A New Electrochemical Biosensor for Determination of Hydrogen Peroxide in Food Based on Well-Dispersive Gold Nanoparticles on Graphene Oxide

A New Electrochemical Biosensor for Determination of HydrogenPeroxide in Food Based on Well-Dispersive Gold Nanoparticles onGraphene Oxide

Bing Zhang, Yuling Cui, Huafeng Chen, Bingqian Liu, Guonan Chen, Dianping Tang*

Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education of China & Fujian Province), Department ofChemistry, Fuzhou University, Fuzhou, 350108, P. R. China*e-mail: [email protected]

Received: March 27, 2011;&Accepted: April 20, 2011

AbstractHerein, we describe a new method for the detection of hydrogen peroxide (H2O2) in food by using an electrochemi-cal biosensor. Initially, ultrafine gold nanoparticles dispersed on graphene oxide (AuNP-GO) were synthesized bythe redox reaction between AuCl4

� and GO, and thionine-catalase conjugates were then assembled onto the AuNP-GO surface on a glassy carbon electrode. With the aid of the AuNP-GO, the as-prepared biosensor exhibited goodelectrocatalytic efficiency toward the reduction of H2O2 in pH 5.8 acetic acid buffer. Under optimal conditions, thedynamic responses of the biosensor toward H2O2 were achieved in the range from 0.1 mM to 2.3 mM, and the detec-tion limit (LOD) was 0.01 mM at 3sB. The Michaelis–Menten constant was measured to be 0.98 mM. In addition, therepeatability, reproducibility, selectivity and stability of the biosensor were investigated and evaluated in detail. Fi-nally, the method was applied for sensing H2O2 in spiked or naturally contaminated samples including sterilizedmilk, apple juices, watermelon juice, coconut milk, and mango juice, receiving good correspondence with the resultsfrom the permanganate titration method. The disposable biosensor could offer a great potential for rapid, cost-ef-fective and on-field analysis of H2O2 in foodstuff.

Keywords: Electrochemical biosensor, Graphene oxide, Gold nanoparticles, Hydrogen peroxide, Food

DOI: 10.1002/elan.201100171

1 Introduction

Food safety is a scientific discipline describing handling,preparation, and storage of food in ways that preventfoodborne illness. Unsafe food causes many acute andlife-long diseases, ranging from diarrhoeal diseases to var-ious forms of cancer [1]. This includes a number of rou-tines to avoid potentially severe health hazards. Accord-ing to the WHO estimation, foodborne and waterbornediarrhoeal diseases taken together kill about 2.2 millionpeople annually and 1.9 million of them children.

Hydrogen peroxide, a powerful oxidizing agent, is usu-ally utilized as an antimicrobial agent in food and as asterilizing agent on the foil lining of aseptic packages con-taining fruit juices and milk products [2]. For example, inUS, Canada, Australia and New Zealand, H2O2 was em-ployed as a bleaching agent in some foods such as wheatflour, edible oil, egg white etc. However, the dosage ofH2O2 should be limited to the amount sufficient for thepurpose in processing food. The Joint FAO/WHO ExpertCommittee on Food Additives (JECFA) considered thatingestion of small amount of H2O2 would produce no tox-icological effects due to rapid decomposition of the chem-ical by the enzyme of the intestinal cells [3]. However,more than of 3 % H2O2 solutions (household strength)

generally would result in vomiting, mild irritation tomucosa and burns in the mouth, throat, oesophagus andstomach [4]. Ingestion of higher concentration, e.g.>10%, would result in more dangerous sequelae such asburns to mucus membranes and gut mucosa [3]. There-fore, quantitative detection of H2O2 in food is very impor-tant.

Various methods and strategies have been exploited forthe detection of H2O2 in foodstuff including titrimetry,spectrophotometry, fluorimetry, chemiluminescence andelectrochemical methods [5–7]. Despite many advances inthese fields, it is still a challenge to find new approachesthat could improve the simplicity and sensitivity of theanalytical methods. Electrochemical biosensor interestsmore and more researchers day by day due to its highsensitivity, low cost, low power requirements and goodcompatibility [8]. Xu reported a highly sensitive ampero-metric biosensor based on MnO2-modified verticallyaligned multiwalled carbon nanotubes for the trace deter-mination of H2O2 in milk with high accuracy [9]. Ping de-signed a Prussian blue and poly(o-phenylenediamine)modified amperometric biosensor for the determinationof hydrogen peroxide in aseptically packaged beverages[10]. Cui and co-workers described a non-enzyme am-perometric biosensor for detection of H2O2 based on cal-

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cined layered double hydroxide [11]. For the successfuldevelopment of an electrochemical biosensor, the con-struction and characteristics of sensors should be crucial.Introducing nanomaterials into the sensing interface wereexpected to improve the analytical properties of the as-prepared biosensors.

