion

An easy, prompt, and sensitive electrochemical detection ofpalmitic acid was designed using rGO/AuNP-modified SPCEthat was successfully fabricated and characterized in thisstudy. The rGO/AuNP-modified SPCE displayed a betterperformance, which was attributed to the excellent conduc-tivity and large active surface area of both reduced grapheneoxide and gold nanoparticles. The developed sensor showed alinear calibration curve towards palmitic acid in the range of0.192-0.833mM with the detection limit of 0.015mM. More-over, rGO/AuNP-modified SPCE also displayed good repeat-ability and reproducibility with RSD of 1.67% and 0.98%,respectively. In addition, the applicability of the developedsensor when tested with spiked real samples was provenand validated with the standard titration method. Cost-effec-tive, green in nature, and coherent methods are among theremarkable features of this designed sensor compared tothe traditional methods; hence, it exhibits good potentialand could be further developed as an alternative way forthe detection of palmitic acid in the future.

y = 9.75x + 5.61R2 = 0.985

0

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Figure 11: (a) Comparison study between the developed method (electrochemical method) with the standard method (titration method); (b)validation curve of the developed method (electrochemical method) with the standard method (titration method), ðn = 3Þ.

Table 1: Comparison of real sample analysis between the developedmethods (electrochemical method) with the standard method(titration method).

Concentration(a.d.)

The standardmethod (% FFA)

(n = 3)

The developedmethod (% FFA)

(n = 3)Calculatedt-test value

5 2:35 ± 0:02 1:80 ± 0:17 2.74

15 3:63 ± 0:03 3:47 ± 0:04 3.04

35 6:04 ± 0:04 6:05 ± 0:13 0.11

Note: t3 = 3:18 (p = 0:05).

12 Journal of Sensors

Data Availability

The data used to support the findings of this study areincluded within the article.

Conflicts of Interest

The authors declare that there is no conflict of interests.

Acknowledgments

The authors would like to thank the Ministry of Higher Edu-cation, Institute of Advanced Technology, Universiti PutraMalaysia, and Department of Chemistry, Faculty of Science,University Putra Malaysia, for all facilities and funds pro-vided (GP-IPS/2018/9652900).

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