amperometric glucose biosensor based on pt-pd nanoparticles supported by reduced graphene oxide and...

DOI: 10.1002/elan.201400018

Amperometric Glucose Biosensor Based on Pt-PdNanoparticles Supported by Reduced Graphene Oxide andIntegrated with Glucose OxidaseM. F. Hossain[a] and Jae Y. Park*[a]

1 Introduction

Diabetes mellitus is a worldwide public health problem,resulting from insulin deficiency and hypoglycemia, whichare characterized by blood glucose concentrations thatare higher or lower than the normal range of 4.4–6.6 mM[1]. The diagnosis and management of diabetes mellitusthus requires an accurate and precise method for moni-toring the blood glucose level. Though glucose biosensorshave been already commercialized for decades, basic rele-vant research is still active [2–5]. Amperometric electro-des based on GOx in particular, which feature simplicity,reliability, high selectivity, and low cost, are widely usedfor the detection of blood glucose concentration [6–8].The glucose level is indirectly monitored by measurementof the current associated with the electrochemical detec-tion of hydrogen peroxide produced during the enzymaticreaction in which glucose is oxidized by oxygen with theaid of GOx.

Bimetallic nanoparticles (NPs) have recently receivedmuch attention as electrocatalysts with enhanced activi-ties and electrochemical reversibility for redox reactions[9–11]. These bimetallic alloys can retain the functionalproperties of each component and possibly offer synergis-tic effects via cooperative interactions, resulting in impor-tant features such as increased surface area, enhancedelectrocatalytic activity, improved biocompatibility, pro-moted electron transfer, and better robustness against in-termediate species. In addition, metal nanoparticles couldeven provide electrochemical reversibility for redox reac-tions, which is not possible with bulk metals [12]. Pt andPd are the most frequently used electrocatalysts for the

oxygen reduction reaction (ORR), with a similar mecha-nism to that of the 4e- reduction of oxygen to water. Pd isamong the most electrocatalytic metals for ORR besidesPt [13]. As palladium is 50 times more abundant on earthand cheaper than Pt, Pd-based catalysts are considered tobe excellent substitutes for Pt in ORR [12, 14]. The addi-tion of Pt to Pd promotes the overall catalytic activity, se-lectivity, and stability of Pd. A simple approach was de-veloped to electrochemically synthesize bimetallic nano-particles (PtPdNPs) on reduced graphene oxide for ex-ploring the application of Pt–Pd alloy nanoparticles tobiosensor preparation. Electrodeposition is the most con-trollable and robust technique for the synthesis of metalNPs, in which the size, density, composition of alloys, andeven the shape of NPs can be well controlled by the elec-trodeposition potential, charge, time, concentration, andcomposition of metal precursor solutions [14].

Pristine graphene obtained from mechanical cleavage islimited by the low-yield production and lack of controlla-bility in film quality, while graphene grown by chemicalvapor deposition (CVD) is compromised by high cost.Graphene oxide (GO), a water-soluble colloidal suspen-sion obtained from the chemical exfoliation of graphite,has become a promising alternative. GO can be chemical-ly or thermally reduced to conductive reduced graphene

Abstract : A novel glucose biosensor was developed basedon the immobilization of glucose oxidase (GOx) on re-duced graphene oxide incorporated with electrochemical-ly deposited platinum and palladium nanoparticles(PtPdNPs). Reduced graphene oxide (RGO) was morehybridized by chemical and heat treatment. Bimetallicnanoparticles were deposited electrochemically on theRGO surface for potential application of the Pd�Pt alloyin biosensor preparation. The as-prepared hybrid elec-trode exhibited high electrocatalytic activities towardH2O2, with a wide linear response range from 0.5 to

8 mM (R2 =0.997) and high sensitivity of 814 �10�6 A/mMcm2. Furthermore, glucose oxidase with active materi-al was integrated by a simple casting method on theRGO/PdPtNPs surface. The as-prepared biosensorshowed good amperometric response to glucose in thelinear range from 2 mM to 12 mM, with a sensitivity of24� 10�6 A/mMcm2, a low detection limit of 0.001 mM,and a short response time (5 s). Moreover, the effect ofinterference materials, reproducibility and the stability ofthe sensor were also investigated.

Keywords: Biosensor · Glucose · Hydrogen peroxide · Pt-Pd nanoparticles · Reduced graphene oxide

[a] M. F. Hossain, J. Y. ParkDepartment of Electronic Engineering, Micro/Nano Devices& Packaging Lab., Kwangwoon University447–1, Wolgye-Dong, Nowon Gu, Seoul, 139-701, Koreatel: +82-2-940-5113; fax: +82-2-942-1502;*e-mail: [email protected]

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 940 – 951 940

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oxide. Compared to pristine graphene or CVD-growngraphene, reduce graphene oxide (RGO) thin films haveoffered competitive results as channel materials in variouselectronic biosensors [15,16]. RGO is a flat monolayer ofgraphite packed into a two-dimensional (2D) honeycomblattice that holds great potential for various biosensor ap-plications. This material has a unique ability to promotefast electron transfer kinetics for a wide range of electro-active species when it is employed as an electrode sub-strate [17,18]. For example, an RGO electrode rich in hy-droxyl and carboxylic groups showed promoted electroca-talytic activity towards the oxidation of paracetamol [19].Kim et al. [20] developed a sensitive sensor for the selec-tive determination of dopamine without the interferenceof ascorbic acid based on an RGO-modified electrode.Shan et al. [21] used graphene as an electrode materialfor low-potential NADH detection and biosensing forethanol. A reduced graphene-oxide-based electrochemi-cal glucose biosensor has also been constructed, demon-strating the potential for applying RGO to biosensors.