Graphene nanosheet, a single layer of carbon atomsbonded together in a hexagonal lattice, has attracted con-siderable attention in the electroanalytical chemistry inrecent years due to its extraordinary properties, such ashigh conductivity, large surface-to-volume ratio, high elas-ticity and electromechanical modulation [12]. Avouris in-vestigated the electroactive actuators based on graphenereinforced Nafion composite electrolytes, and the protonconductivity and the water-uptake ration could be greatlyimproved [13]. Graphene nanosheets offer intriguingelectronic, thermal and mechanical properties and are ex-pected to find a variety of application in high-perfor-mance nanocomposite materials. Recently, several re-views on the application of graphene for the electrochem-ical biosensing have been reported [14,15]. Pumera inves-tigated carbon hybrid materials of graphene sheets deco-rated with metal or metal oxide nanoparticles of gold,silver, copper, cobalt, or nickel from cation exchangedgraphite oxide [16]. The as-prepared hybrid nanomateri-als based on nanosized inorganic particles and clustersrepresent an attractive field of research activity becauseof the possibility to tailor and optimize the properties ofthe resulting materials for various applications. Goldnanoparticles with high volume-to-surface ratio and goodbiocompatibility have been extensively employed for thepreparation of hybrid nanomaterials. To the best of ourknowledge, gold nanoparticles were immobilized on thesurface of graphene nanosheets mainly through covalentconjugation [17], aminated functionalization [18], electro-chemical deposition [19], and layer-by-layer technique[20]. Among these methods, gold nanoparticles were hy-bridized with graphene by the aid of external cross-link-age reagents or chemicals. These materials might behinder or decrease the electron transfer rate from theelectrochemical point of view. Therefore, exploring a newgold nanoparticles and graphene-based platform for sens-ing H2O2 is of great interest.

The aim of this study is to construct a new biosensorfor the determination of H2O2 based on in situ synthesisof gold nanoparticles on graphene nanosheets. Initially,ultrafine gold nanoparticles monodispersed on the surfacegraphene oxide were successfully prepared by the redoxreaction between AuCl4

� and graphene oxide, and the as-prepared AuNP-GO was then utilized as an affinity sup-port for the conjugation of catalase. The biosensor wasused for amperometric detection of H2O2 in food. Theuse of AuNP-GO was expected to enhance the sensitivityof the biosensor, and improved the analytical propertiesof the biosensor.

2 Experimental

2.1 Reagents and Apparatus

Catalase from bovine liver (EC 1.11.1.6, 2000–5000 unitsmg�1 protein), thionine acetate salt (Dye content �85%),graphite powder and sodium citrate tribasic hydrate wereobtained from Sigma-Aldrich (USA). HAuCl4·4H2O waspurchased from Sinopharm Chem. Re. Co. Ltd. (Shang-hai, China). All other reagents were of analytical gradeand were used without further purification. Ultrapurewater obtained from a Millipore water purificationsystem (�18 MW, Milli-Q, Millipore) was used in all runs.0.1 M acetic acid-buffered saline (ABS) with various pHswere prepared by mixing the stock solutions of 0.1 MHAc, 0.1 M NaAc and 0.1 M KCl. Phosphate buffer (PB,0.01 M) solution containing Na2HPO4 and NaH2PO4 wasused as the supporting electrolytes. To spike samples ofsterilized milk, apple juices, watermelon juice, coconutmilk and mango juice, 1 mL of sample was directly trans-ferred to a 2-mL centrifuge tube and different concentra-tions of H2O2 were added.

Electrochemical measurements were carried out with aMPI-E Electrochemiluminescent (Xi�an, Ruimai, China).Electrochemical impedance spectroscopy (EIS) was per-formed on a CHI 604D Electrochemical Workstation(Shanghai, China). Ultraviolet-vis absorption (UV-vis)spectra were recorded with an 1102 UV-vis spectropho-tometer (Techcomp, China). Nanostructures were charac-terized by transmission electron microscopy (TEM, Hita-chi 7650, Japan). Scanning electron microscopy (SEM)measurements were performed using a digital JSM-T220scanning electron microscope (Japan).

