Hindawi Publishing CorporationISRN ElectrochemistryVolume 2013 Article ID 367872 9 pageshttpdxdoiorg1011552013367872

Research ArticleAn Immunosensor for Pathogenic Staphylococcus aureusBased on Antibody Modified Aminophenyl-Au Electrode

Amani Chrouda12 Mohamed Braiek1 Karima Bekir Rokbani3 Amina Bakhrouf3

Abderrazak Maaref2 and Nicole Jaffrezic-Renault1

1 Institute of Analytical Sciences University of Lyon UMR CNRS 5280 5 rue de la Doua 69100 Villeurbanne France2 Laboratory of Interfaces and Advanced Materials Faculty of Sciences University of Monastir Avenue de lrsquoEnvironnement5019 Monastir Tunisia

3 Laboratory of Analysis Treatment and Valorisation of Pollutants of Environment and of Products Faculty of PharmacyUniversity of Monastir 5019 Monastir Tunisia

Correspondence should be addressed to Nicole Jaffrezic-Renault nicolejaffrezicuniv-lyon1fr

Received 8 August 2013 Accepted 9 September 2013

Academic Editors S Arya and E M Richter

Copyright copy 2013 Amani Chrouda et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this work is to elaborate an immunosensing system which will detect and quantify Staphylococcus aureus bacteriaA gold electrode was modified by electrografting of 4-nitrophenyl diazonium in situ synthesized in acidic aqueous solutionThe immunosensor was fabricated by immobilizing affinity-purified polyclonal anti S aureus antibodies on the modified goldelectrode Cyclic voltammetry (CV) and Faradaic Electrochemical Impedance Spectroscopy (EIS) were employed to characterizethe stepwise assembly of the immunosensor The performance of the developed immunosensor was evaluated by monitoring theelectron-transfer resistance detected using Faradaic EISThe experimental results indicated a linear relationship between the relativevariation of the electron transfer resistance and the logarithmic value of S aureus concentration with a slope of 040 plusmn 008 perdecade of concentration A low quantification limit of 10 plusmn 2CFU per ml and a linear range up to 10

7plusmn 2 times 10

6 CFU per mL wereobtained The developed immunosensors showed high selectivity to Escherichia coli and Staphylococcus saprophyticus

1 Introduction

Staphylococcus aureus is a major human pathogen respon-sible for a broad range of diseases because it is able toproduce heat-resistant toxins in food [1] Foods that arefrequently incriminated in staphylococcal poisoning includemeat and meat products milk and water The toxic chemicalreleased by S aureus is enterotoxin that has been foundto be produced at a hazardous level for 108 cells per kg offood [2] Therefore rapid sensitive and reliable methods todetect foodborne pathogens are increasingly necessary inhealth care today Traditionalmethods for foodborne bacteriadetection including polymerase chain reaction (PCR) [3]enzyme-linked immunosorbent assay [4] and those based onculture and colony counting [5] have been widely applied dueto their high reliability However these methods suffer from

obvious disadvantages they are time consuming and requireexpensive equipment and complicated pretreatment so theyare difficult to use on site

Biosensor technologies play an increasingly importantrole in the detection of pathogenic bacteria because theypresent the great potential of satisfying the practical needfor rapid wearable and low-cost detection [6] Amongbiosensors immunosensors are widely investigated for bac-teria detection due to their specific advantages such ashigh affinity and simple fabrication [6 7] In the literaturemany recent studies focus on E coli and Salmonella bacteriadetection with different transducing techniques QCM [8]SPR [9] electrochemical techniques capacitive [10] andamperometric measurements [11] Although several electro-chemical immunosensors for the detection of foodbornepathogenic bacteria have been reported in the literature

2 ISRN Electrochemistry

only a few of them have been used for the quantificationof S aureus [12ndash14] Escamilla-Gomez et al reported anamperometric immunosensor for the determination of Saureus cells obtaining a detection limit of 37 times 10

2 cellsper mL [12] Tan et al have developed a PDMS-basedmicrofluidic immunosensor chip for the detection of food-borne pathogens S aureus and E coli O157 the detectionlimit of this impedimetric immunosensor modified with ananoporous aluminamembrane was around 102 CFU per mL[13] Braiek et al [14] have used a self-assembledmonolayer ofalkanethiols for the antibody immobilization on gold surfaceand EIS for the immunodetection of S aureus and havereported a sensor sensitivity of 127Ω per decade of CFU witha detection limit of 10 CFU per mL [14]

The originality reported in this present study is thedevelopment of an immunosensor based on a diazoniumelectrografted gold electrode for the detection of S aureuswhich has never been tested until now The method for elab-orating stable thin organic layers on conductive substratessuch as glassy carbon (GC) carbon nanotubes HOPG goldmetals and on semiconductive substrates is now recognizedas being an efficient procedure [15ndash22] The mechanism bywhich aryl groups attach themselves to gold is not fully under-stood although there is a good evidence for the formationof Au-C bonds [23] which in the case of aryldiazoniumresults in quite a stable layer By comparison alkanethiollayers that are highly stable [24] can only be applied toa rather limited number of materials (AuAg ) whereasaryldiazonium layers can also be applied to carbonaceousmaterialsThe use of electroreduction of aryl diazonium saltsprovides higher stability with respect to long-term storagein air potential use in cyclic voltammetry under acidicconditions and a wider potential window for subsequentelectrochemistry [25] Diazonium salt molecules modifiedwith various functional groups have been used for theimmobilization of nanoparticles [26] DNA [27] enzymes[28] carbon nanotubes [29 30] proteins antibodies andelectroactive species [31ndash34] on GC Si and Au surfacesMoreover applying an electrochemical potential to the spe-cific electrode (chip) allows biomolecule immobilizationwhich should be compatible with the production of biochipmicroarrays for multidetection