Hydrazine and hydrazine hydrate are widely used re-ducing agents for converting GO to RGO, but these re-ducing agents are toxic and produce a high sheet resist-ance in RGO, because of the nitrogen impurities incorpo-rated during the reduction process [22]. Another possibleway to reduce graphene oxide is using sodium borohy-dride (NaBH4) in aqueous solution. NaBH4 is more effec-tive than hydrazine as a reductant of graphene oxide, as itcan reduce the sheet resistance of RGO [23]. However, itcan be slowly hydrolyzed by water. This issue is overcomeby using ethylene glycol and a small amount of waterduring the reduction reaction, and by limiting the dippingtime. Ethylene glycol not only works as a chelating agent,but also serves as a reducing agent that widens the rangeof GO dispersion in solution [24]. In addition, for maxi-mum performance of RGO, it is important to control thedensity of oxygen-containing groups.

In this work, examination of the RGO modifiedPtPdNPs electrode was conducted for electrocatalytic ac-tivities towards H2O2. Glucose oxidase was immobilizedonto the RGO-modified PtPdNPs sensing-electrode sur-face, and was characterized using cyclic voltammogramsand amperometric techniques for glucose sensor applica-tions.

2 Experimental Sections

2.1 Chemicals and Apparatus

Palladium(II) chloride (PdCl2) (reagent plus grade), hexa-chloroplatinic acid (H2PtCl6) (ACS reagent grade) graph-ite powder (44-mm size), ascorbic acid (AA), uric acid(UA), acetaminophen (AP), b-d(+) glucose, bovineserum albumin (BSA), glucose oxidase (GOx) (Aspergil-lus niger, X-S type) and hydrogen peroxide (30 %) (ACSreagent) were purchased from Aldrich Co. (St. Louis,USA). All other chemicals used were of analytical grade.The b-d(+) glucose (99.5 %, Sigma) stock solution was

prepared by dilution, and H2O2 was mixed in a 50 mMPBS (pH 7.4) solution and all other solutions were pre-pared with deionized water (resistivity�18 MWcm). Allother electrochemical measurements of the fabricatedelectrodes were performed in a three-electrode systemusing an electrochemical analyzer (Model 600D series,CH Instruments Inc., USA). A flat Pt bar and an Ag/AgCl electrode with 3 mM NaCl were utilized as thecounter electrode (CE) and reference electrode (RE), re-spectively. Electrochemical impedance spectroscopy(EIS) was carried out in a frequency range from 0.1 Hz to100 kHz using the same electrochemical analyzer andthree-electrode configuration in a supporting electrolytesolution containing 5 mM [Fe(CN)6]

4�/3�. The physicalcharacteristics of the graphite oxide, RGO, and developedelectrode were investigated by high-resolution X-ray pho-toelectron spectroscopy (XPS), Fourier transform infra-red spectroscopy (FTIR), field emission scanning electronmicroscopy (FESEM), and energy-dispersive X-ray spec-troscopy (EDX). For surface morphological analysisunder FESEM, the samples (size 4 mm �5 mm) werebonded with conductive carbon tape on an aluminumstub using an acceleration voltage of 15 kV. For elementalanalysis under EDX, the samples were bonded with con-ductive carbon tape on an aluminum stub and 20 kV ofbeam voltage and acquisition time 300 s were conducted.FTIR measurements were carried out on a SPECTRUM100 Fourier-transform infrared spectrophotometer at a res-olution of 4 cm�1 with an accumulation of 100 scans foreach spectrum. The infrared spectra were recorded in ab-sorbance units in the 4000–400 cm�1 range by using KBrpellets containing 1 % finely powder samples. X-ray pho-toelectron spectroscopy (XPS) was used for characteriz-ing GO and RGO by using Al Ka radiation (1486.7 eV)with a power of 23.4 W and pass energy of 23.5 eV.