2.2 Preparation of Graphene Oxide (GO)

Graphene oxide nanosheets were prepared according to amodified Hummers method as follows [21]. 0.5 g ofgraphite powder was initially dispersed in 23 mL ofH2SO4 at 0 8C, and 0.5 g of NaNO3 and 3 g of KMnO4

were then dropwise added. The well-mixed slurry wasstirred for 1 h at a 35 8C water bath. Following that,140 mL of H2O was added in the mixture, and the tem-perature was raised to 90 8C. Afterwards, 3 mL of H2O2

(30 wt%) was injected, and the mixture changed to alight brown color. Consequently, graphene oxide was ob-tained by filtering, washing and centrifugation at 4000rpm. The obtained GO was characterized by TEM, whichhas lateral dimensions between 300 nm and 12 mm withan average size of ~3 mm.

2.3 In Situ Synthesis of Gold Nanoparticles on GO(AuNP-GO)

The well-dispersive gold nanoparticles on graphene oxidewere synthesized consulted to the literature with somemodification [22]. Prior to experiment, GO aqueous solu-tion was prepared by dispersing 50 mg of the as-prepared

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GO into 100 mL of distilled water. Following that, 5.0 mLof GO aqueous solution and 0.5 mL HAuCl4 aqueous so-lution (10 mM) were added into a 10-mL volumetricflask, and the mixture was vigorously stirred for 2 h at90 8C. Afterwards, the mixture was washed twice with dis-tilled water and centrifuged for 10 min at 8000 rpm. Theobtained AuNP-GO nanostructures were dispersed into1.0 mL of distilled water for further use (C[GO]

�0.5 mgmL�1).

2.4 Preparation of Thionine-Conjugated CatalaseEnzyme (TCA)

500 mL of catalase (200 mg mL�1) and 500 mL of thionine(30 mM) were initially dissolved into 1 mL of 0.01 M PBSand the pH was adjusted to 9.5 by using 10 wt% K2CO3,and then 100 mL of glutaraldehyde solution was addedinto the mixture [23]. After stirred for 2 h, the mixturewas adjusted to pH 7.0 by using 1.0 M NaH2PO4. Duringthis process, the amino group on the catalase was conju-gated with the amino group on the thionine by the gluta-raldehyde cross-linking [24,25]. The unbound thioninewas removed using ultrafiltration for 12–15 times untilthe peak corresponding to thionine in the elution disap-peared. Finally, the obtained TCA was dispersed into1.0 mL pH 7.4 PB buffer.

2.5 Preparation of Hydrogen Peroxide Biosensors

A glassy carbon electrode (GCE, 2 mm in diameter) waspolished repeatedly with 1.0 and 0.3-mm alumina slurry,followed by successive sonication in bi-distilled water andethanol for 5 min and dried in air. After the cleaned elec-

trode was thoroughly rinsed with water and absolute eth-anol, 3 mL of the as-prepared AuNP-GO suspension wasthrown to the electrode surface, which was left dry atroom temperature (RT). Following that, the modifiedGCE was immersed into the prepared TCA solutionabove, and incubated for 6 h at 4 8C. After washing withpH 7.4 PB buffer, the as-made biosensor was stored at4 8C when not in use. The biosensor is schematically illus-trated in Scheme 1.

2.6 Electrochemical Measurement

The electrochemical cell consisted of a conventionalthree-electrode system with a modified electrode as work-ing electrode, a platinum wire as a counter electrode, anda saturated calomel electrode (SCE) as a reference elec-trode. Prior to the determination of hydrogen peroxide,the biosensor was placed in 2 mL of 0.1 M ABS (pH 5.8)at RT (Note: deaeration with N2 for 10 min), and cyclicvoltammetry (CV) was performed between 300 and�600 mV vs. SCE at 50 mVs�1. When the biosensor hadreached a steady state, hydrogen peroxide with variousconcentrations was added. The response of the biosensortoward H2O2 was measured as the cathodic peak currentat �220 mV vs. SCE and corrected for the baseline fromCV. All experiments were performed in triplicate at RT(25�1.0 8C) controlled by air-condition. The reaction pro-cesses occurring at the surface of the modified electrodefor the determination of H2O2 is illustrated in Scheme 1.

Scheme 1. Schematic illustration of thionine-conjugated catalase, gold nanoparticles-dispersed graphene oxide, and the as-preparedbiosensor.

