Np

Aa

b

a

ARRAA

KNHSCCN

1

pstebsoHaHao

(

h0

Sensors and Actuators B 201 (2014) 51–58

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo ur nal home page: www.elsev ier .com/ locate /snb

onenzymatic H2O2 sensing based on silver nanoparticles cappedolyterthiophene/MWCNT nanocomposite

del A. Abdelwahaba,∗, Yoon-Bo Shimb,∗∗

Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, EgyptDepartment of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, South Korea

r t i c l e i n f o

rticle history:eceived 2 March 2014eceived in revised form 22 April 2014ccepted 1 May 2014vailable online 10 May 2014

eywords:onenzymatic sensorydrogen peroxideilver nanoparticlesarbon nanotubes

a b s t r a c t

A novel method for highly sensitive H2O2 sensor is proposed using silver nanoparticles (AgNPs)modified oxidized poly-2,2′:5′,2′ ′-terthiophene-3-p-benzoic acid/multi wall carbon nanotube (Ox-pTTBA/MWCNT). The Ox-pTTBA/MWCNT nanocomposite film was prepared via electropolymerizationof a TTBA monomer and MWCNT mixture solution, followed by in situ electrooxidation of thepTTBA/MWCNT film. Then, AgNPs were formed on the Ox-pTTBA/MWCNT layer through immersing thefreshly prepared Ox-pTTBA/MWCNT electrode in AgNPs solution. The characterization of sensor probeand experimental parameters affecting its activity were investigated employing UV–vis spectroscopy,transition electronic microscopy (TEM), scanning electron microscopy (SEM), electrochemical impedancespectroscopy (EIS), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CV). The AgNPs/Ox-pTTBA/MWCNT nanocomposite showed an excellent electrocatalytic activity to H2O2 by significantly

onducting polymeranocomposite

increasing the reduction peak current and completely inhibiting the effect of other interfering species.The sensor probe displays a fast response time less than 5 s with a linear range from 10 to 260 �Mand detection limit of 0.24 �M. The sensitive, stable and specific response to H2O2 demonstrates thatthe present sensor is potentially suitable for monitoring H2O2 concentrations in biological system. Theapplication was conducted for the determination of H2O2 in human urine real samples.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Hydrogen peroxide (H2O2) is an essential intermediate thatlays an important role in both environmental and biologicalystems [1]. Numerous analytical techniques, such as spectropho-ometry [2], fluorescence [3], and chemiluminescence [4] have beenmployed for the determination of H2O2. Because of the redoxehavior of H2O2, the electrochemical techniques have received aignificant interest in the determination of H2O2 over other meth-ds due to its simplicity, selectivity, and high sensitivity. Generally,2O2 reduced to water via two-electron transfer process in anqueous solution. However, the direct electrochemical reduction of2O2 with the conventional electrodes is not effective for analytical

pplication due to slow electrode kinetics. Therefore, modificationf the electrode surface is of practically important to enhance the

∗ Corresponding author. Tel.: +20 88 231 2193; fax: +20 88 232 5436.∗∗ Corresponding author. Tel.: +82 51 510 2244; fax: +82 51 514 2430.

E-mail addresses: adel aa@pusan.ac.kr (A.A. Abdelwahab), ybshim@pusan.ac.krY.-B. Shim).

ttp://dx.doi.org/10.1016/j.snb.2014.05.004925-4005/© 2014 Elsevier B.V. All rights reserved.

rate of electron transfer and hence minimize the overpotential ofredox reactions.

Metals nanoparticles have received considerable interest dueto their conducting nature and biocompatibility that makes thesematerials excellent to develop a wide variety of sensors and biosen-sors. Electrochemical reduction of H2O2 has been studied usingdifferent metals and metal oxides nanoparticles, such as gold [5],platinum [6], palladium [7], copper oxide [8], ruthenium oxide [9],and iron oxide [10]. Among them, silver nanoparticles were alsoused to investigate the electrochemical behavior and kinetics ofH2O2 reduction [11–13]. Although, some studies have shown suc-cess in this direction, there is still much effort to develop moresensitive and selective H2O2 sensor is needed for monitoring H2O2release in biological systems.