2.2 Synthesis of Graphite Oxide and RGO

Graphite oxide was prepared with the modified Hummersmethod [25]. Briefly, 2 g of graphite powder and 1.5 g ofNaNO3 were added to 150 mL of 98% H2SO4 solution ina flask immersed in an ice bath. With the stirring still inprogress, 9 g of KMnO4 was added slowly to the mixtureto prevent a sudden accumulation of the heat evolved.After that, the mixture was stirred for 6 days at roomtemperature. Then, 10 mL of 30 % H2O2 was added to thesolution in order to completely react with the remainingKMnO4, leading to a bright yellow solution. To purify thegraphene oxide, the resulting mixture was washed by 3 %H2SO4 and H2O2 until the pH value of the solution wasapproximately 5–6. Graphite oxide platelets were ob-tained after drying the suspension. Then, 50 mg of graph-ite oxide was added to 30 mL of ethylene glycol, followedby sonication for 2 hours (h). After that, 40 mL of waterwas added to the solution, followed by stirring for 1 h.Under stirring, 270 mg of sodium borohydride (NaBH4)was slowly added, and the mixture was heated at 110 8Cfor 2 h. After finishing the reduction reaction, the reac-


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tion mixture was centrifuged and washed five times withdoubly distilled water and then dried in an oven at 95 8Cunder vacuum overnight.

2.3 Preparation of RGO-Modified Gold Electrode

A Ti/Au thin film was sputtered on a Si/SiO2 substrate asa template layer for fabricating sensors. An RGO suspen-sion was cast onto the template layer and dried at roomtemperature, and then placed in an oven to control sur-face oxygen groups and to prevent moisture from beingincorporated into the RGO. The Au electrode greatly re-duces the contact resistance of the RGO and NPs throughthe substrate, and maintains a high background current.To form the proposed nanostructured electrode, a 70-nmtitanium (Ti) layer was sputtered on top of an Si/SiO2

substrate, with which it had good adhesion, as well asa diffusion barrier of gold (Au) and Si and a 200-nm goldlayer to offer low resistivity layer of the overall system.The Si/SiO2 substrate used for deposition was thoroughlycleaned with acetone for 5 min, then in methanol for5 min, and followed by repeated rinsing with deionizedwater. The substrate was then dried and baked in a hotplate. Thin films of Ti, and Au were deposited by usingDC sputtering system and Ti and Au targets with 99.99 %purity, respectively. The sputtering for Ti was carried outat 314.3 W DC power in Ar ambient at a pressure of3.5 mTorr and a fixed target-to-substrate separation of45 mm. Similarly, the sputtering for Au was performed at200 W DC power in Ar ambient at a pressure of 10mTorr and at the same separation between target andsubstrate.

20 mg of RGO powder was placed into a centrifugetube, and then 1 mL/mg of dimethylformamide (DMF)and doubly distilled water (1 : 1) was added. After that,the solution was ultrasonicated at 200 W for 3 h. Then,0.01 mL of the as-prepared RGO suspension was droppedonto the template electrode and dried under ambientconditions. After drying, the RGO-modified Au electrodewas rinsed with PBS solution and distilled water severaltimes, and dried by nitrogen gas. The electrode was thenplaced in a vacuum oven at 150 8C during 10 h to remove

oxygen functional groups and moisture as well as toobtain good adhesion on the Au surface.

2.4 Preparation of RGO/PtPdNPs-Modified GoldElectrode

A conventional three-electrode system was used with anAg/AgCl reference electrode, a platinum sheet auxiliaryelectrode, and the modified Au electrode as the workingelectrode. The PdPtNPs were formed on top of the RGOby electrodeposition under a constant potential of �0.2 Vin a deaerated precursor solution consisting of 2.5 mM ofPdCl2, H2PtCl6 (1 :1), and 100 mM KCl for 90 s and with45, 50 and 55 mC control charges. After that, these elec-trodes were rinsed by distilled water and dried by nitro-gen gas. In this work, we select 55 mC control charge dueto high catalytic activity of the nanoparticles.

2.5 Preparation of Au/RGO/PtPdNPs/GOx

GOx was prepared according to the following procedures.2 mg of GOx and 4 mg of BSA were dissolved in 0.2 mLof deoxygenated phosphate buffer solution (PBS)(50 mM, pH 7.4) and ultrasonicated for 5 min. After that,mixing solution was stirred gently for 5 min and 0.01 mLof glutaraldehyde (1.8 %) was added and stirred for1 min. Afterward, 0.01 mL of the mixture was cast ontothe surface of the Au/RGO/PtPdNPs, and then the sol-vent was allowed to dry at ambient condition. The Au/RGO/GOx, Au/RGO/PtNPs/GOx and Au/RGO/PdNPs/GOx were prepared in the same way. Both bioelectrodeswere stored in PBS with pH 7.4 at 4 8C in a refrigeratorwhen not in use. Figure 1 shows schematic diagrams ofthe RGO/PtPdNPs/GOx-modified Au electrode fabrica-tion process.

3 Results and Discussion

3.1 Characterization of RGO

Figure 2 shows the surface morphology and elementalanalysis of the as-prepared RGO according to FESEM

Fig. 1. Fabrication sequences of the proposed glucose biosensor.