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Determination of Hydrogen Peroxide in Food


3 Results and Discussion

3.1 TEM and UV-vis Absorption Characteristics of GOand AuNP-GO

In this work, the biosensors were fabricated by means ofimmobilization of catalase on nanogold particles-attachedgraphene. The formation of gold nanoparticles on the sur-face of graphene was based on the graphene oxidetoward in situ reduction of Au(III) under the wild condi-tion. To verify the successful synthesis of GO and AuNP-GO, UV-vis absorption spectroscopy was utilized at vari-ous steps. As shown in Figure 1a, there was an absorptionpeak at the UV region (227 nm) for the synthesized GOsuspension, while no absorption peak was observed at thevisible region. After the formation of AuNP-GO nano-structures, a new absorption peak at 516 nm was ap-peared in addition to that of GO (Figure 1b). As indicat-ed from our previous reports [26,27], the peak mainly de-rived from gold nanoparticles. The results revealed thatgraphene oxide could reduce the Au(III) to zero value(Au0) under the wild condition.

To further monitor the formation of the AuNP-GO, thesynthesized AuNP-GO was characterized by using TEM.

As indicated from Figure 2a, no particles were observedon the graphene. The graphene was planar sheet-like, sug-gesting that the nanostructures were neither carbon nano-tubes nor graphite powder. Significantly, we could ob-serve that many nanoparticles were attached onto the sur-face of graphene nanosheets after the formation ofAuNP-GO (Figure 2b). The results further demonstratedthe feasibility of the synthesized method of AuNP-GO.With the aid of gold nanoparticles, the catalase biomole-cules could be immobilized on the surface of AuNP-GOdue to the strong interaction between gold nanoparticlesand proteins [28]. Furthermore, an obvious nanoparticleinterface could be observed at the TCA/AuNP-GO-modi-fied surface, as shown from the SEM image of Figure 2c.

3.2 Cyclic Voltammetric Characteristics of the Biosensors

Figure 3 displays the cyclic voltammograms of differentlymodified electrodes in pH 5.8 ABS at 50 mV s�1. Noredox waves were observed at the cleaned GCE in theapplied potential range (Figure 3a). When AuNP-GO wasmodified on the GCE, the background currents of thecyclic voltammogram were increased (Figure 3b). Thereason might be the fact that the as-prepared AuNP-GOwith good conductivity could increase the surface area ofthe electrode. Importantly, a couple of redox peaks at�170 mV and �220 mV with diffusion-controlled processwas obtained after thionine-conjugated catalase mole-cules were assembled on the AuNP-GO/GCE (Figure 3c).The results indicated that the formation of TCA did notchange the redox properties of thionine, and could beused as a good mediator for electron transfer. In general,graphene oxide with a large number of �OH and �COOH groups exhibited weaker conductivity than that ofreduced graphene [15]. To further clarify the merit ofgold nanoparticles over the conductivity of AuNP-GO,we prepared two modified electrodes, i.e. GO/GCE andAuNP-GO/GCE. The inset of Figure 3 represents theelectrochemical impedance spectroscopy (EIS) of GO/GCE and AuNP-GO/GCE, respectively. As indicatedfrom the inset, the resistance of the AuNP-GO/GCE waslargely lower than that of GO/GCE. Therefore, the exis-

Fig. 1. UV-vis absorption spectra of (a) graphene oxide and (b)gold nanoparticles-dispersed graphene oxide.

Fig. 2. TEM images of (a) GO and (b) AuNP-GO, and (c) SEM image of TCA/AuNP-GO-modified surface.




tence of gold nanoparticles could obviously improve theconductivity of graphene oxide. The reason might be at-tributed to the fact that gold nanoparticles might serve asan intervening “spacer” matrix to penetrate through thenanosheets, and improve the conductivity of grapheneoxide.

Figure 4 shows the cyclic voltammograms of the biosen-sor in pH 5.8 ABS at various scan rates ranging from 10to 100 mVs�1. With the increase of scan rate, the anodicpeak potential shifted to a more positive value and thecathodic peak potential shifted in a negative direction.The redox peak currents were proportional to the scanrate, v (inset of Figure 4), indicating that the redox reac-tion is a surface process.

