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Research ArticleReceived: 13 August 2011 Revised: 13 September 2011 Accepted: 14 September 2011 Published online in Wiley Online Library: 5 December 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2753

Amperometric biosensor for nitriteand hydrogen peroxide based on hemoglobinimmobilized on gold nanoparticles/polythionine/platinum nanoparticlesmodified glassy carbon electrodeYu Zhang, Ruo Yuan,∗ Yaqin Chai, Jinfen Wang and Huaan Zhong

Abstract

BACKGROUND: This paper describes a convenient and effective strategy to construct a highly sensitive amperometric biosensorfor nitrite (NO2

−) and hydrogen peroxide (H2O2). First, Pt nanoparticles (PtNPs) were electrodeposited on a glassy carbonelectrode (GCE) surface, which promoted electron transfer and enhanced the loading of poly-thionine (PTH). Subsequently,thionine (TH) was electropolymerized on the PtNPs/GCE, and gold nanoparticles (AuNPs) were assembled onto the PTH film toimprove the absorption capacity of hemoglobin (Hb) and further facilitate electron transfer. Finally, Hb was immobilized ontothe electrode through the AuNPs.

RESULTS: Cyclic voltammetry (CV) and scanning electron microscopy (SEM) were used to characterize the fabrication process ofthe sensing surface. Under optimum conditions, the biosensors can be used for the determination of NO2

− in the concentrationrange 70 nmol L−1 to 1.2 mmo L−1 and of H2O2 in the range 4.9 µmol L−1 to 6.8 mmol L−1. The detection limits (S/N = 3) were20 nmol L−1 and 1.4 µmol L−1, respectively.

CONCLUSION: The biosensor exhibits good analytical performance, acceptable stability and good selectivity.c© 2011 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: hemoglobin; Au-nanoparticles; poly-thionine; Pt-nanoparticles; nitrite; hydrogen peroxide

INTRODUCTIONNitrite (NO2

−) exists widely in the environment in beveragesand in food products as a preservative.1 However, NO2

− is animportant precursor in the formation of N-nitrosamines, manyof which have been shown to be potential carcinogens in thehuman body.2 Hydrogen peroxide (H2O2) is an essential mediatorin food, pharmaceutical, clinical, industrial and environmentalanalyses,3 thus, its accurate determination is very important.Various methods have been developed to detect NO2

− andH2O2, such as spectrophotometry,4,5 capillary electrophoresis6,7

and electrochemical methods.8 – 10 Among them, electrochemicalmethods have attracted more and more interest for their highsensitivity, relatively good selectivity, fast response, and low cost.Generally, electrochemical determination of NO2

− reported inthe literature is by either oxidation or reduction.11,12 However,oxidative determination of nitrite has attracted increasing interestsince the major limitations to cathodic determination of nitrite,namely interferences from reduction of nitrate and molecularoxygen, are avoided13. NO2

− is electroactive at a bare glassycarbon electrode, whereas, the application of bare electrodes isgenerally limited because several species can poison the electrodesurface and decrease the sensitivity and accuracy.14 Therefore,

many chemically modified electrodes have been developed todecrease the overpotential and to increase the sensitivity, such ascodeposited Pt nanoparticles and Fe (III),15 hemoglobin absorbedhollow CdS nanospheres,16 and polyaniline modified carbonnanotubes.17 Furthermore, the surface of the modified electrodecould provide a means of extending the dynamic range in analyticaldeterminations.13

Modification of an electrode surface with conducting filmsby electropolymerization of redox monomers has receiveda great deal of attention. Polyer-modified electrodes with athree-dimensional distribution of mediators are preferable to amonolayer for the design of biosensors because of the much largercatalytic response of polymer coatings due to the volume effect.18

Moreover, the long-term operational stability of the polymer filmis much higher than that of the adsorbed mediator.

∗ Correspondence to: Ruo Yuan, Key Laboratory of Analytical Chemistry(Chongqing), College of Chemistry and Chemical Engineering, SouthwestUniversity, Chongqing 400715, PR China. E-mail: yuanruo@swu.edu.cn

Key Laboratory of Analytical Chemistry (Chongqing), College of Chemistry andChemical Engineering, Southwest University, Chongqing 400715, PR China

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Scheme 1. The stepwise fabrication processes of the modified electrode.