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and EDX, respectively. Figure 2A shows the FESEMimage of RGO formed after 3 h of ultrasonication. Theimage shows that several layers of RGO sheet were castonto the Au seed layer, as well as the typical wrinkling ofthe RGO sheet formed during chemical reduction. Thegeometric wrinkling not only minimizes the surfaceenergy, but also provides mechanical integrity with a highYoung�s modulus and tensile strength, as well as goodfilm-forming ability due to nanoscale sheet interlocking[26]. The chemical reduction was presumed to cause theobserved corrugation and crumpling of GO, and thereforeprovided a good membrane-forming ability with largersurface coverage on the substrate, which is suitable forthe large-scale production of RGO [27,28]. All of theRGO became covered with PtPdNPs, making it rougherto a greater extent, with more electroactive sites andlarger surface area, as shown in Figure 2B. The PtPdNPswere well dispersed in the RGO network, forming an in-terpenetrating network for favorable conduction path-ways and electron transfer kinetics. Figure 2B showsa fairly smooth and fine dispersion of NPs that providesa good platform for biosensing. The surface roughness ofthe electrode allows for enzyme immobilization. These

characteristics indicate that the electrode is a suitableplatform for enzymatic sensors. BSA and GOx were casttogether onto the RGO/PtPdNPs surface, as shown inFigure 2C. In Figure 2C, two types of traces exist on thesurface. We believe that glucose oxidase was immobilizedon the surface by crosslinking with BSA [50] and main-tained long-term stability.

The elemental compositions of the RGO-modifiedPtPdNPs (55 mC controlled charge) were analyzed byEDX, as shown in Figure 2C. Signature peaks for C, O,Pt, and Pd were observed for the RGO/PtPdNPs. Theweight percentages of C, O, PtNPs, and PdNPs were61.24 % 15.19 %, 15.47 %, and 8.1 %, respectively. Theanalysis showed that a greater percentage of PtNPs com-pared to PdNPs was deposited due to the electrostatic in-teraction from the negatively charged RGO sheet. Theprobable reason is that the negatively charged PtCl6

2�

ions were easy to form, but the positively charged Pd2+

ions were not easy to form in the concentrated chloridemedia. These ions would then be reduced to form thePtPd nanoparticles [14]. This indicates that the PtPdNPscan be successfully synthesized under the given condi-

Fig. 2. FESEM images of (A) Au/RGO, (B) Au/RGO/PtPdNPs, (C) Au/RGO/PtPdNPs/GOx, and (D) corresponding EDX spectrumof Au/RGO/PtPdNPs.


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Fig. 3. FTIR spectra of (A) graphite oxide and RGO, and XPS spectra of (B) graphite oxide and (C) RGO.


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tions, and contribute equally towards the formation of themodified electrode for biosensing.

FTIR is used to obtain an infrared spectrum of the ab-sorption of a solid. Graphite oxide and RGO powderwere characterized by FTIR analysis. The spectra revealthe presence of various types of bonds in the graphiteoxide and RGO. Figure 3A shows the FTIR spectra ofgraphite oxide and RGO, respectively. The major peaksof graphite oxide were formed at 3410.0 cm�1,1709.4 cm�1, 1629.5 cm�1, 1382.2 cm�1, and 1080.6 cm�1.The peak at 3410.0 cm�1 arises from the �OH stretchingvibration, whereas the peak at 1709.4 cm�1 arises from C=O stretching vibration. The peak at 1629.5 cm�1 is attrib-uted to the O�H bending vibration of absorbed watermolecules and the contributions from the vibration of ar-omatic C=C. The peaks at 1382.2 cm�1 and 1080.6 cm�1

arise from the deformation vibration of �OH and the C�O stretching vibration of alkoxy groups [29,30]. Themajor peaks of RGO were formed at 3421.7 cm�1,1718.5cm�1, 1624.2 cm�1, 1577.7 cm�1, and 1419.9 cm�1.The peaks at 3421.7 cm�1 and 1718.5 cm�1 arise from the�OH stretching vibration and the C=O stretching vibra-tion, respectively. The peak at 1624.2 cm�1 is attributed tothe O�H bending vibration of absorbed water moleculesand the contributions from the vibration of aromatic C=C. The peak at 1419.9 cm�1 arises from the deformationvibration of �OH. The peak at 1577.7 cm�1 for the RGOis assigned to the C=C skeletal vibration of graphenesheets [31, 32], which confirms the successful reduction ofthe GO sheets.

Figures 3B and C show the C1s XPS spectra of graphiteoxide and RGO, respectively. The spectra clearly indicatea considerable degree of oxidation corresponding to

carbon atoms in different functional groups: the non-oxy-genated ring C (284.6 eV) that includes C=C bonds tomake sp2 hybridized carbon, the C in C�O bonds(286.6 eV) that include hydroxyl and epoxy groups, andthe C in C=O bonds (288.0 eV) that include carbonylgroups [33,34]. A new peak (285.7 eV) arises after the re-duction of GO, which is sp3 hybridized carbon and in-cludes C�C bonds [35]. The oxygen groups of GO werereduced substantially during the reduction reaction andheat treatment on substrate surface that successfullymade RGO.