Conducting polymers have received considerable interestdue to their conducting nature and high stability that makesthem extremely attractive for immobilizing nanomaterials andbiomolecules to develop a wide variety of sensors and biosensors

[14–18]. Of these, poly-2,2′:5′,2′ ′-terthiophene-3-p-benzoic acid(pTTBA), has been recently employed for electrode modificationand successfully showed a great ability for biosensors applications[19,20]. The purpose of using conducting polymer pTTBA herein,

52 A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 201 (2014) 51–58

F bare Gp −/4− soA r in th

iisfcdsastctat[

lfnOtsgtpt

ig. 1. (i) UV–vis spectra and (ii) TEM image of AgNPs solution. SEM images of (iii)lots of bare GC (�) and AgNPs/Ox-pTTBA/MWCNT/GC (�) in a 5.0 mM [Fe(CN)6]3

gNPs/Ox-pTTBA/MWCNT/GC surfaces. (For interpretation of the references to colo

s to introduce negative charge in the polymer film via electroox-dation of pTTBA. The anionic polymeric film can offer exchangeites and serve as a charge selective compound, which can be usedor strong interaction with Ag nanoparticles. On the other hand,arbon nanotube (CNT) has received much attention over the pastecade as suitable materials for electrode modification and biosen-or applications [21–23]. Due to their unique characteristics, suchs large active surface area, high electrical conductivity, chemicaltability and biocompatibility, CNT has been used to improve elec-rocatalytic performance [24–26]. The nanocomposites of CNT withonducting polymers have recently been employed to enhancehe electrical conductivity of the polymer film by increasing thective surface area and hence facilitating the rate of electronransfer reactions between target analyte and electrode surface14,16,23].

In the present study, an electrochemical sensor for direct ana-ytical detection of H2O2 is proposed. The sensor was successfullyabricated by immobilizing AgNPs on the oxidized pTTBA/MWCNTanocomposite layer. The electrochemical oxidation of pTTBA intox-pTTBA plays an important role in the present sensor fabrica-

ion. Since this process introduces active sites in the Ox-pTTBAurface through the formation of negatively charged carboxylate

roups and conjugated � electron rich-polymer backbone afterhe oxidation of pTTBA. Thus, the Ox-pTTBA film becomes cationicermselective and more conductive which may therefore act asemplates for strong adsorption of AgNPs. Additionally, MWCNT

C, (iv) Ox-pTTBA/MWCNT/GC, and (v) AgNPs/Ox-pTTBA/MWCNT/GC. (vi) Nyquistlution. XPS spectra of (vii) S2p peak and (viii) Ag3d peaks for (a) bare GC and (b)is figure legend, the reader is referred to the web version of the article.)

was used in the nanocomposite to increase sensor conductiv-ity and then amplified electron transfer process on the electrodesurface. The mechanism of nanocomposite sensor formation andexperimental parameters affecting its electrochemical activity areinvestigated and discussed in details.

2. Materials and methods

2.1. Materials

H2O2, ascorbic acid (AA), dopamine (DA), uric acid (UA), glu-tamic acid (GA), glucose (Glu), acetaminophen (AP), AgNO3, sodiumcitrate and NaBH4 were purchased from Sigma and Aldrich (USA).Multi wall carbon nanotubes (MWCNT) was purchased from IljinNanotech (South Korea). Tetrabutylammonium perchlorate (TBAP)was received from Fluka (USA). A 2,2′:5′,2′ ′-terthiophene-3-p-benzoic acid monomer (TTBA) was recently synthesized as areported method [27]. All other chemicals were of extra pure ana-lytical grade and used without further purification. Nitrogen gas(99.99%) was used for maintain deoxygenating in the measurementcell solution before and during the experiments.

2.2. Instruments

Cyclic voltammograms (CV) and chronoamperograms wererecorded using a Potentiostat/Galvanostat, Kosentech model PT-1

sors an

(3H(otE(qvsuaAGep

2

rmcscsTw

2

tcwwmFa

2

1

A.A. Abdelwahab, Y.-B. Shim / Sen

Busan, S. Korea). A UV–vis spectrum was obtained using a UV-101PC (Shimadzu). A JEOL JEM-2010 electron microscope (Jeoligh-Tech. Co.) was used to obtain transition electronic microscopy

TEM) image. Scanning electron microscopy (SEM) images werebtained using a Cambridge Stereoscan 240 (KBSI at Busan). Elec-rochemical impedance spectroscopy (EIS) was recorded with theG&G PAR 273A Potentiostat/Galvanostat and a lock-in amplifierPAR EG&G, Model 5210) linked to a personal computer. The fre-uency was scanned from 100 kHz to 10 Hz at the open circuitoltage, acquiring five points per decade. The amplitude of sinu-oidal voltage of 10 mV was used. XPS experiments were performedsing a VG scientific ESCA lab 250 XPS spectrometer coupled with

monochromated Al K� source having charge compensation. ThegNPs/Ox-pTTBA/MWCNT/GCE, Ox-pTTBA/MWCNT/GCE and bareCE, with an electrode area of 0.07 cm2, were used as workinglectrodes. Reference and counter electrodes were Ag/AgCl andlatinum wire, respectively.

.3. Preparation of colloidal AgNPs

AgNPs were prepared by borohydride reduction of AgNO3 aseported previously [28]. Briefly, a 10 mL of 0.25 mM AgNO3 wasixed with 10 mL of 0.25 mM trisodium citrate. Next, 0.6 mL of ice-

old 0.1 M NaBH4 solution was added to the mixture solution whiletirring. Upon this addition, the solution turned to yellow, indi-ating the formation of AgNPs. An adsorption band in the UV–vispectrum of AgNPs solution was observed at 389 nm (Fig. 1i) andEM images confirmed that the nanoparticles size in the solutionas about 3.5 nm (Fig. 1ii).