TH is a small planar molecule with two –NH2 groupssymmetrically distributed on each side and can be easily dissolvedin water and ethanol.19 It can be electropolymerized by constantor cyclic potential oxidation of a TH-containing solution onelectrode surfaces to produce a stable redox-active layer poly-thionine (PTH).20 TH and PTH are both electron mediators andhave excellent electrocatalytic activity toward the redox of smallmolecular compounds.21 Use of TH and PTH as electron mediatorshas been reported, but PTH used as both electron mediator andcatalyzer to fabricate biosensors has very rarely been reported.

In this work, we constructed a novel biosensor to determineNO2

− and H2O2 with Hb, PTH, PtNPs and AuNPs. First, a glassycarbon electrode (GCE) was modified with PtNPs, which have goodelectron transfer ability, by electrodepositing H2PtCl6 at −0.22 V.The result showed that the use of PtNPs increased the loadingof TH. Second, the electrode was immersed in 0.2 mmol L−1 THsolution and a cyclic voltammetric method used to construct aPTH film. An experiments was used to show that PTH also has theability to catalyze NO2

−. The use of PTH improved the capability ofthe modified electrode. AuNPs was adsorbed onto the PTH film bystatic adsorption. Finally, Hb was immobilized on the electrode byelectrostatic interaction among AuNPs and Hb The AuNPs exhibit astrong adsorption ability and good biocompatibility, which makesthem stable on the electrode. On the other hand, many studieshave shown that enzymes immobilized on gold nanoparticles canretain their biocatalytic and electrochemical activity.3,22 Comparedwith the other methods for the determination of NO2

− and H2O2,the method is inexpensive and sensitive. The prepared biosensorsexhibited fast responses to both NO2

− and H2O2, and possess highsensitivity and good reproduction.

EXPERIMENTALReagents and materialsA stock solution of 5 mg mL−1 Hb (Sigma, USA) was freshlyprepared with 0.1 mol L−1 pH 6.0 phosphate buffer solution (PBS).H2O2 (30%, m/v, solution) and TH were purchased from ShanghaiChemical Reagent Co. (China), and stock solutions of H2O2 wereprepared daily. H2PtCl6 and nitrite sodium were bought fromChongqing Chemical Reagent Co. (China). PBS, 0.1 mol L−1 atvarious pHs were prepared using the stock solution of Na2HPO4,NaH2PO4, and the supporting electrolyte was 0.1 mol L−1 KCl.AuNPs with a diameter about 16 nm were prepared by reducinggold chloride tetrahydrate with sodium citrate at 100 ◦C for half anhour.23 All the other reagents were of analytical grade and used asreceived. Doubly distilled water was used throughout this study.

Apparatus and measurementsAll electrochemical measurements were carried out with aCHI 660A electrochemical workstation (Chenhua Co., Shanghai,China). Scanning electron micrographs were taken with ascanning electron microscope (SEM, S-4800, Japan, Hitachi) at an

acceleration voltage of 5.0 kV. Transmission electron microscopy(TEM) was carried out using a TECNAI 10 (Philips, Holland).All the electrochemical measurements were performed in thethree-electrode system consisting of a modified electrode asworking electrode, a Pt wire as an auxiliary electrode anda saturated calomel electrode (SCE) as reference electrodeat room temperature. All solutions were deoxygenated bybubbling highly pure nitrogen for at least 10 min and maintainedunder nitrogen atmosphere during the measurements. Theamperometric experiments were carried out applying a potentialof −0.3 V for H2O2 and 0.8 V for NO2

− on a stirred cell at roomtemperature.

Preparation of different modified electrodesThe GCE (4 mm diameter) was polished with 0.3 and then0.05 µm alumina slurry, and successively cleaned by sonicatingin ethanol and water. PtNPs were electrochemically deposited onthe bare GCE with constant potential at −0.22 V for 12 s in a 5 mLsolution containing 1 mmol L−1 H2PtCl6 and 0.5 mol L−1 H2SO4.After drying at room temperature, the modified GCE (PtNPs/GCE)was immersed in 0.2 mmol L−1 TH and cycled between −0.4and 1.2 V at 50 mV s−1 for 15 consecutive cycles, to constructPTH/PtNPs/GCE. Following this, it was inserted in a prepared goldcolloids solution for 8 h to form an AuNPs layer. Then, the modifiedelectrode (AuNPs/PTH/PtNPs/GCE) was immersed in Hb solutionat 4 ◦C for 12 h to fabricate Hb/AuNPs/PTH/PtNPs/GCE by physicaladsorption. The finished biosensor was then stored in pH 6.0 PBSat 4 ◦C when not in use. The biosensor fabrication process is shownin Scheme. 1.