3.2 Electrochemical Impedance Spectroscopy (EIS) ofDifferent Modified Electrodes

The electronic transfer properties of the different modi-fied electrodes were characterized by electrochemical im-pedance spectroscopy (EIS). In Particular, EIS spectracontain two portions i.e. semicircle at higher frequenciescorrespond to the electron transfer limited process andlinear line relatively at lower frequencies correspond tothe diffusion process. Figure 4 shows the EIS spectra ofthe bare and modified electrodes recorded in 50 mM PBS(pH 7.4) containing 5 mM [Fe(CN)6]

3�/4� fitted with theRandles equivalence circuit model. From Figure 4 it isseen that there are no well-defined semicircle in the de-sired frequency range which indicates that they possessgood electron transfer kinetics. The electron transfer re-sistance (Ret) of the bare Au electrode was higher thanthat of the Au/RGO (Figure 4 a and b), suggesting thatthe layer of RGO could form on the electrode surface,and enhance the electron transfer from the redox probeof [Fe(CN)6]

3�/4�, to the electrode surface. For the

Fig. 4. EIS of (a) bare Au, (b) Au/RGO, (c) Au/RGO/PtPdNPs and (d) Au/RGO/PtPdNPs/GOx recorded in 50 mM PBS (pH 7.4)containing 5 mM Fe(CN)6

3�/4�. Amplitude: 5 mV, frequency: 0.1 Hz to 100 kHz. Inset: Randles equivalence circuit used to fit the EISdata obtained at all the modified electrodes.


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PtPdNPs modified RGO on Au (Figure 4c), the Ret re-duced much more which reveals that nanoparticles haveimproved the conductivity and electron transfer rate. Butwhen BSA-GOx modified with nanoparticles containingelectrode, Ret value was increased, suggesting that causinga little bit resistance of the electron transfer of the redoxcouple shown in Figure 4d.

3.3 Electrochemical Performance of RGO-ModifiedPtPdNPs Electrode Towards H2O2

The CVs of a typical bare Au and RGO-modified Auelectrode in 50 mM PBS solution are presented in Fig-ure 5A. In Figure 5Aa, there is no anodic peak in the re-spective potential region, but there are two cathodicpeaks around 0.5 V and �0.2 V. There is a cathodic peakat 0.5 V, which has involved the reduction of monolayergold oxide, and another cathodic peak around �0.2 V dueto reduction of recalcitrant metal oxide which may beformed during anodic sweep [49]. In Figure 5Aa andb show that the RGO-modified Au substrate electrodeexhibited higher background current than bare Au sub-strate electrode, which was ascribed to the good electronpropagation within the Au/RGO electrode. Besides, theapparent surface area of the Au/RGO electrode is largerthan that of the bare Au electrode that reported previous-ly [13,36]. Figure 5B shows the CV of the 55 mC con-trolled charge NPs-modified RGO/Au electrode. Fig-ure 5Ba shows the PtNPs modified Au/RGO electrode,there is an anodic peak around �0.6 V, and two cathodicpeaks around 0.15 V and �0.8 V. For the anodic peak at�0.6 V, PtOH was produced due to the oxidation of Pt.There is a broad cathodic peak above 0.15 V, which mayhave involved the reduction of platinum oxide to Pt0, andanother cathodic peak around �0.8 V due to the hydro-gen (H) adsorption on the electrode surface [37]. Similar-ly, there is an anodic peak at around �0.4 V the onanodic curve of RGO/PdNPs-modified Au electrode inFigure 5Bb, corresponding with a single Pd(OH)2 mono-layer formation during the oxidation reaction in PBS. Thecurrent peaks associated with the reduction of palladiumoxide in the CV obtained from the catalyst are at about�0.04 V. The cathodic peak at �0.04 V is ascribed to thereduction palladium oxide and regeneration of catalyst[38,39]. Peaks at �0.48 V and �0.74 V occurred due tothe desorption of H on the surface of the RGO/PdNPs-modified electrode. In Figure 5Bc, the RGO/PtPdNPs-modified Au electrode displays broad and weak peaks,with anodic peaks lying at around �0.84 V, �0.6 V, and�0.35 V, and cathodic peaks at around 0.0 V, �0.5 V, and�0.8 V due to the contribution of the redox reaction ofPt and Pd nanoparticles. The oxidation peak at �0.6 Vmay be existed to Pt, which formed PtOH, while thepeak lying around �0.35 V corresponds to the formationof Pd(OH)2, which is clear from Figures 5Ba,b. The peakat �0.84 V on the anodic curve is contributed by desorp-tion of H on the RGO/PtPdNPs-modified surface. Thecathodic peak at �0.8 V might be occurred due to the ad-

sorption of H on the PtPd surface. The reduction peak at0.0 V was appeared due to the contribution of the oxidereduction of alloying elements. The peak at �0.5 V corre-sponds to only the Pd electrocatalyst, as shown in Fig-ure 5Bb. From Figure 5B, it is also clear that the currentdensity of the RGO/PtPdNPs-modified Au electrode ishigher than those of the RGO/PtNPs and RGO/PdNPsdue to the synergistic effect of the two elements. Thiseffect is probably due to the change of the geometricalligand (e.g. a decrease in the Pt–Pt bond distance) or anelectronic effect (e.g. an increase of Pt d-electron vacan-cies), so one of the elements alters the electronic proper-ties of the other to yield a more active catalytic surface[14]. It is also clearly seen that the onset potential of theRGO/PtPdNPs-modified Au electrode is 0.3 V, which in-dicates that the as-prepared material has good ORR ac-tivity. These results might be occurred due to the bimetal-lic nature of the Pt and Pd nanoparticles.