In addition, the stability of the TCA/AuNP-GO/GCEwas also investigated. The GCE coated with TCA/AuNP-GO was stored in pH 7.4 PB buffer, and measured bycyclic voltammetry periodically. Alternatively, the TCA/AuNP-GO/GCE was dried at 4 8C over pH 7.4 PBS formost of the storing time, and cyclic voltammograms wererun occasionally. For both storage conditions, the peakpotentials were essentially unchanged during 4-week stor-age, and the peak current decreased 10.3% of the initialcurrents at 28th day. The results indicated that TCA andAuNP-GO could firmly immobilize on the GCE.

Fig. 3. Cyclic voltammograms of (a) bared GCE, (b) AuNP-GO/GCE, (c) TCA/AuNP-GO/GCE in pH 5.8 ABS at 50 mV s�1. Inset:EIS of AuNP-GO/GCE and GO/GCE in 5 mM Fe(CN)6

4�/3� solution.

Fig. 4. Cyclic voltammograms of TCA/AuNP-GO/GCE at various scan rates from 10 to 100 mV s�1. Inset: Peak currents vs. scanrate.




3.3 Electrocatalytic Characteristics of the Biosensors

To monitor the bioactivity of the immobilized catalase onthe electrode, the modified electrode was utilized for thereduction of H2O2 in the solution. Figure 5A displays thecyclic voltammograms of the as-prepared biosensor inpH 5.8 ABS at the absence and presence of H2O2. In theblank ABS, the modified electrode only gave the electro-chemical behavior of the TCA/AuNP-GO/GCE with apair of quasireversible anodic and cathodic waves (Fig-ure 5A-a). Upon addition of 500 mM H2O2 into pH 5.8ABS, an obvious catalytic behavior with the increase ofthe cathodic peak and the decrease of the anodic peakwas appeared (Figure 5A-b). Whether could the as-madeAuNP-GO catalyze the reduction of H2O2, however? Toclarify this issue, AuNP-GO/GCE and TCA/AuNP-GO/GCE were used for the determination of 5 H2O2 concen-trations between 0.5 mM and 1 mM. Curve a of Figure 5Brepresents the current responses of the AuNP-GO/GCEtoward H2O2. Experimental results indicated that the syn-thesized AuNP-GO could almost not catalyze the reduc-tion of H2O2 (Note: 0.1 M pH 5.8 Fe(CN)6

4�/3�–PB solu-tion). Therefore, the catalytic current mainly derivedfrom the immobilized catalase toward the reduction ofH2O2. To further clarify the merits of the as-preparedAuNP-GO, three types of biosensors, i.e. TCA/GO/GCE,TCA/AuNP/GCE and TCA/AuNP-GO/GCE were usedfor the detection of the above mentioned analytes (Note:TCA/GO/GCE was prepared by directly casting GO onthe GCE surface, while the AuNPs on the TCA/AuNP/GCE was obtained by using the electrodeposited methodas described in our previous report [28]). As indicatedfrom curves b–d of Figure 5B, the TCA/AuNP-GO/GCEexhibited higher amperometric response and sensitivitythan those of other sensing platforms. The results indicat-ed that the immobilized catalase molecules could remaintheir native bioactivity, and the synthesized AuNP-GOacted as the bridge to provide an electrical contact or apathway of electron transfer between the immobilizedcatalase and the base electrode. The electrocatalytic re-

duction mechanism of catalase towards H2O2 might beexplained as the following cycles:

H2O2 þ Catalase! H2Oþ Compound I ð1Þ

Compound Iþ thionineðredÞ ! Compound IIþ thionineðoxÞ

ð2Þ

Compound IIþ thionineðoxÞ þ 2Hþ !Catalaseþ thionineðoxÞ þH2O

ð3Þ

ThionineðoxÞ þ 2e� þ 2Hþ ! thionineðredÞ ð4Þ

Therefore, the reduction current was proportional tothe H2O2 concentration in the solution, as long as theH2O2 concentration is not limiting. That is, catalase couldbe recycled at the electrode, resulting in an increase of itsreduction current.

3.4 Optimization of External Conditions

In general, the acidity of the supporting electrolyte influ-enced the catalytic activity of the enzyme. Figure 6ashows the effect of pH of ABS on the currents of the bio-sensor in 0.1 M ABS toward 0.5 mM H2O2 (as an exam-ple). Experimental results indicated that the cathodic cur-rents increased with the increase of pH from 3.0 to 5.5,and then decreased from 6.0 to 8.0. The low responses inhigh or low pH solutions might be ascribed to the chargechange of mercapto or primary groups in enzyme, whichweakened the electrostatic interaction between biomole-cules and nanogold particles. However, there was no sig-nificant change at the range of pH 5.5–6.0. Consideringthe deviation of operation or experiments, pH 5.8 ABSwas chosen for the detection of H2O2 in this study.