RESULTS AND DISCUSSIONSEM characterizationThe morphologies of the PtNPs were investigated using scanningelectronic microscopy (SEM), and images are shown in Fig. 1. It canbe seen clearly that PtNPs are evenly distributed on the electrodesurface. The diameter of the nanospheres is about 220 nm. Thisfeature endows the modified electrode with a high surface area.Thus, compared with bare GCE, PtNPs-modified GCE could increasethe loading of PTH.

Cyclic voltammetric behaviors of different modified electrodesThe cyclic voltammetric experiments were conducted to furthercharacterize the stepwise assembly of the biosensors. Figure 2shows the cyclic voltammograms of different modified electrodesin 0.1 mol L−1 pH 6.0 PBS at 50 mV s−1. No peak was observedat the bare (Fig. 2(a)) electrode. After electropolymerizing TH, themodified GCE (PTH/PtNPs/GCE) (Fig. 2(c)) exhibited two redoxpeaks (A and B). Peak A, which was studied in this work,was attributed to the redox of PTH, and peak B was causedby conjugation between thionine monomers.18 After adsorbing

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Figure 1. SEM images of PtNPs electrodeposited onto GCE.

-0.4 -0.2 0.0 0.2 0.4-20

-15

-10

-5

0

5

10B

A

e dc

b

a

I/µA

E/V(vs.SCE)

Figure 2. CVs of bare GCE (a), PtNPs/GCE (b), PTH/PtNPs/GCE (c),AuNPs/PTH/PtNPs/GCE (d), Hb/AuNPs/PTH/PtNPs/GCE (e) in 0.1 mol L−1

pH 6.0 PBS. Scan rate: 50 mV s−1.

AuNPs, a remarkable peak current increase was observed (Fig. 2(d)),owing to the AuNPs playing an important role similar to an electronconduction tunnel. As a result of the added Hb (Fig. 2(e)), the peakcurrent was reduced rapidly, indicating that the non-conductiveHb was successfully incorporated onto the AuNPs/PTH/PtNPs/GCE.

Optimization of the experimental parameterThe influence of deposition time of the PtNPs onto the biosensorwas investigated (Fig. 1S in Supplementary Material). It can beseen that the optimal time was 12 s. The effect of pH on thebiosensor is shown in Fig. 2S in Supplementary Material. It can beseen that the highest response currents of NO2

− and H2O2 wereobtained at pH 6.0 and pH 7.0. Therefore, we selected pH 6.0 PBSas the supporting electrolyte for the determination of NO2

− andpH 7.0 PBS for H2O2.

The effect of applied potential on the biosensor was alsoinvestigated (Fig. 3S in Supplementary Material). With the appliedpotential decreasing the chronoamperometric current responseof H2O2 increased gradually and the chronoamperometric currentresponse of NO2

− decreased gently. Considering the interferenceof many coexisting potentially interfering species at too negativeand too positive potential, an applied potential of −0.3 Vwas chosen to detect H2O2 and 0.8 V was chosen to detectNO2

−.

0.5 0.6 0.7 0.8 0.9 1.0-5

0

5

10

15

20

I/µA

E/V(vs.SCE)

d

c

ba

Figure 3. CVs of the Hb/AuNPs/PTH/PtNPs modified GCE in 0.1 mol L−1 pH6.0 PBS containing 0 (a), 0.2 mmol L−1 (b), 0.5 mmol L−1 (c), 0.8 mmol L−1

(d) NaNO2 at a scan rate of 50 mV s−1.

200 400 600 800 10000

4

8

12

16

0.0 0.2 0.4 0.6 0.8 1.00

4

8

12

16 d

c

bI/µA

C/mM

a

Cur

rent

/µA

Time/s

a

b

c

d

Figure 4. Amperometric response of different modified electrodes toNO2

− in 0.1 mol L−1 pH 6.0 PBS at an applied potential of 0.8 V uponsuccessive addition of NO2

− of the same concentration at time inter-vals of 40 s. (a) PTH/GCE, (b) PTH/PtNPs/GCE, (c) AuNPs/PTH/PtNPs/GCE,(d) Hb/AuNPs/PTH/PtNPs/GCE.

