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Sensors and Actuators B 192 (2014) 628– 633

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locate /snb

lectrochemical biosensor for assessment of the total antioxidantapacity of orange juice beverage based on the immobilizing DNA on aoly l-glutamic acid doped silver hybridized membrane

ueliang Wanga,∗, Cuiling Jiaob, Zhangyu Yua,b,∗∗

Department of Chemistry and Chemical Engineering, Heze University, Heze 274015, ChinaCollege of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China

r t i c l e i n f o

rticle history:eceived 17 July 2013eceived in revised form 31 October 2013ccepted 9 November 2013vailable online 18 November 2013

a b s t r a c t

An electrochemical DNA damage biosensor for assessment of the antioxidant capacity in orange juicebeverage was fabricated. The biosensor was constituted with a hybridized membrane of poly l-glutamicacid and Ag and an outside layer of chitosan (CS)/double stranded DNA (ds-DNA). The Fenton solution(Fe2+/H2O2) was used to generate hydroxyl radical (•OH) and induce ds-DNA damage. The orange juicebeverage and ascorbic acid (AA) could scavenge •OH and protect ds-DNA from damage effectively. Based

eywords:NA damagentioxidant capacityscorbic acidrange juice beverage

on this, the antioxidant capacities of the orange juice beverage and AA were studied by linear sweepvoltammetry using Ru(NH3)6

3+ as indicator. The ultraviolet visible absorption spectrometry was alsoused to detect the ds-DNA damage and the antioxidant capacity with almost the same conclusions as theelectrochemical method. The proposed biosensor exhibited good stability and reproducibility and wassuitable for the assessment of the antioxidant capacity in beverages.

iosensor

. Introduction

Reactive oxygen species (ROS) including superoxide anionO2

−), hydrogen peroxide, and hydroxyl radical (•OH), are gener-ted naturally in vivo during metabolic processes and keeps in aalance level in normal living organisms [1]. However, when a body

s subjected to the environmental or behavioral stressors (pollution,unlight exposure, cigarette smoking, excessive alcohol consump-ion, etc.), excess ROS are generated [2]. If the excess ROS cannote scavenged in time, they would attack and induce DNA, proteinsnd lipids damage, and impede normal cell functions [3]. Therefore,verproduction of ROS is associated with numerous diseases likeancer and Alzheimer’s disease, as well as aging. In living systems,he deleterious effects of ROS can be neutralized by the endogenousnd exogenous antioxidant systems. Antioxidants act as reduc-ants (free radical terminators), metal chelating and singlet oxygenuenchers [4]. Fruits are good sources of exogenous antioxidantsecause they are rich in vitamins A, E, C and �-carotene and other

ioactive compounds, such as phenolic compounds, flavonoids androteins [5]. Vitamin C or ascorbic acid (AA), is a powerful exoge-ous antioxidant and widely exists in many fruits and vegetables.

∗ Corresponding author. Tel.: +86 530 5668162; fax: +86 530 5668162.∗∗ Corresponding author at: Department of Chemistry and Chemical Engineering,eze University, Heze 274015, China. Tel.: +86 530 5668162; fax: +86 530 5668162.

E-mail addresses: qust-1977wxl@163.com (X. Wang), yuzywxl@163.com (Z. Yu).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.11.025

© 2013 Elsevier B.V. All rights reserved.

It is thought to plays a key role in the protection against biologicaloxidation processes by scavenging free radicals as a reducing agent.Previous reports have proved that the reducibility of AA preventsBSA oxidative damage effectively [3]. Fruit oranges are rich sourcesof vitamin C which is essential for the overall health of a person, andtherefore the orange juice has been widely used in flavored wateras additive.

Several analytical methodologies have been proposed in orderto quantify antioxidants in food, beverages and biological fluids.