The immobilized amount of AuNP-GO on the GCEalso affected the analytical performance of the biosensor.If the AuNP-GO was too much on the GCE, the formedfilm became thick, and it was easier to fall off from theGCE. If the dropped AuNP-GO was too little, it could

Fig. 5. A) Cyclic voltammograms of TCA/AuNP-GO/GCE in pH 5.8 ABS at the (a) absence and (b) presence of 0.5 mM H2O2 at50 mV s�1, and B) electrochemical responses of (a) AuNP-GO/GCE (b) TCA/AuNP/GCE, (c) TCA/GO/GCE and (d) TCA/AuNP-GO/GCE toward various concentrations of H2O2.




not favor the immobilized of catalase molecules, thus af-fecting the sensitivity of the biosensor. As seen from Fig-ure 6b, the optimal amount of AuNP-GO was 3 mL. So,3 mL of AuNP-GO was applied for the preparation of bio-sensor.

3.5 Electrochemical Response of the Biosensors TowardH2O2 Standards

Under optimal conditions, the as-prepared biosensor wasutilized for the detection of H2O2 in pH 5.8 ABS by usingcyclic voltammetry. As seen from Figure 7, the cathodiccurrents increased with addition of an aliquot of H2O2 inpH 5.8 ABS, and the anodic currents decreased. Signifi-cantly, we found that the cathodic currents increased line-arly with the increase of H2O2 concentration rangingfrom 0.1 mM to 2.3 mM. The linear regression equationswere obtained as i (mA)=�0.0009� C[H2O2] (mM)�0.4214(n=10, R2 =9967). The detection limit (LOD) of the bio-sensor was found to 0.01 mM estimated at 3sB, which waslower than those of graphene and ZnO nanocomposites-based amperometric biosensor (0.6 mM) [29], magnetite-graphene-based biosensor (0.5 mM) [30], graphene-modi-fied electrode (0.11 mM) [31], graphene-Pt nanoparticleshybrid material-based biosensor (0.1 mM) [32], graphene/Prussian blue-based biosensor (1.9 mM) [33], prussianblue nanocubes on reduced graphene oxide (45 nM) [34],single-layer graphene nanoplatelet-enzyme compositefilm (0.105 mM) [35], single-stranded DNA/graphenenanocomposite-based biosensor (38.5 mM) [36], and cat-ionic polyelectrolyte-functionalized graphene nanosheetsand gold nanoparticles (0.44 mM) [20].

When the concentration of H2O2 was higher than2.3 mM, a response plateau is observed, showing the char-acteristic of Michaelis–Menten kinetic mechanism. Theapparent Michaelis–Menten constant (Km

app), which is anindication of the enzyme–substrate kinetics, could be cal-culated from the Lineweaver–Burk equation:

1=Iss ¼ 1=Imax þKmapp=Imaxc ð5Þ

where Iss is the steady-state current after the addition ofthe substrate, c the bulk concentration of the substrate,and Imax the maximum current measured under saturatecondition. Consequently, the Km

app for the biosensor wasobtained as 0.98 mM by an analysis of the slope and inter-cept for the double reciprocal plot of the steady-state cur-rent versus the H2O2 concentration. The 0.98 mM ofKm

app is lower than those of graphene and ZnO nanocom-posites-modified electrode (1.46 mM) [29], graphene-Ptnanoparticles hybrid material-based biosensor (5.0 mM)[32], and single-layer graphene nanoplatelet-enzyme com-posite film (11.2 mM) [35], which suggested a higher ac-cessibility of catalase and a lower limitation due to diffu-sion.

3.6 Reproducibility, Repeatability, Selectivity, Stability ofthe Biose nsors

The repeatability of the biosensor was evaluated by usingthe relative standard deviation (RSD) of 7 assays basedon the same biosensor at 10 mM and 1.0 mM H2O2, re-spectively. The RSDs were 7.8 % and 5.9 % at the above

Fig. 6. The effect of (a) pH of ABS and (b) volume of AuNP-GO on the electrochemical responses of the biosensor.