.4. Functionalization of MWCNT

The MWCNT was functionalized by acid treatment according tohe previous procedure [14]. Firstly, 50 mL of a mixture solution ofoncentrated HNO3 and H2SO4 (1:3) containing 50 mg of MWCNTas sonicated for about 8 h. Thereafter, the mixture was washedith distilled water and filtrated for several times using the filterembrane (0.2 �m) until the pH of the filtrates became neutral.

inally, the carboxylated MWCNT was dried in the oven at 80 ◦C forbout 12 h and stored at room temperature until use.

.5. Sensor fabrication

For the preparation of AgNPs/Ox-PTTBA/MWCNT sensor, a.0 mM TTBA monomer was mixed with 1.0 mg/mL carboxylated

Scheme 1. Schematic representation of the fabrication and overall de

d Actuators B 201 (2014) 51–58 53

MWCNT in a 0.1 M TBAP/CH2Cl2 solution and sonicated for 30 min.The Ox-pTTBA/MWCNT nanocomposite film were formed on theelectrode surface by cycling the potential from 0.0 to +1.6 V in theabove mixture solution for five cycles at the scan rate of 0.1 V/s[14]. After that, an electrochemical oxidation of pTTBA/MWCNTfilm to produce Ox-pTTBA/MWCNT was occurred in PBS pH 7.0from 0.0 to +1.8 V for three cycles at 0.1 V/s. This process introducesactive sites on the Ox-pTTBA/MWCNT film, and may therefore actas templates for strong adsorption of AgNPs from the solution.This is due to the cationic selectivity of the negatively charged car-boxylate groups of the Ox-pTTBA/MWCNT nanocomposite [14,29].In order to incorporate AgNPs onto the Ox-pTTBA/MWCNT film,the freshly prepared Ox-pTTBA/MWCNT electrode was immersedat open circuit in a well stirred colloidal AgNPs solution for 2 h.Then, the sensor was rinsed thoroughly with distilled water andstored until use. Scheme 1 shows the schematic of the AgNPs/Ox-pTTBA/MWCNT nanocomposite sensor fabrication.

3. Results and discussion

3.1. Characterization of AgNPs/Ox-pTTBA/MWCNTnanocomposite

Fig. 1 shows the SEM images of stepwise of the sensorfabrication: (iii) GC, (iv) Ox-pTTBA/MWCNT/GC, (v) AgNPs/Ox-pTTBA/MWCNT/GC. As shown, the morphology of the Ox-pTTBA/MWCNT layer shows a homogeneous nanocomposite film.This might be due to the incorporation of MWCNT into pTTBAduring the electropolymerization process [14]. The diameter ofMWCNT in the nanocomposite film was determined to be about10 nm. In addition, the SEM image of the AgNPs/Ox-pTTBA/MWCNTsurface shows the formation of AgNPs on the Ox-pTTBA/MWCNTfilm, indicating successful fabrication of the nanocomposite sensorprobe.

EIS was carried out to study the conductivity of the elec-trode surfaces after modification. Fig. 1vi shows the Nyquist plotsrecorded for bare and AgNPs/Ox-pTTBA/MWCNT electrodes in a5.0 mM [Fe(CN)6]3−/4− solution. A Randle circuit was employed toanalyze the obtained impedance results (Inset of Fig. 1vi). Where,Rs is the solution resistance, Rp1, Rp2 are the polarization resis-

tances, W is the Warburg element, and CPE1, CPE2 are the constantphase elements. The parameter values were obtained by fitting theresults to the equivalent circuit using Zview 2 impedance software.A plot of bare electrode shows the charge transfer resistance in

tection of H2O2 by AgNPs/Ox-pTTBA/MWCNT nanocomposite.

54 A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 201 (2014) 51–58

-10 0 10 0 30 0 500 70 0 90 0 1100-40

-30

-20

-10

0

10

20

(C)

I/µA

E/mV

-400 -200 0 200 400 600 800-15

-10

-5

0

5

10

15

20

(B)

E/mV

I/µA

-600 -400 -200 0 200-20

-10

0

10

20

30

E/mV

I/µA

(A)

F CNT

1 and O

Rv4tte

paatsAwtaoAwcO

3

nsatOeewo

ig. 2. CVs recorded for the AgNPs/Ox-pTTBA/MWCNT (dotted lines) Ox-pTTBA/MW.0 mM K3[Fe(CN)6] solutions. (C) CVs of the AgNPs/Ox-pTTBA/MWCNT (solid line)

p1 and Rp2 to be 1020 and 22,920 �, respectively. While, thesealues decreased with the AgNPs/Ox-pTTBA/MWCNT electrode to50 and 10,060 � for Rp1 and Rp2, respectively. This result indicateshat, the AgNPs/Ox-pTTBA/MWCNT nanocomposite layer improveshe conductivity by facilitating the electron transfer process on thelectrode surface.