Recently, a lot of attention has been paid to determination offree radicals and antioxidants in the food technology and humanhealth fields, and a substantial number of methods have beendevoted to the evaluation of antioxidant activity in vivo and in vitro[6,7]. These studies can provide important data for evaluation ofthe efficacy of an antioxidant. Compared with bioassays in vivo,assays in vitro are attractive for their simplicity, convenience, repro-ducibility, and low cost. Test of the direct antioxidant capacityin vitro is useful, because if a substance that is poorly effective invitro will not be better in vivo. In vitro evaluation techniques includespectrophotometry [8], fluorescence [9], chemiluminescence [10],chromatography [11], electron spin resonance [12] and electro-chemistry techniques [13,14]. The electroanalytical approaches forassessment of the total or individual antioxidant capacity have been

reviewed by Barroso et al. [15]. Among of electrochemical tech-niques, DNA-based electrochemical biosensors have been proved tobe one of the most efficient methods for measuring the antioxidantscapacity [16]. Most of these electrochemical DNA biosensors are

Actuators B 192 (2014) 628– 633 629

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X. Wang et al. / Sensors and

ased on immobilized ds-DNA on the surface of electrode and therotective effect of the antioxidants induced by •OH generated via aenton-type reaction. In living systems, Fenton chemistry, as one ofhe important ways to generate ROS, is involved in oxidative dam-ge in vivo. Therefore, it is significant to study the protective effectf antioxidants for the ds-DNA damage induced by Fenton-typeeaction in vitro.

For electrochemical DNA-based biosensor fabrication, immobi-ization of ds-DNA on different kinds of transducer surfaces is a keyor stability, reproducibility, and sensitivity [17]. Silver has beensed widely as sensing material to modified electrode because of

ts excellent conductivity, good electrocatalytic activity and chemi-al stability [18,19]. When silver is modified on the electrode, highlyeactive silver oxides are generated and alter the conductivity of themmobilized ds-DNA molecules [20]. Amino acids as the most basicorm of biology protein have been widely applied in electrochemi-al biosensors [21,22] due to their good biocompatibility, stabilitynd the easily available materials. Conducting polymer incorpo-ated with metallic particles provide an exciting system and holdotential application in electronics, sensors and catalysis [23,24]ecause these hybridizeds have synergistic chemical and physicalroperties.

In this work, a membrane of poly l-glutamic acid hybridizedith silver was formed on a glassy carbon electrode (GCE) surface

y electrochemical co-deposition techniques. The modified elec-rode was used to immobilize ds-DNA molecules and fabricate anlectrochemical DNA-based biosensor for assessment of the antiox-dant capacities of AA and orange juice beverage. The biosensor hadood properties for sensing antioxidant capacities of orange juiceeverage and AA. It has potential use in assessment of antioxidantroperty in foodstuff.

. Materials and procedures

.1. Chemicals and apparatus

Herring sperm DNA (double stranded DNA, ds-DNA) was pur-hased from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China).u(NH3)6Cl3 was obtained from Aldrich. 0.1 mol L−1 phosphateuffer solution (PBS) was used as supporting electrolyte and wasrepared by mixing the solution of 0.1 mol L−1 Na2HPO4 and.1 mol L−1 NaH2PO4. The appropriate pH values were adjustedith 0.1 mol L−1 H3PO4 or 0.1 mol L−1 NaOH. Single-stranded DNA

ss-DNA) was produced from ds-DNA as the procedures reportedn literature of [25]. Hydroxyl radical (•OH) was produced by Fen-on reagents (FeSO4 and H2O2). Ascorbic acid (AA) was obtainedrom Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China).range juice beverage (Trade mark: Meizhi Yuan, Producer: Coca-ola Co.) was purchased from supermarket and filtered with filteraper before use. Redistilled water purified by Quartz sub-boilingater distiller (SYZ-550, Jintan Jingbo Experimental Instrument

actory, China) was used in this work. All other chemicals weref analytical grade and were used without further purifica-ion.

All electrochemical experiments were carried out on a CHI 660 electrochemical workstation (Shanghai CH Instruments Co., Ltd.,hina) with a three-electrode arrangement consisting of a glassyarbon electrode (GCE) or film-coated GCE working electrode, aaturated calomel reference electrode (SCE) and a platinum wireuxiliary electrode. All the following potentials reported in thisork were against the SCE. The scanning electron microscopy

SEM) images and energy-dispersive X-ray spectroscopy (EDX) ofhe membrane were obtained using a Carl Zeiss LEO SUPRA 55 fieldmission scanning electron microscope (Germany). The pH valuesf all solutions were measured with a pHS-25 acidometer (Shanghai

Fig. 1. The SEM images (A) and EDX (B) of the poly l-glutamic acid hybridized withAg composite membrane.

Leici Instrument Factory, China). The UV–vis spectroscopy exper-iments were performed with a T1900 UV-Vis spectrophotometer(Beijing Purkinje General Instrument Co., Ltd. China).