The CVs of a typical RGO/PtPdNPs-modified elec-trode in 50 mM PBS with and without 2 mM H2O2 solu-tion are presented in Figure 5C. Figure 5Ca shows the CVof the RGO/PtPdNPs-modified Au electrode without2 mM H2O2. The CV curve of the RGO/PtPdNPs-modi-fied Au electrode in 50 mM PBS with 2 mM H2O2 isshown in Figure 5Cb. These figures indicate that the oxi-dation of RGO/PtPdNPs in hydrogen peroxide at from�0.65 to 0.4 V is lower than that of RGO/PtPdNPs with-out hydrogen peroxide. In contrast, the reduction ofRGO/PtPdNPs from 0.2 to �0.65 V in hydrogen peroxideis greater than that of RGO/PtPdNPs without hydrogenperoxide. This result reveals that the RGO/PtPdNPs hasgood electrocatalytic properties in hydrogen peroxide so-lution by means of reduction.

The current responses measured under different con-centrations of H2O2 at a given potential are a significantfactor for evaluating an amperometric sensor, as shown inFigures 5D and E. Figures 5D and E show the ampero-metric measurement of Au/RGO at �0.2 V and +0.55 Vupon successive additions of H2O2 in PBS solution, andlinear curve is shown in the inset of Figure 5D. Compara-tively high linearity, sensitivity, and stability were ob-tained at �0.2 V. This may be happened for more reduc-tion of hydrogen peroxide at this potential by the catalyt-ic effect of this electrode. From Figure 5D, it is clear thatalong with the addition of H2O2, the reduction current in-creased linearly. The corresponding calibration plot isshown in the inset in this figure. Linear detection from0.5 mM to 8 mM and a response time of 7 s are also ob-served. Figure 5E shows that with the addition of H2O2 tothe PBS, the current was decreased and later saturated.This result implies that intermediate poison attacks thecatalytic surface and hinders the current through the elec-trode. Similar to the Au/RGO electrode, Figures 5F andG show the amperometric responses of Au/RGO/PtPdNPs at �0.2 V and +0.55 V upon successive addi-tions of H2O2 to the PBS solution and the inset calibra-tion curve. The response time of this sensor is 2 s, withlinear detection in the range of 0.5–8 mM, and a sensitivity


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of 814�10�6 A/mMcm2 at �0.2 V. A response time of 3 s,linear detection in the range of 0.5–6.5 mM, and a sensitiv-

ity of 486� 10�6 A/mMcm2 were found at 0.55 V. Hydro-gen peroxide is oxidized at 0.55 V and reduced at �0.2 V.

Fig. 5. Cyclic voltammograms (CVs) of (A) fabricated electrodes: (a) bare Au and (b) Au/RGO. (B) CVs of different modified elec-trodes: (a) Au/RGO/PtNPs, (b) Au/RGO/PdNPs, and (c) Au/RGO/PtPdNPs electrodes in 50 mM PBS (pH, 7. 4), scan rate: 50 mV/s.(C) CVs of (a) as-prepared Au/RGO/PtPdNPs electrodes in 50 mM PBS only and (b) in 50 mM PBS with 2 mM H2O2. Amperometricresponse of (D) and (E) for Au/RGO whereas (F) and (G) for Au/RGO/PtPdNPs electrodes in PBS (pH, 7.4, 50 mM) to the succes-sive injection of the concentration of H2O2 in 0.5 mM at �0.2 V and 0.55 V respectively, with inset calibration curve.


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So, the reduction of H2O2 at �0.2 V is stronger than oxi-dation of H2O2 at 0.55 V. In Figures 5 (D) through (G),the amperometric response of the RGO/PtPdNP-modi-fied electrode is higher than that of the RGO-modifiedelectrode. This result indicates that the nanoparticles ex-hibit strong electrocatalytic activity on the RGO-modi-fied electrode, which might be related to the higher spe-cific surface area of the deposited PtPdNPs compared tothe bulk material. The high surface area of the RGO pro-vided a large amount of anchoring sites for the depositionof PtPdNPs during the synthesis of the RGO/PtPdNPs byelectrodeposition and it might also be attributed to thesynergistic effect of RGO and the nanoparticles [2].