Fig. 7. Dynamic responses of the biosensor toward H2O2 inpH 5.5 ABS. Insets: Calibration curve of the biosensor, n=3.




mentioned analyte, respectively. The sensor-to-sensor re-producibility was estimated according to the cathodic cur-rents toward 0.5 mM H2O2 at 5 different biosensors. TheRSD was 8.1 %. Therefore, the repeatability and reprodu-cibility of the biosensor were acceptable.

Next, the specificity of the biosensor was studied by as-saying other interfering reagents, such as ascorbic acid,glucose, uric acid, and acetic acid. As indicated fromFigure 8, the biosensors displayed low current responsestowards these interfering reagents except ascorbic acid.The reason might be attributed to the fact that ascorbicacid could reduce to the (C5H5

�)Fe3+ produced in the cat-alyzed reaction. Significantly, the existence of glucose,uric acid, and acetic acid did not affect the current re-sponse of the biosensor for H2O2.

The stability of the biosensor was investigated over a60-day period. When the biosensor was stored dry at 4 8Cand measured intermittently (every 2–3 days), no obviouschange in the cathodic current to 0.5 mM H2O2 was foundover 35-day period. At 60th day, the cathodic current ofthe biosensor was 76.8 % of the initial current. We specu-late that the slow decrease of response mainly attributedto the gradual deactivation of the immobilized catalaseon the surface of the nanoparticles.

3.7 Analysis of H2O2 in Food and Method Validation

To investigate the feasibility of applying the biosensor tomeasure H2O2 in a complex matrix, 5 naturally contami-nated samples including sterilized milk, apple juices, wa-termelon juice, coconut milk and mango juice (i.e. in thepresence of H2O2) were assayed by the biosensor and theclassical KMnO4 titration method [37,38], respectively.Following that, H2O2 standards with random concentra-tions were spiked into these specimens, and the contentsof H2O2 were assayed by using the above mentionedmethods. The results are listed in Table 1. As seen fromin Table 1, the RSD was less than 9.5 %, and the recoverywas 86.5–117 %, indicating a good correlation betweenboth analytical methods.

4 Conclusions

This work describes a facile and sensitive electrochemicalbiosensing strategy for the detection of H2O2 in food sam-ples by using biomolecules-functionalized AuNP-GOsensing platform. The presence of nanogold particles im-proved the conductivity of graphene oxide, increased theimmobilized amount of catalase, and enhanced the sensi-tivity of the biosensor. Compared with the conventionalgraphene-based biosensors, the prepared AuNP-GOcould avoid the use of other cross-linkage reagents.Meanwhile, the in situ synthesized gold nanoparticlescould homogeneously disperse on the surface of grapheneoxide, thus enabled for the preparation of the biosensorwithin the relatively short time. The precision, selectivityand stability of the biosensor were acceptable. The lowcost and large production scale of AuNP-GO makes it apromising material for the construction of biosensor andbioelectronic devices. Further investigation is to enhancethe signal of the electrochemical responses.

Acknowledgements

Support by the National Natural Science Foundation ofChina (21075019, 20735002), the Research Fund for theDoctoral Program of Higher Education of China(20103514120003), the “973” National Basic ResearchProgram of China (2010CB732403), and Program for Re-

Fig. 8. The specificity of the biosensor toward various interfer-ing reagents.

Table 1. Comparison of the assayed results for naturally contaminated or spiking food samples obtained by the biosensor and the per-manganate titration method (n=3).

Sample type Contaminated samples (mM) Mean�SD (mM) RSD (%) Spiking samples (mM) Recovery (%)

By biosensor By titration Added By biosensor

Sterilized milk 1.3 1.1 1.2�0.1 8.3 50 54.8 106.8Apple juices 3.5 4.2 3.9�0.4 9.1 100 89.5 86.5Watermelon juice 13.2 11.8 12.5�0.7 5.6 200 192.3 90.2Coconut milk 7.8 6.9 7.4�0.5 6.1 300 342.1 111.1Mango juice 9.3 10.2 9.8�0.5 4.6 400 478.9 117.0




turned High-Level Overseas Chinese Scholars of FujianProvince (XRC-0929) is gratefully acknowledged.

References

[1] S. Millour, L. Noel, A. Kadar, R. Chekri, C. Vastel, B. Sirot,J. Leblanc, T. Guerin, Food Chem. 2011, 126, 1787.

[2] E. Gomez-Plaza, M. Cano-Lopez, Food Chem. 2011, 125,1131.