In this work anti-S aureus antibody is covalentlyanchored onto a gold electrode surface through graftingonto p-aminophenyl groups in situ generated in the aqueousdeposition solution The aryl diazonium covalently bindsto the surface by the spontaneous release of nitrogen [3536] and the nitro group is then reduced The differentsteps for immunosensor elaboration have been characterizedusing electrochemical measurements The EIS technique isan efficient electrochemical technique for the transductionof biosensing events on electrodes [37 38] and is there-fore used here for affinity assays antibody-cell binding Bymodeling Nyquist plots a calibration curve for S aureus hasbeen determined and the analytical characteristics of thisimmunosensor have been deduced and compared to those ofprevious relevant immunosensors

2 Experimental

21 Reagents Polyclonal antibodies (developed in rabbit)against Staphylococcus aureus were obtained from BIOtechRDP (Sfax Tunisia) P-nitroaniline (PNA) hydrochlo-ric acid (37) sodium nitrite phosphate buffered saline(PBS) potassium ferrocyanide [K

4Fe(CN)

6] potassium ferri-

cyanide [K3Fe(CN)

6] hydrogen peroxide (30) glutaralde-

hyde (25) and bovine serum albumin (BSA) were pur-chased from Sigma Aldrich All solutions were prepared withultra-pure Milli-Q water

22 Bacteria Cultivation S aureus reference strain(ATCC25923) was used in the present study The bacteriastrain was cultured in a tryptic soy broth [TSB (Difco)] oron TSB agar plates for 24 hrs at 37∘C A highly concentratedbacteria suspension was prepared as follows the liquidculture medium was inoculated with 100 120583L of pre-culturesolution and then cultivated at 37∘C for 18ndash24 hrs Thebacterial culture was centrifuged for 5min at 6400 rpm andthen washed twice Finally it was resuspended in sterilephosphate-buffered saline (PBS) The proportions of viablecells and bacterial concentration were determined by thespread-plate technique Optical density (OD) measuredusing a spectrophotometer was used as a measure of theconcentration of bacteria in a suspension The cultures ofS aureus strain were grown to the stationary growth phasefor OD

600= 06 At the stationary growth phase bacterial

concentration did not change with time and subsequentlyall the operations to determine bacterial concentration canbe performed accurately [14]

23 Electrochemical Instrumentation Electrochemical mea-surements were performed using a Voltalab 40 potentiostat-galvanostat with a standard three-electrode configurationThe measurement set-up consisted of a three-electrode sys-tem with a gold electrode (surface area 119860 = 007 cm2) asthe working electrode a saturated calomel electrode (SCE)as the reference electrode and a platinum plate as the counterelectrode

All electrochemical measurements were carried out ina measuring chamber of volume 119881 (chamber) = 5mLand inside a Faraday cage at room temperature 119879 =

296(plusmn3) 119870(23 plusmn 3∘C)

Two-electrochemical techniques were used in this workas follows

(i) Cyclic voltammetry was performed in 5mM ferroferricyanide PBS solution (8mM) at a scan rate of100mVs

(ii) Faradaic EIS was used by applying a small sinusoidalsignal (amplitude 10mV frequency range 100mHz to100 kHz) to the system at open circuit potential in5mM ferroferricyanide PBS solution (8mM)

(iii) The ZviewZplot modeling software compatible withWindows (provided by Scribner Associate Inc South-ern Pines NC USA) was used for fitting the Faradaicimpedance spectra

ISRN Electrochemistry 3

minus04 minus02 00 02 04 06minus1600

minus1200

minus800

minus400

0

400

123

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

minus14 minus12 minus10 minus08 minus06 minus04 minus02 00 02 04 06minus50

minus40

minus30

minus20

minus10

0

10

1

2345

EV (versus SCE)

J(m

Amiddotcm

minus2)

(b)

Figure 1 (a) Three cyclic voltammograms (numbered) for in situ-generated p-nitrophenyl diazonium salt (p-NP) at gold electrode in acidicsolution HCl (05M) scan rate 100mVs (b) Five successive cyclic voltammograms (numbered) of p-nitrophenyl-modified gold electrodein 01M KCl scan rate 100mVs

3 Results and Discussion

31 Functionalization of the Electrode Surface withp-Nitrophenyldiazonium Cation

311 Formation of p-Nitrophenyl Diazonium Cation Thegold surface was cleaned in acetone in an ultrasonic bathfor 10min dried under a nitrogen flow and then dipped for2min into a piranha solution 4 1 (vv) [98 H

2SO430

H2O2] rinsed with ultrapure water and dried under a

nitrogen flowThe diazonium cations were synthesized in situin accordance according to previously described procedure[35] 20mM of NaNO

2was added to the electrolytic solution

containing 20mM p-nitroaniline (PNA) in 05M HCl understirring at room temperature The solution was cooled in anice bath (the temperature did not exceed 0∘C)

312 Grafting of p-Nitrophenyl Diazonium Salt The electro-chemical grafting on golf electrode was immediately per-formed in the mixture above described by cyclic voltamme-try (three cycles from 06 to minus04VSCE at a scan rate of100mVs) The consecutive cyclic voltammograms (CVs) ofp-nitrophenyl diazonium at the gold electrode are presentedin Figure 1(a)

The CVs are characterized by the first cycle exhibiting awell-defined reproducible and irreversible reduction peaklocated at 04VSCE This feature corresponds to the typicalelectro-reduction reaction of the diazonium function leadingto the elimination of a nitrogen molecule and the productionof highly reactive radicals This radical was previously shownto attack the surface and to form a covalent bond between thearyl group and the Au electrode [39 40]

Very low currents were observed during the second andthird voltammetric cycles evidencing that surface saturationof grafted molecules has been achieved Although Brooksbyand Downard have pointed out that the electrochemicaldetermination of surface concentration must be interpreted

with caution [41 42] they assume a surface concentration ofnmolcm2 for one monolayer [43]

The surface concentration ΓNP of the grafted nitrophenyl(NP) groups can be calculated according to the followingformula