The characterization of AgNPs/Ox-pTTBA/MWCNT nanocom-osite was further studied using XPS. All XPS spectra were takenfter Ar ion gas etching for 50 s and corrected using a C1s peakt 284.6 eV as an internal standard. Fig. 1vii shows the S2p spec-ra observed for (a) bare GC and (b) AgNPs/Ox-pTTBA/MWCNT/GCurfaces. As shown, no peak appeared for a bare GC, where thegNPs/Ox-pTTBA/MWCNT/GC surface shows a peak at 163.5 eV,hich due to the S C bond in the polymer structure suppor-

ing the formation of nanocomposite layer on the GC surface. Inddition, Fig. 1viii (b) shows the appearances of the two peaksf Ag 3d5/2 and Ag3d3/2 at 368.8 and 374.7, respectively for thegNPs/Ox-pTTBA/MWCNT/GC nanocomposite. While, these peaksere not observed with a bare GC as shown in Fig. 1viii (a), indi-

ating that the AgNPs have been successfully immobilized onto thex-pTTBA/MWCNT film.

.2. Electrochemical characterization and performance

The electrochemical properties of the Ox-pTTBA/MWCNTanocomposite layer was investigated employing CV. Fig. 2Ahows the CVs recorded for the Ox-pTTBA/MWCNT (solid line)nd bare (dashed line) electrodes in 1.0 mM [Ru(NH3)6]Cl3 solu-ion. As shown, the enhanced redox peaks appeared for thex-pTTBA/MWCNT nanocomposite as compared with the bare

lectrode. However, when the Ox-pTTBA/MWCNT electrode wasxposed to a solution containing 1.0 mM K3[Fe(CN)6] (Fig. 2B), aell-defined redox peak of the [Fe(CN)6]4−/[Fe(CN)6]3− couple was

bserved with the bare electrode (dotted line), while no peak was

(solid lines) and bare (dashed lines) electrodes in (A) 1.0 mM [Ru(NH3)6]Cl3 and (B)x-pTTBA/MWCNT (dashed line) electrodes in 0.1 M H2SO4.

observed for the CV recorded for the Ox-pTTBA/MWCNT (solidline). This result indicates the formation of a negatively chargedcarboxylate groups of the polymeric nanocomposite film whichfacilitates the electron transfer process of [Ru(NH3)6]3+ ions dueto the electrostatic interaction. While, the electron transfer pro-cess of [Fe(CN)6]3− ions completely inhibited and hence the redoxpeak current was negligible [30,31]. In order to characterize thepositively charged AgNPs, the CVs of the AgNPs/Ox-pTTBA/MWCNTelectrode in [Ru(NH3)6]Cl3 and K3[Fe(CN)6] solutions are recordedin Fig. 2A and B (dotted lines), respectively. As shown, well-defined redox peaks of both [Ru(NH3)6]4+/[Ru(NH3)6]3+ and[Fe(CN)6]4−/[Fe(CN)6]3− couples were observed. This clearly indi-cated that, the AgNPs interact with the Ox-pTTBA/MWCNTelectrostatically and covered wholly and uniformly the elec-trode surface and hence the effect of the negatively chargedof the Ox-pTTBA/MWCNT nanocomposite was completely pre-vented. In addition, to investigate the formation of AgNPs onto theOx-pTTBA/MWCNT nanocomposite film, the CV of the AgNPs/Ox-pTTBA/MWCNT electrode in 0.1 M H2SO4 solution was recorded(Fig. 2C). A well-defined oxidation peak of the Ag/Ag+ at 0.45 Vis obtained with the AgNPs/Ox-pTTBA/MWCNT (solid line), andno signal was observed for the Ox-pTTBA/MWCNT (dotted line).This result conformed the successful fabrication of nanostructuredAgNPs on the Ox-pTTBA/MWCNT nanocomposite layer and henceit can be applied for further analytical detection of H2O2.