[3] http://www.cfs.gov.hk/english/programme/programme_rafs/programme_rafs fa_02_02.html.

[4] http://www.gasdetection.com/TECH/h2o2.html.[5] J. Song, J. Xu, P. Zhao, L. Lu, J. Bao, Microchim. Aca 2011,

172, 117.[6] Q. Wang, Y. Yun, J. Zheng, Microchim. Acta 2009, 167, 153.[7] R. Bou, R. Codony, A. Tres, E. Decker, F. Guardiola, Anal.

Biochem. 2008, 377, 1.[8] X. Luo, I. Hsing, Biosens. Bioelectron. 2009, 25, 803.[9] B. Xu, M. Ye, Y. Yu, W. Zhang, Anal. Chim. Acta 2010, 674,

20.[10] J. Ping, J. Wu, K. Fan, Y. Ying, Food Chem. 2011, 126, 2005.[11] L. Cui, H. Yin, J. Dong, H. Fan, T. Liu, P. Ju, S. Ai, Biosens.

Bioelectron. 2011, 26, 3278.[12] B. Su, J. Tang, J. Huang, H. Yang, B. Qiu, G. Chen, D. Tang,

Electroanalysis 2010, 22, 2720.[13] P. Avouris, Nano Lett. 2010, 10, 4285.[14] B. Su, J. Tang, H. Yang, G. Chen, J. Huang, D. Tang, Elec-

troanalysis 2011, 23, 832.[15] D. Brownson, C. Banks, Analyst 2010, 135, 2768[16] M. Pumera, Chem. Soc. Rev. 2010, 39, 4146[17] W. Song, D. Li, Y. Li, Y. Li, Y. Long, Biosens. Bioelectron.

2011, 26, 3181.[18] K. Huang, D. Niu, X. Liu, Z. Wu, Y. Fan, Y. Chang, Y. Wu,

Electrochim. Acta 2011, 56, 2947.

[19] M. Du, T. Yang, K. Jiao, J. Mater. Chem. 2010, 20, 9253.[20] Y. Fang, S. Guo, G. Zhu, Y. Zhai, E. Wang, Langmuir 2010,

26, 11277.[21] W. Hummers, R. Offeman, J. Am. Chem. Soc. 1958, 80,

1339.[22] X. Chen, G. Wu, J. Chen, X. Chen, Z. Xie, X. Wang, J. Am.

Chem. Soc. 2011, 133, 3693.[23] D. Tang, R. Niessner, D. Knopp, Biosens. Bioelectron. 2009,

24, 2125.[24] S. Koev, P. Dykstra, X. Luo, G. Rubloff, W. Bentley, G.

Payne, R. Ghodssi, Lab Chip 2010, 10, 3026.[25] P. Dharmapandian, S. Rajesh, S. Rajasingh, A. Rajendran,

C. Karunakaran, Sens. Actu. B 2010, 148, 17.[26] D. Tang, B. Su, J. Tang, J. Ren, G. Chen, Anal. Chem. 2010,

82, 1527.[27] D. Tang, J. Ren, Anal. Chem. 2008, 80, 8064.[28] D. Tang, R. Yuan, Y. Chai, Anal. Chem. 2008, 80, 1582.[29] J. Xu, C. Liu, Z. Wu, Microchim. Acta 2011, 172, 425.[30] Y. He, Q. Sheng, J. Zheng, M. Wang, B. Liu, Electrochim.

Acta 2011, 56, 2471.[31] M Li, S. Xu, M. Tang, L. Liu, F. Gao, F. Wang, Electrochim.

Acta 2011, 56, 1144.[32] R. Dey, C. Raj, J. Phys. Chem. C 2010, 114, 21427.[33] E. Jin, X. Lu, L. Cui, D. Chao, C. Wang, Electrochim. Acta

2010, 55, 7230.[34] L. Cao, Y. Liu, B. Zhang, L. Lu, ACS Appl. Mater. Interf.

2010, 2, 2339.[35] Q. Lu, X. Dong, L. Li, X. Hu, Talanta 2010, 82, 1344.[36] Q. Zhang, Y. Qiao, F. Hao, L. Zhang, Z. Wu, Y. Li, J. Li, X.

Song, Chem. Eur. J. 2010, 16, 8133.[37] N. Klassen, D. Marchington, H. McGowan, Anal. Chem.

1994, 66, 2919.[38] M. Bowman, J. Chem. Educ. 1949, 26, 103.




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