ΓNP =119876

119899119865119860

(1)

119876(119862) is the charge corresponding to the integral 119869(119905)119889119905 whichis the area of the current time reduction peak located at04VSCE (cf Figure 1(a)) It was calculated using Origin 8software 119899 is the number of electrons exchanged per reactantmolecule (119899 = 1) 119865 is the Faraday constant (96500Cmol)and 119860 is the surface area of the working electrode

A surface concentration (mole coverage) of p-NPG(NP) = 34(plusmn03) nmolcm2 was found when the experi-ments were repeated three times in the same conditions Thesurface concentration of p-NPon theAu surface is then foundas a significantly higher coverage than one monolayer for thefirst voltammetric half cycle

313 Reduction of Nitrophenyl Group to Aminophenyl GroupThe electrode was then washed and transferred to 01MKCl solution and subjected to five potential scans between04 and minus125VSCE at 100mVs in order to reduce thenitro group and obtain a modified film of 4-aminophenylon the electrode surface Figure 1(b) shows a cathodicsweep of the p-nitrophenyl modified gold electrode with anirreversible reduction peak at aroundminus09VSCEDuring thesecond scan the reduction peak is drastically diminishedindicating that nearly all the electroactive -Ph-NO

2groups

are reduced in the first scan However as pointed out inprevious works [15ndash17] the reduction of -Ph-NO

2to -Ph-

NH2is incomplete as evidenced by the reversible couple that

appears at 11986412

= minus026VSCEThis couple is assigned to thehydroxyaminophenylnitroso phenyl interconversion

4 ISRN Electrochemistry

minus04 minus02 00 02 04 06minus8

minus6

minus4

minus2

0

2

4

6

A

B

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 400

4

8

12

16

A

B

00 05 10 15 20 25 30 35 40 45 5000051015202530

Z998400(120596) (kΩ)

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

minusZ998400998400(120596

) (kΩ

)

(b)

Cf

Rs

Rf RCT

CPE

Zw

(c)

Figure 2 (a) Cyclic voltammograms of 5mM Fe(CN)6

3minus4minus at scan rate of 100mVs (A) on bare electrode (B) on p-AP-Au electrode (b)Nyquist plots in 5mM Fe(CN)

6

3minus4minus at open circuit potential using frequency range of 10 kHz to 10mHz (A) on bare electrode (B) on p-AP-Au electrode (c) An electrical equivalent circuit for both a bare and a modified electrode which comprises a series resistance 119877

119878(resistance

of solution and of ohmic contacts) an interfacial film resistance 119877119891 an interfacial film capacity 119862

119891 a resistance related to the charge transfer

rate of redox reactions at the functionalized electrode 119877CT a constant phase element (CPE) related to the capacitance of the functionalizedAu electrodeelectrolyte interface

Once the p-aminophenyl layer had been formed theelectrochemical behavior of the modified gold electrode sur-face was investigated by cyclic voltammetry in the presenceof the Fe2+Fe3+ redox couple Figure 2(a) shows the cyclicvoltammogram for bare Au electrode and for aminophenyl-Au electrode in the presence of the Fe2+Fe3+ redox coupleThis voltammogram is highly affected by the deposited layerthe redox peaks have entirely disappeared as expected forcompletely blocked surfaces The aminophenyl (pAP) groupis assumed to be covalently grafted to the Au surface throughthe formation of an Au-C bond [43 44] From the literatureafter diazonium cation reduction the stability of a diazoniumcompound on gold electrodes is weaker than on glassy carbonelectrodes [45]

Electrochemical impedance spectroscopy measurementswere also performed to further characterize the modifiedsurfaces Impedance119885(120596) is a complex number according to(2)

119885 (120596) = 1198851015840(120596) + 119895 sdot 119885

10158401015840(120596) (2)

where 120596 is the angular frequency 119895 = radicminus1 is the imaginarynumber and120596 the angular frequency is equal to 2120587119891119891 beingac-frequency

Impedance 119885(120596) is represented in the complex planeaccording to a Nyquist plot imaginary part (minus11988510158401015840(120596)) versusReal part (1198851015840(120596)) Figure 2(b) represents the Nyquist plots forbare Au electrode and for aminophenyl-Au electrode Indeedthe charge-transfer resistance estimated from the diameter ofthe semi-circle of the Nyquist plot is higher for the p-AP-Auelectrode than for the bare gold electrode This is due to thelower rate of electron transfer through the film showing highcoverage at the electrode

An electrical equivalent circuit for both a bare and amodified electrode shown in Figure 2(c) comprises a seriesresistance 119877

119878(resistance of solution and of ohmic contacts) a

resistance related to the charge transfer rate of redox reactionsat the functionalized electrode 119877CT a constant phase element(CPE) related to the capacitance of the functionalized Auelectrodeelectrolyte interface The capacitance of the inter-facial film 119862

119891in parallel with the resistance of the interfacial

film 119877119891are also included in the equivalent circuit

It is worth noting that the CPE reflects the nonide-ality of the double-layer at the functionalized gold elec-trodeelectrolyte interface due to the roughness and porosityof the interfacial film

ISRN Electrochemistry 5

minus04 minus02 00 02 04 06

minus3

minus2

minus1

0

1

2 A

B

C

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 40 50 60 700

8

16

24

32

C

B

A

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(b)

Figure 3 (a) Cyclic voltammograms of (A) p-AP-Au electrode (B) p-AP+GA-Au electrode and (C) antibody modified Au-p-AP+GA-Auelectrode recorded in the presence of Fe(CN

6)3minus4minus as a redox probe at scan rate of 100mVs (b) Nyquist plots of impedance spectra of (A)

Au-p-AP (B) with Au-p-AP+GA and (C) with antibody modified Au-p-AP+GA recorded in the presence of Fe(CN6)3minus4minus at a frequency

range between 01Hz and 100 kHz at open circuit potential

The CPE is defined as

119895 sdot 11988510158401015840(120596) =

1

119876

119895120596minus119899 (3)

where 119876 is the CPE constant and has the quality of asusceptance or 119876-capacitance 120596 is the angular frequency0 lt 119899 lt 1 is the factor describing deviation from idealcapacitor [46]