3.3. Electrochemical detection of H2O2

Metal nanoparticles-based sensor electrodes often gave theenhanced current response and higher sensitivity and selectivity

for H2O2 detection [32,33]. Thus, the AgNPs/Ox-pTTBA/MWCNTelectrode was employed for electrochemical detection of H2O2.Fig. 3A shows the CVs of a (a) bare, (b) Ox-pTTBA/MWCNT and(c) AgNPs/Ox-pTTBA/MWCNT electrodes in PBS containing 100 �M

A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 201 (2014) 51–58 55

-90 0 -70 0 -50 0 -300 -10 0 10 0

0

50

100

150 (B)

cbI/

μA

d

a

E/mV-90 0 -70 0 -50 0 -300 -10 0 10 0

-20

0

20

40

60

80

100 (A)

bI/μA

c

a

E/mV

-90 0 -70 0 -50 0 -300 -10 0 100

0

50

100

150

200

250 (C)I/μA

E/mV

5 10 15 20 250

50

100

150

200

250

I/μA

υ1/2/(mV/ s)1/2

F BA/MWp 0, (c) 1c rents

HsieAte

dpcgtsp

ssrrr

3

wistopfH

ig. 3. (A) CVs recorded for (a) bare, (b) Ox-pTTBA/MWCNT and (c) AgNPs/Ox-pTTTTBA/MWCNT electrode in PBS contains various concentrations of H2O2 (a) 0, (b) 5ontains 100 �M H2O2 at various scan rates. Inset shows a plot of cathodic peak cur

2O2. In comparison, a large enhanced current response can beeen with the AgNPs/Ox-pTTBA/MWCNT electrode, while no signif-cant signals were observed with either bare or Ox-pTTBA/MWCNTlectrodes. This might be attributed to the hybrid nanomaterials ofgNPs/Ox-pTTBA/MWCNT nanocomposite integrating the advan-

age properties of nanometal AgNPs and may therefore significantlynlarged the catalytic peak current of H2O2 reduction (Scheme 1).

In order to investigate the suitability of this method for theetermination of H2O2, the catalytic activities of the AgNPs/Ox-TTBA/MWCNT electrode toward H2O2 were examined at variousoncentrations (Fig. 3B). It was found that, the catalytic peak currentradually increased with the increasing concentration of H2O2 inhe solution. This indicates that the proposed sensor is potentiallyuitable for the electrochemical detection of H2O2, which wouldrobably behave well in the amperometric experiments.

Furthermore, the scan rate dependency of the CV peaks of theensor probe was also studied in PBS containing 100 �M H2O2 ashown in Fig. 3C. The cathodic peak current increased as the scanate increased from 10 to 500 mV/s. Inset of Fig. 3C shows a linearelationship of cathodic peak currents vs. the square root of scanates, indicating the process is diffusion-controlled [34].

.4. Optimization of H2O2 detection

The effect of pH on the response of the sensor probe to H2O2as studied in the range from pH 5.0 to 9.0 (Fig. 4A). The results

ndicated that, the current increased with the increasing pH of theolution until it reached 7.0. However, further increase over pH 7,he current response decreased. The maximum peak current was

btained at pH 7.0, indicating the catalytic activity of the sensorrobe was more effective at pH 7.0 and hence a higher peak currentor H2O2 reduction was observed. Therefore, the optimum pH for

2O2 sensor was selected to be pH 7.0.

CNT electrodes in PBS containing 100 �M H2O2. (B) CVs recorded for AgNPs/Ox-00 and (d) 150 �M. (C) CVs recorded for AgNPs/Ox-TTBA/MWCNT electrode in PBS

vs. the square root of scan rates.

The effect of adsorption time of AgNPs on the Ox-pTTBA/MWCNT nanocomposite was investigated (Fig. 4B). Thecatalytic peak current of H2O2 increased as the adsorption timeincreased from 0.5 to 2.0 h. Thereafter, the current response didnot significantly increase when the adsorption time increasedfurther up to 4.0 h. This might be attributed to that, AgNPs hasbeen capped entirely the surface of Ox-pTTBA/MWCNT electrodeafter 2 h. Hence, 2.0 h was used as the optimum adsorption time ofAgNPs.

The effect of applied potential on the amperometric currentresponse was also studied (Fig. 4C). The current response increasedas the applied potential shifted from −0.1 V to a more negativevalue, where the maximum current response was observed at−0.6 V. Applying more negative potential up to −0.9 V, the currentresponse did not significantly increase. Hence, −0.6 V was chosenas the optimum applied potential for H2O2 detection.

3.5. Amperometric response, selectivity and stability of H2O2sensor

Fig. 5A shows the typical current-time response of the sensorprobe upon successive addition of varying concentrations of H2O2.The sensor showed a fast amperometric response time less than5 s after each addition of H2O2 with a linear relationship from10 to 260 �M. The linear regression equation was expressed as:Ip (�A) = 2.42 (±0.42) + 0.58 (±0.01) [H2O2] (�M), with the corre-lation coefficient of 0.998. The relative standard deviation (RSD)was determined to be 4.8% and the detection limit was estimatedto be 0.24 ± 0.04 �M. The lower detection limit was obtained as

compared with other recently reported nonenzymatic H2O2 sen-sors based nanometals (Table 1), suggesting that the proposedsensor might be effective for the detection of H2O2 in practicalapplications.