2.2. Construction of the biosensor

Before modified, the glassy carbon electrode (GCE) was abradedmanually on a 2000-grit Sic paper, ultra-sonicated in ethanol for30 s and in water for 1 min before being rinsed. The pre-treatedelectrode was placed in a solution containing 1.0 mmol L−1 silvernitrate and 4.0 mmol L−1 l-glutamic acid monomer, and cyclic vol-tammetry was performed with a scan rate of 100 mV S−1 in thepotential range of −0.8–2.0 V for six cycles. The Ag+ and l-glutamicacid monomer were co-deposited on the surface of the GCE andformed a hybridized membrane. The SEM and EDX of the membranewere showed in Fig. 1. The poly l-glutamic acid formed many wrin-kles on the GCE surface. The nanoAg (the bright dot) scattered onthe surface of the poly l-glutamic acid and the EDX (Fig. 1B) showedthat the nanoAg was about 2.5% in the membrane. The large areaand good biocompatibility of poly l-glutamic acid and good con-ductivity of nanoAg were very suitable to immobilize ds-DNA toconstruct biosensor.

After the modified electrode was rinsed with water and driedin air, 8.0 �L ds-DNA of 2.0 mg mL−1 layered onto the modifiedelectrode. To avoid the ds-DNA falling off, 8.0 �L chitosan (CS) of1.0 g L−1 were casted on the most outside. The final modified elec-trode was named as CS/ds-DNA/Ag-PGL/GCE.

2.3. Determination of DNA damage and antioxidant capacity

The processes of determination of DNA damage were as follows:the biosensor was immersed in a freshly prepared 2.0 mL Fentonsolution (1.0 mmol L−1 FeSO4 and 4.0 mmol L−1 H2O2) of pH 5.5 for a

630 X. Wang et al. / Sensors and Actuators B 192 (2014) 628– 633

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ig. 2. Cyclic voltammograms of 1.0 mmol L−1 [Fe(CN)6]3−/4− containing 0.1 M KClt (a) GCE, (b) Ag-PGL/GCE,(c) CS/ds-DNA/Ag-PGL/GCE, scan rate: 100 mV S−1.

iven time. The Fenton solution generated •OH to induce DNA dam-ge. Then the biosensor was rinsed with water and immediatelymmersed into PBS pH 4.0 containing 50 mmol L−1 Ru(NH3)6

3+ toerform the linear sweep voltammetry (LSV) in the potential rangerom +0.6 to +1.1 V.

The antioxidant capacity of orange fruit beverage or ascorbiccid was detected by the same procedures as above except fordding 0.5 mL orange fruit beverage or 0.5 mL 7.5 �mol L−1 ascorbiccid into 2.0 mL Fenton solution.

Ipt/Ip0 was used as a detection signal to assess the degree ofNA damage and antioxidant capacity. Ipt was the peak currentf 50 mmol L−1 Ru(NH3)6

3+ after the biosensor treated with theenton solution in the absence or in the presence of an antioxidant;

p0 was measured when no treatment was done.

.4. Cyclic voltammetry

Cyclic voltammetry (CV) was performed in.0 mmol L−1 K3[Fe(CN)6] and 1.0 mmol L−1 K4[Fe(CN)6] (1:1)olution containing 0.1 mol L−1 KCl at a scan rate of 100 mV S−1.

.5. Ultraviolet visible absorption spectrometry (UV–vis)

The UV–vis spectroscopy of 20 �mol L−1 methylene blue (MB)n the absence or in the presence of 0.01 mg mL−1 ds-DNA wasecorded in the wavelength range of 540–700 nm using the reagentlank as reference solution, where the ds-DNA had been treatedith Fenton solution for a given time in the absence or in theresence of antioxidant. The absorbance change of MB before orfter adding ds-DNA was used as signals to investigate the ds-DNAamage and antioxidant capacity.