A specific electrochemical element of diffusion the War-burg element (119885

119908) is defined as

(119885119908) = 120590 (1 minus 119895) 120596

minus05 (4)

Here 120590 denotes the Warburg coefficient This equationrequires that 1198681198851015840(120596)119868 = 119868119885

10158401015840(120596)119868

32 Electrochemical Monitoring of Antibody Immobilization

321 Procedure of Covalent Grafting of the Antibody Follow-ing deposition of the p-aminophenyl (p-AP) film on the goldelectrode the terminal amine was activated by incubationfor 60min with glutaraldehyde (25) at pH 4 at roomtemperature [39] The value of pH was chosen close to pKaof aminophenyl group

After rinsing with PBS the electrode was incubated for 1hour in a 02mgmL solution of anti-S aureus antibodiesTheamino groups on the antibody molecule enable the covalentbonding with the glutaraldehyde activated amino functionson Au electrode The total time necessary for the wholeprocess of preparation of the immunosensor is 3 h

Finally after rinsing with PBS the electrodes were incu-bated for 20min in BSA (1) to block the unreacted aldehydegroups Cyclic voltammetry (CV) and EIS were employedto characterize the p-AP gold electrode (a) p-AP-GA goldelectrode (b) and p-AP-GA-Ab gold

Figure 3(a) shows the CV curves and Figure 3(b) Nyquistplots obtained with these surfaces

The Faradaic EIS measurements are in good agreementwith CV measurements the current density decreases (Fig-ure 3(a)) and the diameter of the semi-circles of the Nyquistdiagrams increases significantly after the different stages ofthe elaboration of the immunosensor (Figure 3(b)) Thisindicates that following the observed blocking effect of thep-AP film a further blocking effect was observed after thebinding of glutaraldehyde and the covalent immobilizationof the antibody

322 Number of Immobilized Antibodies (Binding Sites) Inorder to calculate the number of binding sites we calculate theantibody surface coverage 120579Ab using the following equation

1 minus 120579Ab =119877CTAP119877CTAb

(5)

119877CTAb Charge transfer resistance after antibody immobiliza-tion 119877CTAP Charge transfer resistance after grafting of theaminophenyl layer55 of the surface is found to be covered with antibodiesConsidering the surface density for a dense antibody layerdetermined in [47] the number of immobilized antibodieson the aminophenyl layer it should be 48 times 10

12 Abcm2

33 Detection of S aureus Cells Using ElectrochemicalImpedance Spectroscopy Having developed our sensor westudied its response towards different concentrations of theS aureus bacteria The electrodes were incubated in different

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

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Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 ISRN Electrochemistry

only a few of them have been used for the quantificationof S aureus [12ndash14] Escamilla-Gomez et al reported anamperometric immunosensor for the determination of Saureus cells obtaining a detection limit of 37 times 10

2 cellsper mL [12] Tan et al have developed a PDMS-basedmicrofluidic immunosensor chip for the detection of food-borne pathogens S aureus and E coli O157 the detectionlimit of this impedimetric immunosensor modified with ananoporous aluminamembrane was around 102 CFU per mL[13] Braiek et al [14] have used a self-assembledmonolayer ofalkanethiols for the antibody immobilization on gold surfaceand EIS for the immunodetection of S aureus and havereported a sensor sensitivity of 127Ω per decade of CFU witha detection limit of 10 CFU per mL [14]

The originality reported in this present study is thedevelopment of an immunosensor based on a diazoniumelectrografted gold electrode for the detection of S aureuswhich has never been tested until now The method for elab-orating stable thin organic layers on conductive substratessuch as glassy carbon (GC) carbon nanotubes HOPG goldmetals and on semiconductive substrates is now recognizedas being an efficient procedure [15ndash22] The mechanism bywhich aryl groups attach themselves to gold is not fully under-stood although there is a good evidence for the formationof Au-C bonds [23] which in the case of aryldiazoniumresults in quite a stable layer By comparison alkanethiollayers that are highly stable [24] can only be applied toa rather limited number of materials (AuAg ) whereasaryldiazonium layers can also be applied to carbonaceousmaterialsThe use of electroreduction of aryl diazonium saltsprovides higher stability with respect to long-term storagein air potential use in cyclic voltammetry under acidicconditions and a wider potential window for subsequentelectrochemistry [25] Diazonium salt molecules modifiedwith various functional groups have been used for theimmobilization of nanoparticles [26] DNA [27] enzymes[28] carbon nanotubes [29 30] proteins antibodies andelectroactive species [31ndash34] on GC Si and Au surfacesMoreover applying an electrochemical potential to the spe-cific electrode (chip) allows biomolecule immobilizationwhich should be compatible with the production of biochipmicroarrays for multidetection

In this work anti-S aureus antibody is covalentlyanchored onto a gold electrode surface through graftingonto p-aminophenyl groups in situ generated in the aqueousdeposition solution The aryl diazonium covalently bindsto the surface by the spontaneous release of nitrogen [3536] and the nitro group is then reduced The differentsteps for immunosensor elaboration have been characterizedusing electrochemical measurements The EIS technique isan efficient electrochemical technique for the transductionof biosensing events on electrodes [37 38] and is there-fore used here for affinity assays antibody-cell binding Bymodeling Nyquist plots a calibration curve for S aureus hasbeen determined and the analytical characteristics of thisimmunosensor have been deduced and compared to those ofprevious relevant immunosensors

2 Experimental

21 Reagents Polyclonal antibodies (developed in rabbit)against Staphylococcus aureus were obtained from BIOtechRDP (Sfax Tunisia) P-nitroaniline (PNA) hydrochlo-ric acid (37) sodium nitrite phosphate buffered saline(PBS) potassium ferrocyanide [K