56 A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 201 (2014) 51–58

4 5 6 7 8 9 1060

70

80

90

100

110(A)

I/µA

pH

0.0 -0.2 -0.4 -0.6 -0.8 -1.010

20

30

40

50(C)

E/V

I/µA

0 1 2 3 4 540

60

80

100

120(B)

I/µA

Time/h

Fig. 4. Optimization of H2O2 detection using the AgNPs/Ox-pTTBA/MWCNT

0 10 0 20 0 30 0 40 0 50 00

5

10

15

UA

DA

AA GA AP

Glu

H2O

2

H2O

2

Time/s

I/µA

(B)

0 200 400 600 8000

50

100

150

200

Time/s

I/µA

(A)

0 100 200 300050100150200

I/µA

H2O2/µM

Fig. 5. Amperometric response obtained using the sensor probe in PBS after multipleadditions of H2O2. Inset shows the corresponding calibration plot. (B) Amperogramof the addition of 5 �M H2O2 and 0.1 mM of other interfering species.

probe: (A) pH, (B) adsorption time of AgNPs and (C) applied potential.

To investigate the selectivity of the sensor probe, the ampero-metric responses to 5 �M H2O2 and 0.1 mM of different biologicalinterfering species was recorded (Fig. 5B). As shown, these com-pounds did not interfere with H2O2 detection, indicating that theproposed sensor completely prevented the diffusion of interfer-ing species. Moreover, the stability of the sensor toward H2O2 wasexamined with respect to the storage time. The sensor retainedabout 95% of its initial response to H2O2 over a period of one month.The repeatability of the sensor-to-sensor variation has been studiedfor five electrodes prepared under the same condition. The rela-tive standard deviation (R.S.D.) of these electrodes was 3.8% forthe current response to H2O2 indicating a good repeatability ofthe nanocomposite sensor. In addition, the stability of the sensorto multiple uses was also studied. The sensor lost only 2.5% fromits initial response to H2O2 after 20 continuous measurements ofH2O2 concentration. Although the AgNPs/Ox-pTTBA/MWCNT sen-sor probe is simple and easy to fabricate, it has also a long time

stability without significant change of its response to H2O2, indi-cates that the present method is an excellent for H2O2 detection.

Table 1Comparison of recently reported nonenzymatic H2O2 sensors based nanometalscomposite.

Nanocomposite Linear range(�M)

Detectionlimit (�M)

References

RuOHCF/MWCNT 100–1000 4.7 [9]Fe2O3/CoO 50–485 0.1 [10]Ag nanocrystals/graphene 20–1000 3 [13]Pt/AuNPs 1–450 1.18 [32]PdNPs/MWCNTs 1–1000 0.3 [33]AgNPs/Ox-pTTBA/MWCNT 10–260 0.24 [This work]

A.A. Abdelwahab, Y.-B. Shim / Sensors an

Table 2Determination of H2O2 in human urine samples at the AgNPs/Ox-pTTBA/MWCNTnanocomposite (n = 5).

Sample Spiked (�M) Found (�M) RSD Recovery

3

sudiuct

4

ioptaesnpssAtbam

A

b

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

1 10 10.46 4.1% 104.62 10 10.25 3.8% 102.53 10 9.82 3.2% 98.2

.6. Real samples application

To study the reliability of the AgNPs/Ox-pTTBA/MWCNT sen-or for practical applications, the determination of H2O2 in humanrine real samples was investigated. The samples was collected andiluted 100 times with 0.1 M PBS (pH 7.0). Table 2 shows the analyt-

cal values obtained for three urine samples. The recovery of spikedrine samples was in the range from 98.2 to 104.6%, which indi-ates the appreciable practicality of the nonenzymatic sensor forhe detection of H2O2 in real samples.

. Conclusion

We developed and characterized a novel sensor for analyt-cal detection of H2O2 based on the incorporation of AgNPsnto the Ox-pTTBA/MWCNT nanocomposite film. The AgNPs/Ox-TTBA/MWCNT probe exhibited high sensitivity and selectivityo H2O2 by significantly enhancing the reduction peak currentnd the effect of other interfering compounds was completelyliminated. The present sensor provides a novel route for synthe-izing hybrid nanomaterials which integrating the advantages ofanometals AgNPs for highly sensitive H2O2 sensor. The stable Ox-TTBA/MWCNT nanocomposite polymer was used as a matrix fortrong capturing of AgNPs. The higher catalytic response of the sen-or to H2O2 might be attributed to the close compaction betweengNPs and electrode for fast electron transfer. The advantages of

he proposed sensor are less expensive, easy to fabricate, highly sta-le, very sensitive, specific response to H2O2, and more convenients compared with other reports demonstrating that the presentethod is an ideally H2O2 sensor.

cknowledgement

This study was financially supported by the NRF grant fundedy the MEST, South Korea (No. 20100029128).

eferences

[1] M.R. Guascito, E. Filippo, C. Malitesta, D. Manno, A. Serra, A. Turco, A new amper-ometric nanostructured sensor for the analytical determination of hydrogenperoxi, Biosens. Bioelectron. 24 (2008) 1057–1063.