3.4 Electrocatalytic Performance of Au/RGO/PtPdNPs/GOx for Glucose Detection

The CVs of different RGO-modified electrodes in 50 mMPBS solution are presented in Figure 6A. Figures 6Aaand b show that the RGO/GOx-modified Au electrodedecreases the current compared to the RGO-modifiedelectrode. This may have happened when glucose oxidasewas deposited on the RGO, since glucose oxidase makesa barrier layer against electron transportation. Asa result, the current decreases after the deposition ofGOx. Figure 6Ba shows the CV of RGO/PtPdNPs-modi-fied electrode for comparison with that of the RGO/PtPdNPs/GOx-modified electrode in Figure 6Bb. In Fig-ures 6Ba and b, the redox peak current and backgroundcurrent of the RGO/PtPdNPs/GOx-modified electrodeare lower than those of the RGO/PtPdNPs-modified elec-trode. There is also no change in the CV curve after load-ing the GOx. The probable reason is that after loadingthe glucose oxidase and BSA on the surface of the elec-trode, a hydrophobic protein layer may be formed on thesurface, which insulates the conductive support and theinterfacial electron transfer [40]. Figures 6Bb and c showthat the background reduction current of RGO/PtPdNPs/

GOx from �0.05 to �0.6 V in glucose is smaller than thatof RGO/PtPdNPs/GOx without glucose, and the oxida-tion background current of RGO/PtPdNPs/GOx from�0.6 to 1.0 V in glucose is higher than that of RGO/PtPdNPs/GOx without glucose. The probable reason isthat the oxidation current is supposed to increase theamount of the adsorbed OH� on the Au/RGO/PtPdNPs/GOx electrode. Moreover, the PtPdNPs-modified elec-trode has good tolerance against poisoning by intermedi-ates by virtue of the novel metal particles used [41,42].This result reveals that RGO/PtPdNPs/GOx has goodelectrocatalytic performance through glucose oxidation. Itis also seen that in Figure 6Bc, when injection of glucosein PBS solution, the reduction current decreased ataround �0.2 V. This peak was formed due to oxidation ofglucose in the cathodic direction. The potential reason isthat the electrical blockage of the electrode surface bynonconductive metal oxide forming at anodic potentials[43,45]. This effect may be much more significant due tothe introduction of nanoparticles integrated glucose ox-idase, because formation of insulating metal oxide on theelectrode surfaces leads to longer tunneling distance andtherefore to higher resistance. This result implies that theGOx immobilized by the RGO/PtPdNPs film retained itsbiocatalyst activity and the interesting electrocatalytic ac-tivity toward the oxidation of glucose.

Figure 6C shows the amperometric measurement ofAu/RGO/GOx at 0.55 V upon successive additions of glu-cose in PBS solution, and the corresponding calibrationplot is shown in the inset of this figure. A linear detectionfrom 2 mM to 10 mM and a response time of 20 s are ob-served in the calibration curve. From this figure, it is clearthat the amperometric response is very poor due to theabsence of nanoparticles. It is worth nothing that nano-particles anchor the RGO and improve catalytic activity.

Amperometric measurement was carried out at �0.2 V,0.3 V, and 0.55 V, but a significant sensitivity was obtainedat 0.55 V, as shown Figure 6D. It can be reasonably inter-preted that intermediates poison the active catalytic sitesof the PtPdNPs/GOx formed, and cause the amperomet-ric response to become saturated and to decrease the cur-rent [41]. Amperometric measurement of the glucose wasconducted at 0.55 V with the successive addition of glu-cose (2 mM), and response was obtained within 5 s. Thecorresponding calibration curve for the fabricated elec-trode is shown in the inset of Figure 5D. It exhibiteda linear sensing range of 2–12 mM with a sensitivity of24� 10�6 A/mMcm2 (R2 =0.996). The detection limit ofglucose was determined as 0.001 mM (the signal threetimes above noise). Similarly, monometallic NPs modifiedreduced graphene oxide based glucose sensors were alsoprepared and examined. The PdNPs based glucose sensorshowed linear range of 2–10 mM with sensitivity of 18.5�10�6 A/mMcm2 and detection limit of 0.005 mM at 0.55 V.Whereas, PtNPs based glucose sensor exhibited linearrange of 2–8 mM with sensitivity of 7.8 � 10�6 A/mMcm2

and detection limit of 0.015 mM at 0.55 V. Various glu-cose sensors are listed in Table 1 with respect to sensitivi-

Fig. 5. (continued)


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ty, the linear range, detection limit and potential. The per-formance of the fabricated sensor is quite good in com-parison with the reported glucose sensors in Table 1.

3.5 Interference Effect on As-Prepared Glucose Sensor

Anti-interference properties are also important factorsfor sensing applications. The amperometric responses of

Fig. 6. Cyclic voltammograms (CVs) of (A) fabricated electrodes: (a) as-prepared Au/RGO and (b) Au/RGO/GOx. (B) CVs of (a)Au/RGO/PtPdNPs, (b) Au/RGO/PtPdNPs/GOx electrodes in 50 mM PBS (pH, 7.4), and (c) as-prepared Au/RGO/PtPdNPs/GOxelectrodes in 50 mM PBS with 4mM glucose and scan rate of 50 mV/s. (C) Amperometric response of Au/RGO/GOx electrode inPBS (pH, 7.4, 50 mM) to the successive injection of the concentration of glucose in 2 mM at 0.55 V, with inset calibration curve. (D)Au/RGO/PtPdNPs/GOx electrode in PBS (pH, 7.4, 50 mM) to the successive injection of the concentration of glucose in 2 mM at0.55 V, with inset calibration curve.