4Fe(CN)

6] potassium ferri-

cyanide [K3Fe(CN)

6] hydrogen peroxide (30) glutaralde-

hyde (25) and bovine serum albumin (BSA) were pur-chased from Sigma Aldrich All solutions were prepared withultra-pure Milli-Q water

22 Bacteria Cultivation S aureus reference strain(ATCC25923) was used in the present study The bacteriastrain was cultured in a tryptic soy broth [TSB (Difco)] oron TSB agar plates for 24 hrs at 37∘C A highly concentratedbacteria suspension was prepared as follows the liquidculture medium was inoculated with 100 120583L of pre-culturesolution and then cultivated at 37∘C for 18ndash24 hrs Thebacterial culture was centrifuged for 5min at 6400 rpm andthen washed twice Finally it was resuspended in sterilephosphate-buffered saline (PBS) The proportions of viablecells and bacterial concentration were determined by thespread-plate technique Optical density (OD) measuredusing a spectrophotometer was used as a measure of theconcentration of bacteria in a suspension The cultures ofS aureus strain were grown to the stationary growth phasefor OD

600= 06 At the stationary growth phase bacterial

concentration did not change with time and subsequentlyall the operations to determine bacterial concentration canbe performed accurately [14]

23 Electrochemical Instrumentation Electrochemical mea-surements were performed using a Voltalab 40 potentiostat-galvanostat with a standard three-electrode configurationThe measurement set-up consisted of a three-electrode sys-tem with a gold electrode (surface area 119860 = 007 cm2) asthe working electrode a saturated calomel electrode (SCE)as the reference electrode and a platinum plate as the counterelectrode

All electrochemical measurements were carried out ina measuring chamber of volume 119881 (chamber) = 5mLand inside a Faraday cage at room temperature 119879 =

296(plusmn3) 119870(23 plusmn 3∘C)

Two-electrochemical techniques were used in this workas follows

(i) Cyclic voltammetry was performed in 5mM ferroferricyanide PBS solution (8mM) at a scan rate of100mVs

(ii) Faradaic EIS was used by applying a small sinusoidalsignal (amplitude 10mV frequency range 100mHz to100 kHz) to the system at open circuit potential in5mM ferroferricyanide PBS solution (8mM)

(iii) The ZviewZplot modeling software compatible withWindows (provided by Scribner Associate Inc South-ern Pines NC USA) was used for fitting the Faradaicimpedance spectra

ISRN Electrochemistry 3

minus04 minus02 00 02 04 06minus1600

minus1200

minus800

minus400

0

400

123

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

minus14 minus12 minus10 minus08 minus06 minus04 minus02 00 02 04 06minus50

minus40

minus30

minus20

minus10

0

10

1

2345

EV (versus SCE)

J(m

Amiddotcm

minus2)

(b)

Figure 1 (a) Three cyclic voltammograms (numbered) for in situ-generated p-nitrophenyl diazonium salt (p-NP) at gold electrode in acidicsolution HCl (05M) scan rate 100mVs (b) Five successive cyclic voltammograms (numbered) of p-nitrophenyl-modified gold electrodein 01M KCl scan rate 100mVs

3 Results and Discussion

31 Functionalization of the Electrode Surface withp-Nitrophenyldiazonium Cation

311 Formation of p-Nitrophenyl Diazonium Cation Thegold surface was cleaned in acetone in an ultrasonic bathfor 10min dried under a nitrogen flow and then dipped for2min into a piranha solution 4 1 (vv) [98 H

2SO430

H2O2] rinsed with ultrapure water and dried under a

nitrogen flowThe diazonium cations were synthesized in situin accordance according to previously described procedure[35] 20mM of NaNO

2was added to the electrolytic solution

containing 20mM p-nitroaniline (PNA) in 05M HCl understirring at room temperature The solution was cooled in anice bath (the temperature did not exceed 0∘C)

312 Grafting of p-Nitrophenyl Diazonium Salt The electro-chemical grafting on golf electrode was immediately per-formed in the mixture above described by cyclic voltamme-try (three cycles from 06 to minus04VSCE at a scan rate of100mVs) The consecutive cyclic voltammograms (CVs) ofp-nitrophenyl diazonium at the gold electrode are presentedin Figure 1(a)

The CVs are characterized by the first cycle exhibiting awell-defined reproducible and irreversible reduction peaklocated at 04VSCE This feature corresponds to the typicalelectro-reduction reaction of the diazonium function leadingto the elimination of a nitrogen molecule and the productionof highly reactive radicals This radical was previously shownto attack the surface and to form a covalent bond between thearyl group and the Au electrode [39 40]

Very low currents were observed during the second andthird voltammetric cycles evidencing that surface saturationof grafted molecules has been achieved Although Brooksbyand Downard have pointed out that the electrochemicaldetermination of surface concentration must be interpreted

with caution [41 42] they assume a surface concentration ofnmolcm2 for one monolayer [43]

The surface concentration ΓNP of the grafted nitrophenyl(NP) groups can be calculated according to the followingformula

ΓNP =119876

119899119865119860

(1)

119876(119862) is the charge corresponding to the integral 119869(119905)119889119905 whichis the area of the current time reduction peak located at04VSCE (cf Figure 1(a)) It was calculated using Origin 8software 119899 is the number of electrons exchanged per reactantmolecule (119899 = 1) 119865 is the Faraday constant (96500Cmol)and 119860 is the surface area of the working electrode

A surface concentration (mole coverage) of p-NPG(NP) = 34(plusmn03) nmolcm2 was found when the experi-ments were repeated three times in the same conditions Thesurface concentration of p-NPon theAu surface is then foundas a significantly higher coverage than one monolayer for thefirst voltammetric half cycle