. Results and discussion

.1. Cyclic voltammograms of [Fe(CN)6]3−/4− at differentlectrodes

The electrochemical sensing characteristics of the sensorere characterized by recording the cyclic voltammograms of

.0 mmol L−1 [Fe(CN)6]3−/4− (1:1) containing 0.1 mol L−1 KCl and

he results were showed in Fig. 2. As many literatures reported,Fe(CN)6]3−/4− had a couple of sensitive redox peak currents on theCE (a). The anodic peak current (ipa) and cathodic peak current

ipc) were −5.041 × 10−5 A and 4.943 × 10−5 A, respectively, with

Fig. 3. LSV of 50 mmol L−1 Ru(NH3)63+ on the CS/ds-DNA/Ag-PGL/GCE after it was

treated with 1.0 mmol L−1 FeSO4/4.0 mmol L−1 H2O2 for different time. (a) 0 min,(b)10 min, (c) 20 min, (d) 30 min,(e) 40 min and on CS/ss-DNA/Ag-PGL/GCE(f).

the peak-to-peak separation (�Ep) of 83 mV. After the GCE wasmodified with a layer of Ag-PGL (b), the ipa and ipc of [Fe(CN)6]3−/4−

increased to −5.277 × 10−5 A and 5.314 × 10−5 A, respectively, andan increase of �Ep was observed, which can be ascribed to syner-gistic effect of the good conductivity of Ag and large area but poorelectron transfer properties of poly l-glutamic acid. However, whenanother layer of ds-DNA was covered on the surface of the Ag-PGL,the redox peak currents of [Fe(CN)6]3−/4− decreased apparently (c)due to the poor conductivity of the outside chitosan.

3.2. Electrochemical determination of the DNA damage

Ru(NH3)63+ was always used as the electroactive indicator to

monitor ds-DNA damage [26]. After CS/ds-DNA/Ag-PGL/GCE wastreated by Fenton reagent for different time, the electrode waswashed and placed into 50 mmol L−1 Ru(NH3)6

3+ which was pre-pared with pH 4.0 PBS solution and LSV signals were recorded. Theresults showed in Fig. 3.

When the CS/ds-DNA/Ag-PGL/GCE was not treated with the Fen-ton reagents (a, 0 min), a very little peak of Ru(NH3)6

3+ was foundat 0.87 V, which was in accordance with Ref. [26]. However, afterthe electrode was treated with Fenton reagents for 10 min (b), thepeak current increased apparently and with the time of the treat-ment prolonging, the peak current increased further (from b toe). The largest peak current was obtained after the electrode wastreated for 40 min (e), which suggested that the ds-DNA damagewas becoming more severe. The hydroxyl radical generated in theFenton reaction attacked the ds-DNA in the film and induced theds-DNA damage, resulted in more guanine was exposed from theprotection of ds-DNA double helix. When the Ru(NH3)6

3+ was usedas electroactive indicator, the Ru(NH3)6

3+ would oxidize guanineand form a redox catalytic cycle (Eqs. (1)–(3)) [27,28].

ds-DNA•OH attack−→ DNA(guanine) (1)

Ru(NH3)3+6 + DNA(guanine) → Ru(NH3)2+

6 + DNA(guanine+) (2)

Ru(NH3)2+6 = Ru(NH3)3+

6 + e− (3)

3+

As control experiments, the LSV of 50 mmol L−1 Ru(NH3)6 onthe CS/ss-DNA/Ag-PGL/GCE (f) was also recorded and found thatthe peak current is larger than that on the CS/ds-DNA/Ag-PGL/GCEwhich was treated with Fenton reagents for 40 min.

X. Wang et al. / Sensors and Actuat

0 10 20 30 400

1

2

3

4

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b

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Fig. 4. Dependence of the ds-DNA damage degree on the incubation time in differ-e −1 −1 −1

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nt solutions. (a)1.0 mmol L FeSO4/4.0 mmol L H2O2; (b) 1.0 mmol L FeSO4,.0 mmol L−1 H2O2 and 0.5 mL orange fruit beverage; (c) 1.0 mmol L−1 FeSO4,.0 mmol L−1 H2O2 and 7.5 �mol L−1 AA

.3. Eelectrochemical determination of the antioxidant capacity

As strong antioxidants, orange fruit beverage and ascorbic acidAA) could scavenge some free radical, including •OH (Wang, Zhang,iong, & Wang, 2010). The protection of orange fruit beverage andA for ds-DNA from damage was investigated by calculating thehange the Ipt/Ip0 values and the results were showed in Fig. 4.

When the biosensor was treated with Fenton solution in thebsence (curve a) or in the presence of antioxidant (orange fruiteverage (curve b) or AA (curve c)) for less than 18 min, the Ipt/Ip0alues had no obvious changes and near to 1, indicating that thes-DNA damage was little. However, with the treatment time pro-

onging, the slope of curve a increased significantly, indicating thathe ds-DNA damage was getting seriously. The slope of curve br curve c had only a little increase, suggesting that orange fruiteverage or AA had good protection effect for the ds-DNA damage

nduced by Fenton reagents. The proposed biosensor was efficientor assessment of the antioxidant capacity.