[2] C. Matsubara, N. Kawamoto, K. Takamura, Oxo[5,10, 15, 20-tetra(4-pyridyl)porphyrinato]titanium(IV): an ultra-high sensitivityspectrophotometric reagent for hydrogen peroxide, Analyst 117 (1992)1781–1784.

[3] L.S. Zhang, G.T.F. Wong, Optimal conditions and sample storage for the deter-mination of H2O2 in marine waters by the scopoletin–horseradish peroxidasefluorometric method, Talanta 48 (1999) 1031–1038.

[4] S. Hanaoka, J.M. Lin, M. Yamada, Chemiluminescent flow sensor for H2O2 basedon the decomposition of H2O2 catalyzed by cobalt(II)-ethanolamine compleximmobilized on resin, Anal. Chim. Acta 426 (2001) 57–64.

[5] A. Liu, W. Dong, E. Liu, W. Tang, J. Zhu, J. Han, Non-enzymatic hydrogen peroxidedetection using gold nanoclusters-modified phosphorus incorporated tetrahe-dral amorphous carbon electrodes, Electrochim. Acta 55 (2010) 1971–1977.

[6] S. Hrapovic, Y. Liu, K.B. Male, J.H.T. Luong, Electrochemical biosensing platformsusing platinum nanoparticles and carbon nanotubes, Anal. Chem. 76 (2004)1083–1088.

[7] J. Huang, D. Wang, H. Hou, T. You, Electrospun palladium nanoparticle-loaded

carbon nanofibers and their electrocatalytic activities towards hydrogen per-oxide and NADH, Adv. Funct. Mater. 18 (2008) 441–448.

[8] W.-Z. Le, Y.-Q. Liu, Preparation of nano-copper oxide modified glassy carbonelectrode by a novel film plating/potential cycling method and its characteri-zation, Sens. Actuators B: Chem. 141 (2009) 147–153.

d Actuators B 201 (2014) 51–58 57

[9] R.C. Pena, V.O. Silva, F.H. Quina, M. Bertotti, Hydrogen peroxide monitoringin Fenton reaction by using a ruthenium oxide hexacyanoferrate/multiwalledcarbon nanotubes modified electrode, J. Electroanal. Chem. 686 (2012) 1–6.

10] J. Wang, H. Gao, F. Sun, Q. Hao, C. Xu, Highly sensitive detection of hydrogenperoxide based on nanoporous Fe2O3/CoO composites, Biosens. Bioelectron. 42(2013) 550–555.

11] X. He, C. Hu, H. Liu, G. Du, Y. Xi, Y. Jiang, Building Ag nanoparticle 3D catalyst viaNa2Ti3O7 nanowires for the detection of hydrogen peroxide, Sens. Actuators B:Chem. 144 (2010) 289–294.

12] F.W. Campbell, S.R. Belding, R. Raron, L. Xiao, R.G. Compton, Hydrogen peroxideelectroreduction at a silver-nanoparticle array: investigating nanoparticle sizeand coverage effects, J. Phys. Chem. C 113 (2009) 9053–9062.

13] L. Zhong, S. Gan, X. Fu, F. Li, D. Han, L. Guo, L. Niu, Electrochemicallycontrolled growth of silver nanocrystals on graphene thin film and applica-tions for efficient nonenzymatic H2O2 biosensor, Electrochim. Acta 89 (2013)222–228.

14] A.A. Abdelwahab, W.C.A. Koh, H.-B. Noh, Y.-B. Shim, A selective nitric oxidenanocomposite biosensor based on direct electron transfer of microperoxidase:removal of interferences by co-immobilized enzymes, Biosens. Bioelectron. 26(2010) 1080–1086.

15] A.A. Abdelwahab, M.-S. Won, Y.-B. Shim, Direct electrochemistry of cholesteroloxidase immobilized on a conducting polymer: application for a cholesterolbiosensor, Electroanalysis 22 (2010) 21–25.

16] Y. Zhu, W.C.A. Koh, Y.-B. Shim, An amperometric immunosensor for IgG basedon conducting polymer and carbon nanotube-linked hydrazine label, Electro-analysis 22 (2010) 2908–2914.

17] T.-Y. Lee, Y.-B. Shim, Direct DNA hybridization detection based on theoligonucleotide-functionalized conductive polymer, Anal. Chem. 73 (2001)5629–5632.

18] M.A. Rahman, P. Kumar, D.S. Park, Y.-B. Shim, Electrochemical sensors basedon organic conjugated polymers, Sensors 8 (2008) 118–141.

19] W.C.A. Koh, P. Chandra, D.-M. Kim, Y.-B. Shim, Electropolymerized self-assembled layer on gold nanoparticles: detection of inducible nitric oxidesynthase in neuronal cell culture, Anal. Chem. 83 (2011) 6177–6183.