313 Reduction of Nitrophenyl Group to Aminophenyl GroupThe electrode was then washed and transferred to 01MKCl solution and subjected to five potential scans between04 and minus125VSCE at 100mVs in order to reduce thenitro group and obtain a modified film of 4-aminophenylon the electrode surface Figure 1(b) shows a cathodicsweep of the p-nitrophenyl modified gold electrode with anirreversible reduction peak at aroundminus09VSCEDuring thesecond scan the reduction peak is drastically diminishedindicating that nearly all the electroactive -Ph-NO

2groups

are reduced in the first scan However as pointed out inprevious works [15ndash17] the reduction of -Ph-NO

2to -Ph-

NH2is incomplete as evidenced by the reversible couple that

appears at 11986412

= minus026VSCEThis couple is assigned to thehydroxyaminophenylnitroso phenyl interconversion

4 ISRN Electrochemistry

minus04 minus02 00 02 04 06minus8

minus6

minus4

minus2

0

2

4

6

A

B

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 400

4

8

12

16

A

B

00 05 10 15 20 25 30 35 40 45 5000051015202530

Z998400(120596) (kΩ)

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

minusZ998400998400(120596

) (kΩ

)

(b)

Cf

Rs

Rf RCT

CPE

Zw

(c)

Figure 2 (a) Cyclic voltammograms of 5mM Fe(CN)6

3minus4minus at scan rate of 100mVs (A) on bare electrode (B) on p-AP-Au electrode (b)Nyquist plots in 5mM Fe(CN)

6

3minus4minus at open circuit potential using frequency range of 10 kHz to 10mHz (A) on bare electrode (B) on p-AP-Au electrode (c) An electrical equivalent circuit for both a bare and a modified electrode which comprises a series resistance 119877

119878(resistance

of solution and of ohmic contacts) an interfacial film resistance 119877119891 an interfacial film capacity 119862

119891 a resistance related to the charge transfer

rate of redox reactions at the functionalized electrode 119877CT a constant phase element (CPE) related to the capacitance of the functionalizedAu electrodeelectrolyte interface

Once the p-aminophenyl layer had been formed theelectrochemical behavior of the modified gold electrode sur-face was investigated by cyclic voltammetry in the presenceof the Fe2+Fe3+ redox couple Figure 2(a) shows the cyclicvoltammogram for bare Au electrode and for aminophenyl-Au electrode in the presence of the Fe2+Fe3+ redox coupleThis voltammogram is highly affected by the deposited layerthe redox peaks have entirely disappeared as expected forcompletely blocked surfaces The aminophenyl (pAP) groupis assumed to be covalently grafted to the Au surface throughthe formation of an Au-C bond [43 44] From the literatureafter diazonium cation reduction the stability of a diazoniumcompound on gold electrodes is weaker than on glassy carbonelectrodes [45]

Electrochemical impedance spectroscopy measurementswere also performed to further characterize the modifiedsurfaces Impedance119885(120596) is a complex number according to(2)

119885 (120596) = 1198851015840(120596) + 119895 sdot 119885

10158401015840(120596) (2)

where 120596 is the angular frequency 119895 = radicminus1 is the imaginarynumber and120596 the angular frequency is equal to 2120587119891119891 beingac-frequency

Impedance 119885(120596) is represented in the complex planeaccording to a Nyquist plot imaginary part (minus11988510158401015840(120596)) versusReal part (1198851015840(120596)) Figure 2(b) represents the Nyquist plots forbare Au electrode and for aminophenyl-Au electrode Indeedthe charge-transfer resistance estimated from the diameter ofthe semi-circle of the Nyquist plot is higher for the p-AP-Auelectrode than for the bare gold electrode This is due to thelower rate of electron transfer through the film showing highcoverage at the electrode

An electrical equivalent circuit for both a bare and amodified electrode shown in Figure 2(c) comprises a seriesresistance 119877

119878(resistance of solution and of ohmic contacts) a

resistance related to the charge transfer rate of redox reactionsat the functionalized electrode 119877CT a constant phase element(CPE) related to the capacitance of the functionalized Auelectrodeelectrolyte interface The capacitance of the inter-facial film 119862

119891in parallel with the resistance of the interfacial

film 119877119891are also included in the equivalent circuit

It is worth noting that the CPE reflects the nonide-ality of the double-layer at the functionalized gold elec-trodeelectrolyte interface due to the roughness and porosityof the interfacial film

ISRN Electrochemistry 5

minus04 minus02 00 02 04 06

minus3

minus2

minus1

0

1

2 A

B

C

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 40 50 60 700

8

16

24

32

C

B

A

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(b)

Figure 3 (a) Cyclic voltammograms of (A) p-AP-Au electrode (B) p-AP+GA-Au electrode and (C) antibody modified Au-p-AP+GA-Auelectrode recorded in the presence of Fe(CN

6)3minus4minus as a redox probe at scan rate of 100mVs (b) Nyquist plots of impedance spectra of (A)

Au-p-AP (B) with Au-p-AP+GA and (C) with antibody modified Au-p-AP+GA recorded in the presence of Fe(CN6)3minus4minus at a frequency

range between 01Hz and 100 kHz at open circuit potential

The CPE is defined as

119895 sdot 11988510158401015840(120596) =

1

119876

119895120596minus119899 (3)

where 119876 is the CPE constant and has the quality of asusceptance or 119876-capacitance 120596 is the angular frequency0 lt 119899 lt 1 is the factor describing deviation from idealcapacitor [46]

A specific electrochemical element of diffusion the War-burg element (119885

119908) is defined as

(119885119908) = 120590 (1 minus 119895) 120596

minus05 (4)

Here 120590 denotes the Warburg coefficient This equationrequires that 1198681198851015840(120596)119868 = 119868119885

10158401015840(120596)119868

32 Electrochemical Monitoring of Antibody Immobilization

321 Procedure of Covalent Grafting of the Antibody Follow-ing deposition of the p-aminophenyl (p-AP) film on the goldelectrode the terminal amine was activated by incubationfor 60min with glutaraldehyde (25) at pH 4 at roomtemperature [39] The value of pH was chosen close to pKaof aminophenyl group