Table 1. Comparison of various glucose sensing hybrid electrodes.

Sample Linear range Detection limit Applied potential Sensitivity References

GCE/RGO/PdNPs/GOx 0.05–4 mM 0.034 mM 0.5 V [a] 14.1�10�6 A/mMcm2 [17]Au/Gr/AuNPs/chitosan/Gox 2–14 mM 0.18 mM 0.5 V [a] 0.55�10�6 A/mM [2]GCE/RGO/ZnO/GOx 0.02–6.2 mM 0.02 mM �0.4 V [b] 18.97�10�6 A/mM [43]GCE/PLL/ERGO/GOx 0.25–5 mM – 0.42 V [b] – [44]GCE/Gr/CdS/GOx 2–16 mM 0.7 mM – [b] 1.76�10�6 A/mMcm2 [46]GCE/Gr/Au/GOx/nafion ~30 mM 0.001 mM 0.8 V [a] – [47]GCE/IL-Gr/AuNPs/GOx 2–20 mM 0.13 mM 0.35 V [a] 0.16�10�6 A/mM [48]Au/RGO/PtNPs/GOx 2–8 mM 0.015 mM 0.55 V [a] 7.8� 10�6 A/mMcm2 This workAu/RGO/PdNPs/GOx 2–10 mM 0.005 mM 0.55 V [a] 18.5�10�6 A/mMcm2 This workAu/RGO/PtPdNPs/GOx 2–12 mM 0.001 mM 0.55 V [a] 24�10�6 A/mMcm2 This work

[a] Glucose detection by chronoamperometry [b] Glucose detection by cyclic voltammetry.


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some possible interfering species on RGO/PdPtNPs/GOx-modified electrode such as ascorbic acid (AA),acetaminophen (AP), and uric acid (UA) has been stud-ied. These interfering oxidation currents were less com-pared to the 6.43�10�6 A value of 1 mM glucose. Currentresponses of 1.28 �10�6 A for 0.1 mMAP, negligible cur-rent for 0.5 mM UA, and 0.12� 10�6 A for 0.1 mM AAwere observed at 0.55 V which depicted in Figure 7A.

For reproducibility test, four glucose biosensors werefabricated as the equivalent process that the respectivebiosensor was fabricated. Each sensor was measured insuccessive injection of 2 mM glucose in the PBS solutionup to 6 mM and the tested result graph is shown in Fig-ure 7B. It is realized that the fabricated sensors had an ac-ceptable reproducibility in terms of the amperometricmeasurement. The stability of the fabricated sensor wasalso observed for three weeks. The glucose sensitivity was

decreased by 13.33 % at that time. These results indicatethat the sensor has long-term stability.

4 Conclusions

Reduced graphene oxide (RGO) was successfully devel-oped by the exfoliation of graphite oxide, and an RGO/PtPdNPs-modified electrode was fabricated. This modi-fied electrode has excellent catalytic activity towards theoxidation and reduction of hydrogen peroxide. It showedgood analytical properties, with a short response time of2 s, sensitivity of 814�10�6 A/mMcm2, and linear range of0.5–8 mM towards hydrogen peroxide reduction, and a re-sponse time of 3 s, sensitivity of 486� 10�6 A/mMcm2, andlinear range of 0.5–6.5 mM towards hydrogen peroxideoxidation. In addition, after the immobilization of GOxon the RGO/PtNPs, RGO/PdNPs as well as RGO/PtPdNPs, novel and biocompatible RGO/PtNPs/GOx,RGO/PdNPs/GOx and RGO/PtPdNPs/GOx-modified Auelectrode were successfully constructed. Among the otherfabricated sensor electrodes, RGO/PtPdNPs/GOx-modi-fied Au electrode showed high electrocatalytic activitytoward glucose oxidation in PBS solution, with a responsetime of 5 s, sensitivity of 24 �10�6 A/mMcm2, and linearrange of 2–12 mM for glucose oxidation. The high sensi-tivity and good stability with the electrode led to the suc-cessful construction of a practical glucose biosensor,which could also be extended to the immobilization ofsome other biomolecules.

Acknowledgements

This research was partially supported by the researchgrant of Kwangwoon University in 2013, the InternationalCollaborative R&D Program of KIAT/MKE, and the ITR&D program of KEIT/MKE. The authors are gratefulto MinDaP group members for their technical support.

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Received: January 14, 2014Accepted: February 21, 2014

Published online: April 1, 2014


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amperometric glucose biosensor based on pt-pd nanoparticles supported by reduced graphene oxide and...

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