When the sensor was treated with Fenton solution for 40 min,pt/Ip0 values decease with the concentration of AA in the rangef 1.0–50 �mol L−1. Others antioxidants, such as gallic acid andesveratrol were also studied. If the sensor was treated with Fen-on solution for 40 min without antioxidant, and the DNA damageegree was considered as 100%, the DNA damage degree in theresence of antioxidants was described as Ipt, presence/Ipt, absenceIpt, presence and Ipt, absence were peak currents in the presence andbsence of antioxidant, respectively). The results showed that inhe presence of 7.5 �mol L−1 AA, resveratrol or gallic acid, the DNAamage degree was 29.68%, 37.6% and 59.6%, respectively, whichere consistent with the results of Ref. [29], and the proposed

ensor had a broader linear range for AA.

.4. Optimizing experimental conditions

The influence of the molar concentration ratio of Fe2+ and H2O2n the degree of ds-DNA damage was investigated by fixing theoncentration of Fe2+ or H2O2 when the sensor was treated withenton solution for 40 min. The results were shown in Fig. 5. Whenhe concentration of Fe2+ was fixed as 1.0 mmol L−1 (Fig. 5A), in the

3+

atio range of 1:0–1:8, the Ipt/Ip0 of Ru(NH3)6 increased at firstnd then declined with the increase of the concentration of H2O2.he biggest value of Ipt/Ip0 was obtained at the ratio of 1:4. Whenhe concentration of H2O2 was fixed as 4.0 mmol L−1 (Fig. 5B), the

ors B 192 (2014) 628– 633 631

values of Ipt/Ip0 decreased with the increase of the concentration ofFe2+ in the ratio range of 1:4–4:4, which were in accordance withFig. 5A. However, if the biosensor only was treated with PBS (Fig. 5B,0:0), 4.0 mmol L−1 H2O2 (Fig. 5B, 0:4) or 1.0 mmol L−1 Fe2+ (Fig. 5A,1:0), respectively, the values of Ipt/Ip0 were almost no differenceand near to 1. The results showed that DNA damage was causedby •OH generated via Fenton reactions. Therefore, when the molarconcentration ratio of Fe2+ and H2O2 was 1:4, the Fenton solutioncould induce the most severe ds-DNA damage.

In the pH range of 4.0–7.0, the influence of the pH value onthe efficiency of ds-DNA damage was investigated by comparingthe LSV response change (�ip) of Ru(NH3)6

3+ on the biosensorafter and before the biosensor was treated with Fenton solutionfor 10 min. The �ip increased with the pH values at first and thendeclined. The biggest value of �ip was obtained at about 5.5. There-fore, the pH 5.5 was used for treatment of the biosensor.

3.5. Repeatability and stability of the CS/ds-DNA/Ag-PGL/GCE

Repeatability and stability were important parameters forevaluating the performance of a modified electrode. The repeat-ability was studied by recording the LSV peak currents change ofRu(NH3)6

3+ (�ip), respectively, on a batch of five biosensors fab-ricated in the same way after and before they were treated withFenton reagents for 30 min. The relative standard deviation (RSD)of �ip was 5.6%, suggesting that the repeatability of the sensor wasgood. At the same modified electrode, LSV peak currents change ofRu(NH3)6

3+ was repetitively detected for 7 times, RSD was 3.91%.The result indicated that the modified electrode exhibited good sta-bility. The long-term stability was investigated according to thereference of [28]. In brief, the �ips were obtained on a new fab-ricated biosensor and a biosensor stored in the air for 15 days atroom temperature, respectively, and then calculated the differenceof them, then the difference was compared with the �ips obtainedon the new fabricated biosensor. The results showed that the meandeviation was less than 5% (n = 5), showing the good stability of thebiosensor.

3.6. UV–vis determination of ds-DNA damage

Methylene blue (MB) is a sort of water-soluble dye. It can beused an electro-active probe in detecting ds-DNA damage and DNAhybridization by electrochemical methods [30–33] and as an opti-cal probe in biophysical systems [34,35]. Many electrochemical andspectroscopic studies have showed that MB can interact with ds-DNA by intercalating into the base pairs of ds-DNA, while the groovebinding and electrostatic attractions were also play some role in theinteractions [30–35] depending on ionic strength, concentrationratio of MB/DNA and base sequence of DNA [31]. MB has become agood model compound for theoretical studies of the binding typesof dyes to DNA due to its high extinction coefficient [36].