After rinsing with PBS the electrode was incubated for 1hour in a 02mgmL solution of anti-S aureus antibodiesTheamino groups on the antibody molecule enable the covalentbonding with the glutaraldehyde activated amino functionson Au electrode The total time necessary for the wholeprocess of preparation of the immunosensor is 3 h

Finally after rinsing with PBS the electrodes were incu-bated for 20min in BSA (1) to block the unreacted aldehydegroups Cyclic voltammetry (CV) and EIS were employedto characterize the p-AP gold electrode (a) p-AP-GA goldelectrode (b) and p-AP-GA-Ab gold

Figure 3(a) shows the CV curves and Figure 3(b) Nyquistplots obtained with these surfaces

The Faradaic EIS measurements are in good agreementwith CV measurements the current density decreases (Fig-ure 3(a)) and the diameter of the semi-circles of the Nyquistdiagrams increases significantly after the different stages ofthe elaboration of the immunosensor (Figure 3(b)) Thisindicates that following the observed blocking effect of thep-AP film a further blocking effect was observed after thebinding of glutaraldehyde and the covalent immobilizationof the antibody

322 Number of Immobilized Antibodies (Binding Sites) Inorder to calculate the number of binding sites we calculate theantibody surface coverage 120579Ab using the following equation

1 minus 120579Ab =119877CTAP119877CTAb

(5)

119877CTAb Charge transfer resistance after antibody immobiliza-tion 119877CTAP Charge transfer resistance after grafting of theaminophenyl layer55 of the surface is found to be covered with antibodiesConsidering the surface density for a dense antibody layerdetermined in [47] the number of immobilized antibodieson the aminophenyl layer it should be 48 times 10

12 Abcm2

33 Detection of S aureus Cells Using ElectrochemicalImpedance Spectroscopy Having developed our sensor westudied its response towards different concentrations of theS aureus bacteria The electrodes were incubated in different

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Electrochemistry 3

minus04 minus02 00 02 04 06minus1600

minus1200

minus800

minus400

0

400

123

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

minus14 minus12 minus10 minus08 minus06 minus04 minus02 00 02 04 06minus50

minus40

minus30

minus20

minus10

0

10

1

2345

EV (versus SCE)

J(m

Amiddotcm

minus2)

(b)

Figure 1 (a) Three cyclic voltammograms (numbered) for in situ-generated p-nitrophenyl diazonium salt (p-NP) at gold electrode in acidicsolution HCl (05M) scan rate 100mVs (b) Five successive cyclic voltammograms (numbered) of p-nitrophenyl-modified gold electrodein 01M KCl scan rate 100mVs

3 Results and Discussion

31 Functionalization of the Electrode Surface withp-Nitrophenyldiazonium Cation

311 Formation of p-Nitrophenyl Diazonium Cation Thegold surface was cleaned in acetone in an ultrasonic bathfor 10min dried under a nitrogen flow and then dipped for2min into a piranha solution 4 1 (vv) [98 H

2SO430

H2O2] rinsed with ultrapure water and dried under a

nitrogen flowThe diazonium cations were synthesized in situin accordance according to previously described procedure[35] 20mM of NaNO

2was added to the electrolytic solution

containing 20mM p-nitroaniline (PNA) in 05M HCl understirring at room temperature The solution was cooled in anice bath (the temperature did not exceed 0∘C)

312 Grafting of p-Nitrophenyl Diazonium Salt The electro-chemical grafting on golf electrode was immediately per-formed in the mixture above described by cyclic voltamme-try (three cycles from 06 to minus04VSCE at a scan rate of100mVs) The consecutive cyclic voltammograms (CVs) ofp-nitrophenyl diazonium at the gold electrode are presentedin Figure 1(a)

The CVs are characterized by the first cycle exhibiting awell-defined reproducible and irreversible reduction peaklocated at 04VSCE This feature corresponds to the typicalelectro-reduction reaction of the diazonium function leadingto the elimination of a nitrogen molecule and the productionof highly reactive radicals This radical was previously shownto attack the surface and to form a covalent bond between thearyl group and the Au electrode [39 40]

Very low currents were observed during the second andthird voltammetric cycles evidencing that surface saturationof grafted molecules has been achieved Although Brooksbyand Downard have pointed out that the electrochemicaldetermination of surface concentration must be interpreted

with caution [41 42] they assume a surface concentration ofnmolcm2 for one monolayer [43]

The surface concentration ΓNP of the grafted nitrophenyl(NP) groups can be calculated according to the followingformula

ΓNP =119876

119899119865119860

(1)

119876(119862) is the charge corresponding to the integral 119869(119905)119889119905 whichis the area of the current time reduction peak located at04VSCE (cf Figure 1(a)) It was calculated using Origin 8software 119899 is the number of electrons exchanged per reactantmolecule (119899 = 1) 119865 is the Faraday constant (96500Cmol)and 119860 is the surface area of the working electrode

A surface concentration (mole coverage) of p-NPG(NP) = 34(plusmn03) nmolcm2 was found when the experi-ments were repeated three times in the same conditions Thesurface concentration of p-NPon theAu surface is then foundas a significantly higher coverage than one monolayer for thefirst voltammetric half cycle

313 Reduction of Nitrophenyl Group to Aminophenyl GroupThe electrode was then washed and transferred to 01MKCl solution and subjected to five potential scans between04 and minus125VSCE at 100mVs in order to reduce thenitro group and obtain a modified film of 4-aminophenylon the electrode surface Figure 1(b) shows a cathodicsweep of the p-nitrophenyl modified gold electrode with anirreversible reduction peak at aroundminus09VSCEDuring thesecond scan the reduction peak is drastically diminishedindicating that nearly all the electroactive -Ph-NO

2groups

are reduced in the first scan However as pointed out inprevious works [15ndash17] the reduction of -Ph-NO