Electrocatalytic oxidation of NO2− on the modified electrode

Figure 3 shows the influence of the concentration of NO2−

on the CVs of the modified electrode. With increase in NO2−

concentration, the anodic peak current increased obviously athigh potential. Moreover, the peak current of 0.8 mmol L−1 NO2

−

at the Hb/AuNPs/PTH/PtNPs/GCE (Fig. 3(d)) is about 15 times thanthat of the bare GCE (Fig. 3(a)), suggesting that the modifiedelectrode strongly mediates the oxidation of NO2

−.Figure 4 shows a comparison of the amperometric re-

sponses of PTH/GCE (Fig. 4(a)), PTH/PtNPs/GCE (Fig. 4(b)),AuNPs/PTH/PtNPs/GCE (Fig. 4(c)), and Hb/AuNPs/PTH/PtNPs/GCE(Fig. 4(d)) to successive additions of different concentrations ofNO2

−. Compared with the PTH/GCE, the PTH/PtNPs/GCE exhib-ited higher electroactivity toward NO2

−, indicating that PTH couldbe solely used to catalyze NO2

−, and that PtNPs increased theloading of PTH and facilitated the electron transfer. Accordingto Zhao K et al.,13 the electrochemical reaction process of NO2

−

and PTH could be expressed as Scheme. 2. In acidic solution, PTHis positively charged due to the protonation of NH2. The NH3

+

in the structure of PTH attracts negatively charged ions such asNO2

−, and it is possible to accumulate NO2− on the electrode

surface. Thus, the use of PTH promoted the oxidation of NO2−

and improved the sensitivity of the determination. Then, with theaddition of AuNPs, the modified electrode showed higher elec-troactivity. This improved analytical performance is attributed toAuNPs, which have good electron transfer ability, high surface-to-volume ratios and good biocompatibility. Finally, compared

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Scheme 2. The electrocatalytic mechanism of thionine to the oxidation of nitrite.

-0.4 -0.2 0.0 0.2 0.4-15

-10

-5

0

5

10

b

a

I/µA

E/V(vs.SCE)

Figure 5. CVs of the Hb/AuNPs/PTH/PtNPs modified GCE in 0.1 mol L−1 pH6.0 PBS containing 0 (a), 0.35 mmol L−1 (b) H2O2 at a scan rate of 50 mV s−1.

with AuNPs/PTH/PtNPs/GCE, Hb/AuNPs/PTH/PtNPs/GCE exhibitedhigher electroactivity toward NO2

−, revealing that Hb has the abil-ity for catalytic oxidation of NO2

− under appropriate conditions.Though the exact mechanism of catalytic oxidation of NO2

− by Hbis not yet clear, according to Geng et al.24 the mechanisms may beas follows:

Hb-heme-Fe(II) → Hb-heme-Fe(III) + e

Hb-heme-Fe(III) → [Hb-heme-Fe(IV)] · +2e

[Hb-heme-Fe(IV)] · +NO2− + H2O → Hb-heme-Fe(III)

+ NO3− + 2H+

Hb-heme-Fe(III) + PTHH → Hb-heme-Fe(II) + PTH+

PTH+ + H+ + 2e → PTHH

First, ferrous Hb was oxidized to ferric Hb at low potential, andthen at high potential, the ferric Hb was oxidized fleetingly to [Hb-heme-Fe(IV)]·, which oxidized NO2

− to NO3− in solution. Lastly,

the ferric Hb was reduced to ferrous Hb by PTHH. The detectionlimit was estimated to be 20 nmol L−1 (S/N = 3) and the linearresponse range of the sensor to NO2

− was from 70 nmol L−1 to1.2 mmol L−1 with a correlation coefficient of 0.995 (n = 23).

Electrocatalytic reduction of H2O2 on the modified electrodeFigure 5 shows the response of the reduction of H2O2. The anodicpeak current reduced and the reduction peak current increasedwith the addition of H2O2. It also reveals that PTH as a mediatoreffectively shuttles electrons between the redox centers of theenzyme and the base electrode.