The ds-DNA damage induced by Fenton reaction was alsodetected by UV–vis spectroscopy using MB as indicator. The resultswere showed in Fig. 6. In the wavelength of 540–700 nm, MB hasan UV–vis absorption peak at wavelength of 585 nm (curve a).As adding 0.1 mg mL−1 ds-DNA, the absorbance of MB decreasedapparently (curve b) and blue shift, which suggested that theMB interacted with ds-DNA. After the ds-DNA reacted with Fen-ton reagents for different time and mixed with MB solution, theabsorbance of MB decrease with the reaction time (curve d–g).Because the ds-DNA was damaged in Fenton solution, more basesin DNA that could bind more MB were exposed, and accordingly,

the absorbance of MB decreased. Curve c was the absorbance curveof MB after adding the ds-DNA which had reacted with Fentonreagents for 10 min in the presence of 0.5 mL orange fruit bever-age. The absorbance of MB was bigger than that obtained in the

632 X. Wang et al. / Sensors and Actuators B 192 (2014) 628– 633

Fig. 5. Dependence of the ds-DNA damage degree on the molar concentration ratio of Fe2

as 1.0 mmol L−1; (B) the concentration of H2O2 was fixed as 4.0 mmol L−1

550 600 65 0 70 00.00

0.02

0.04

0.06

0.08

0.10

Abs

Wavelength/nm

a

g

Fig. 6. UV–vis spectroscopy of MB after and before it interacted with ds-DNA.20 �mol L−1 MB in 0.1 mol L−1 PBS of pH 8.0 (a); 20 �mol L−1 MB in 0.1 mol L−1 PBSof pH 8.0 containing 0.1 mg mL−1 ds-DNA and Fenton reagent reacted for 0 min(b); 10 min (d); 20 min (e); 30 min (f); 40 min (g), respectively. 20 �mol L−1 MBib

stac

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n 0.1 mol L−1 PBS of pH 8.0 containing 0.1 mg mL−1 ds-DNA, 0.5 mL orange fruiteverage and Fenton reagent reacted for 10 min (c).

ame condition without orange fruit beverage. The results showedhat orange fruit beverage had protection effect for ds-DNA damagend the results were in accordance with that obtained by electro-hemical method.

. Conclusion

In this work, the antioxidant capacities of orange juice bever-ge and AA were investigated based on an electrochemical ds-DNAamage biosensor using Ru(NH3)6

3+ as indicators. The study of LSVf Ru(NH3)6

3+ showed that this biosensor had good properties inetection of ds-DNA damage induced by •OH generated via a clas-ical Fenton reaction and the antioxidant capacities. The optimalarameters governed the performance of the biosensor was stud-

ed. The ds-DNA damage and the antioxidant capacities of orangeruit beverage and AA were also detected by UV–vis spectroscopy.ntioxidants of orange fruit beverage and AA implied an effective

rotection effect for ds-DNA damage in a certain range of con-entration. The experiment procedure exhibited good stability andeproducibility.

[

+ and H2O2 when incubation time is 40 min. (A) The concentration of Fe2+ was fixed

Acknowledgments

We are grateful to the financial support of the National NaturalScience Foundation of China (21105023), Shandong Young Scien-tists Award Fund (BS2013HZ027) and Natural Science Foundationof Shandong Province, China (ZR2009BM003).

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Biographies

Xueliang Wang, a doctor of engineering major in applied chemistry. Now he worksin the Department of Chemistry and Engineering as an associate professor, HezeUniversity, Heze, China. His research interests include electroanalytical chemistry,biosensors, nanotechnology, immunoassay and detection of the transgenic plantproducts.

Cuiling Jiao, a master major in the analytical chemistry, now studying in the Collegeof Chemistry and Chemical Engineering, Qufu Normal University, Qufu, China.

Zhangyu Yu, a professor and a PhD supervisor of Shandong University. Now heworks as a president of Heze University, Heze, China. His research work focuseson boi-electrochemistry, electro-analytical chemistry, quantum chemistry andsupramolecular chemistry.