20] P. Chandra, H.-B. Noh, M.-S. Won, Y.-B. Shim, Detection of daunomycinusing phosphatidylserine and aptamer co-immobilized on Au nanoparticlesdeposited conducting polymer, Biosens. Bioelectron. 26 (2011) 4429–4442.

21] J. Wang, M. Musameh, Y. Lin, Solubilization of carbon nanotubes by nafiontoward the preparation of amperometric biosensors, J. Am. Chem. Soc. 125(2003) 2408–2409.

22] Y. Xu, P.E. Pehrsson, L. Chen, R. Zhang, W. Zhao, Double stranded DNA-singlewalled carbon nanotube hybrids for optical hydrogen peroxide and glucosesensing, Phys. Chem. C 111 (2007) 8638–8643.

23] Y. Zhu, J.I. Son, Y.-B. Shim, Amplification strategy based on gold nanoparticle-decorated carbon nanotubes for neomycin immunosensors, Biosens. Bioelec-tron. 26 (2010) 1002–1008.

24] B. Wu, Y. Kuang, X. Zhang, J. Chen, Noble metal nanoparticles/carbon nanotubesnanohybrids: synthesis and applications, Nano Today 6 (2011) 75–90.

25] J.-J. Zhang, M.-M. Gu, T.-T. Zheng, J.-J. Zhu, Synthesis of gelatin-stabilizedgold nanoparticles and assembly of carboxylic single-walled carbon nano-tubes/Au composites for cytosensing and drug uptake, Anal. Chem. 81 (2009)6641–6648.

26] B.-S. Kong, D.-H. Jung, S.-K. Oh, C.-S. Han, H.-T. Jung, Single-walled carbonnanotube gold nanohybrids: application in highly effective transparent andconductive films, Phys. Chem. C 111 (2007) 8377–8382.

27] D.-M. Kim, J.-H. Yoon, M.-S. Won, Y.-B. Shim, Electrochemical characteriza-tion of newly synthesized polyterthiophene benzoate and its applications toan electrochromic device and a photovoltaic cell, Electrochim. Acta 67 (2012)201–207.

28] R.C. Doty, T.R. Tshikhudo, M. Brust, D.G. Fernig, Extremely stable water-solubleAg nanoparticles, Chem. Mater. 17 (2005) 4630–4635.

29] A.A. Abdelwahab, O.-S. Jung, Y.-B. Shim, Enhanced electrocatalytic reductionof oxygen with a molecule having multi-quinone moieties adsorbed in thenanofiber film, J. Electroanal. Chem. 632 (2009) 102–108.

30] A.A. Abdelwahab, H.-M. Lee, Y.-B. Shim, Selective determination of dopaminewith a cibacron blue/poly-1,5-diaminonaphthalene composite film, Anal. Chim.Acta 650 (2009) 247–253.

31] A.A. Abdelwahab, D.-M. Kim, N.M. Halappa, Y.-B. Shim, A selective catalyticoxidation of ascorbic acid at the aminopyrimidyl functionalized-conductivepolymer electrode, Electroanalysis 25 (2013) 1178–1184.

32] Y. Li, Q. Lu, S. Wu, L. Wang, X. Shi, Hydrogen peroxide sensing using ultra-thin platinum-coated gold nanoparticles with core@shell structure, Biosens.Bioelectron. 41 (2013) 576–581.

33] J.M. You, Y.N. Jeong, S.K. Kim, H.C. Choi, S. Jeon, Reductive determination ofhydrogen peroxide with MWCNTs-Pd nanoparticles on a modified glassy car-bon electrode, Biosens. Bioelectron. 26 (2011) 2287–2291.

34] R.L. McCreery, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 17, MarcelDekker, New York, 1991.

Biographies

Adel A. Abdelwahab received his Ph.D. in analytical chemistry from Departmentof Chemistry at Pusan National University, South Korea, in 2010. He is working asa lecturer at Department of Chemistry, Al-Azhar University. His current research

5 sors an

ific

YU

8 A.A. Abdelwahab, Y.-B. Shim / Sen

nterests are the development of electrochemical sensors and biosensors, modi-

ed electrodes, study of electron transfer reaction of enzymes and proteins, andharacterization of conducting polymers and their applications.

oon-Bo Shim received his Ph.D. in Department of Chemistry at Pusan Nationalniversity, South Korea, in 1985. He is working as a professor at Department of

d Actuators B 201 (2014) 51–58

Chemistry and a director at Institute of BioPhysio Sensor Technology (IBST), Pusan

National University. His current research interests are the development of bio-(protein, DNA, enzyme, etc.)/chemical-sensors, electroanalytical method of tracebiological, organic species with modified electrodes, electron transfer of organiccompounds and proteins on the biomembranes, and characterization of conductingpolymers and their applications.