Figure 6 shows the amperometric responses of Hb/AuNPs/PTH/PtNPs modified electrode to H2O2 in 0.1 mol L−1 pH 7.0PBS at an applied potential of −0.3 V. The linear response rangeof the biosensor to H2O2 concentration was from 4.9 µmol L−1

0.00 0.15 0.30 0.45-8

-6

-4

-2

I/µA

CH2O2/mM

200 400 600 800 1000

-10

-8

-6

-4

-2

I/µA

Time/s

Figure 6. Amperometric response of the Hb/AuNPs/PTH/PtNPs/GCE toH2O2 in 0.1 mol L−1 pH 7.0 PBS at an applied potential of −0.3 V.

to 6.8 mmol L−1 with a correlation coefficient of 0.994 (n = 11).The detection limit was 1.4 µmol L−1. The simplified mechanismfor the electrochemical catalytic reaction may be expressed as thefollowing schemes:18

H2O2 + Hb-heme-II → Hb-heme-III + H2O

Hb-heme-III + PTHH → Hb-heme-II + PTH+

At first, ferrous Hb was oxidized to ferric Hb via H2O2, and thenthe oxidized Hb oxidized PTHH to PTH+. The overall reductionreaction of Hb-heme-III included two separate steps:25

Hb-heme-III + PTHH → compound + PTH

compound + PTH → Hb-heme-II + PTH+

Finally, the oxidized PTH+ was reduced to PTHH to produce thecathodic catalytic current at the sensor: PTH+ + H+ + 2e → PTHH.

The performance of the biosensor developed in this studywas compared with other NO2

− and H2O2 biosensors as listed inTable 1S. This reveals that our proposed biosensor shows excellentperformance in terms of low detection limit and long linear range.

InterferenceInterference can be evaluated by the value of the current ratio.In this work, the possible interference by Na+, Mg+, K+, Zn2+,SO4

2−, NO3−, Cl−, PO4

3−, HPO42− and H2PO4

− was investigated.100-fold concentrations were added into 0.1 mol L−1 pH 6.0 PBScontaining 0.5 mmol L−1 NO2

−. The interference currents werevery small (less than 5%). And in the same way, the toleranceratio (less than 10%) was observed with the addition of 20-folddopamine, L-tryptophane, and L-cysteine.

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Stability, repeatability and real sample analysisof the biosensorThe modified electrode was suspended above 0.1 mol L−1 pH 6.0PBS at 4 ◦C. Every 2 days it was investigated by measuring thecurrent response to NO2

− and H2O2. After a month, the biosensorretained 90% of its initial response to NO2

− and 87.3% of its initialresponse to H2O2. The fabrication reproducibility of six electrodes,made independently, showed an acceptable repeatability withthe RSD of 5.2% and 6.7% for the current determination of0.5 mmol L−1NO2

− and 0.35 mmol L−1 H2O2. Thus, the biosensorshave good stability and excellent repeatability.

In order to examine the capability of the proposed electrodein practical applications, experiments were investigated in realwater samples for determination of NO2

− using the standardaddition method. Water samples were from Jialing River, whichwas an important tributary of the Yangtze River and a vital watersource for Chongqing residents. The RSD for each sample andthe recoveries obtained were displayed in Table 2S. The resultsindicate that the modified electrode could be used efficiently todetermine nitrite in real samples.

CONCLUSIONSIn this work, PtNPs and PTH, which had the ability to accelerateelectron transmission, were first used together to fabricate anitrite and hydrogen biosensor. Also, AuNPs were used toimprove the characteristics of the biosensor because of its goodbiocompatibility and large effective surface area. By combiningthe merits of PtNPs, PTH and AuNPs, the biosensor showed fastresponse, high sensitivity, good selectivity, a wide linear range,low limit of detection and acceptable storage stability. In addition,this novel method is convenient and versatile.

ACKNOWLEDGEMENTSThis work is supported by National Natural Science Foundationof China (21075100), Ministry of Education of China (708073),Specialized Research Fund for the Doctoral Program of Higher Edu-cation (20100182110015), State Key Laboratory of ElectroanalyticalChemistry (SKLEAC 2010009) and Natural Science FoundationProject of Chongqing City (CSTC-2011BA7003, CSTC-2009BA1003).

Supporting informationSupporting information may be found in the online version of thisarticle.

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