2to -Ph-

NH2is incomplete as evidenced by the reversible couple that

appears at 11986412

= minus026VSCEThis couple is assigned to thehydroxyaminophenylnitroso phenyl interconversion

4 ISRN Electrochemistry

minus04 minus02 00 02 04 06minus8

minus6

minus4

minus2

0

2

4

6

A

B

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 400

4

8

12

16

A

B

00 05 10 15 20 25 30 35 40 45 5000051015202530

Z998400(120596) (kΩ)

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

minusZ998400998400(120596

) (kΩ

)

(b)

Cf

Rs

Rf RCT

CPE

Zw

(c)

Figure 2 (a) Cyclic voltammograms of 5mM Fe(CN)6

3minus4minus at scan rate of 100mVs (A) on bare electrode (B) on p-AP-Au electrode (b)Nyquist plots in 5mM Fe(CN)

6

3minus4minus at open circuit potential using frequency range of 10 kHz to 10mHz (A) on bare electrode (B) on p-AP-Au electrode (c) An electrical equivalent circuit for both a bare and a modified electrode which comprises a series resistance 119877

119878(resistance

of solution and of ohmic contacts) an interfacial film resistance 119877119891 an interfacial film capacity 119862

119891 a resistance related to the charge transfer

rate of redox reactions at the functionalized electrode 119877CT a constant phase element (CPE) related to the capacitance of the functionalizedAu electrodeelectrolyte interface

Once the p-aminophenyl layer had been formed theelectrochemical behavior of the modified gold electrode sur-face was investigated by cyclic voltammetry in the presenceof the Fe2+Fe3+ redox couple Figure 2(a) shows the cyclicvoltammogram for bare Au electrode and for aminophenyl-Au electrode in the presence of the Fe2+Fe3+ redox coupleThis voltammogram is highly affected by the deposited layerthe redox peaks have entirely disappeared as expected forcompletely blocked surfaces The aminophenyl (pAP) groupis assumed to be covalently grafted to the Au surface throughthe formation of an Au-C bond [43 44] From the literatureafter diazonium cation reduction the stability of a diazoniumcompound on gold electrodes is weaker than on glassy carbonelectrodes [45]

Electrochemical impedance spectroscopy measurementswere also performed to further characterize the modifiedsurfaces Impedance119885(120596) is a complex number according to(2)

119885 (120596) = 1198851015840(120596) + 119895 sdot 119885

10158401015840(120596) (2)

where 120596 is the angular frequency 119895 = radicminus1 is the imaginarynumber and120596 the angular frequency is equal to 2120587119891119891 beingac-frequency

Impedance 119885(120596) is represented in the complex planeaccording to a Nyquist plot imaginary part (minus11988510158401015840(120596)) versusReal part (1198851015840(120596)) Figure 2(b) represents the Nyquist plots forbare Au electrode and for aminophenyl-Au electrode Indeedthe charge-transfer resistance estimated from the diameter ofthe semi-circle of the Nyquist plot is higher for the p-AP-Auelectrode than for the bare gold electrode This is due to thelower rate of electron transfer through the film showing highcoverage at the electrode

An electrical equivalent circuit for both a bare and amodified electrode shown in Figure 2(c) comprises a seriesresistance 119877

119878(resistance of solution and of ohmic contacts) a

resistance related to the charge transfer rate of redox reactionsat the functionalized electrode 119877CT a constant phase element(CPE) related to the capacitance of the functionalized Auelectrodeelectrolyte interface The capacitance of the inter-facial film 119862

119891in parallel with the resistance of the interfacial

film 119877119891are also included in the equivalent circuit

It is worth noting that the CPE reflects the nonide-ality of the double-layer at the functionalized gold elec-trodeelectrolyte interface due to the roughness and porosityof the interfacial film

ISRN Electrochemistry 5

minus04 minus02 00 02 04 06

minus3

minus2

minus1

0

1

2 A

B

C

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 40 50 60 700

8

16

24

32

C

B

A

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(b)

Figure 3 (a) Cyclic voltammograms of (A) p-AP-Au electrode (B) p-AP+GA-Au electrode and (C) antibody modified Au-p-AP+GA-Auelectrode recorded in the presence of Fe(CN

6)3minus4minus as a redox probe at scan rate of 100mVs (b) Nyquist plots of impedance spectra of (A)

Au-p-AP (B) with Au-p-AP+GA and (C) with antibody modified Au-p-AP+GA recorded in the presence of Fe(CN6)3minus4minus at a frequency

range between 01Hz and 100 kHz at open circuit potential

The CPE is defined as

119895 sdot 11988510158401015840(120596) =

1

119876

119895120596minus119899 (3)

where 119876 is the CPE constant and has the quality of asusceptance or 119876-capacitance 120596 is the angular frequency0 lt 119899 lt 1 is the factor describing deviation from idealcapacitor [46]

A specific electrochemical element of diffusion the War-burg element (119885

119908) is defined as

(119885119908) = 120590 (1 minus 119895) 120596

minus05 (4)

Here 120590 denotes the Warburg coefficient This equationrequires that 1198681198851015840(120596)119868 = 119868119885

10158401015840(120596)119868

32 Electrochemical Monitoring of Antibody Immobilization

321 Procedure of Covalent Grafting of the Antibody Follow-ing deposition of the p-aminophenyl (p-AP) film on the goldelectrode the terminal amine was activated by incubationfor 60min with glutaraldehyde (25) at pH 4 at roomtemperature [39] The value of pH was chosen close to pKaof aminophenyl group

After rinsing with PBS the electrode was incubated for 1hour in a 02mgmL solution of anti-S aureus antibodiesTheamino groups on the antibody molecule enable the covalentbonding with the glutaraldehyde activated amino functionson Au electrode The total time necessary for the wholeprocess of preparation of the immunosensor is 3 h

Finally after rinsing with PBS the electrodes were incu-bated for 20min in BSA (1) to block the unreacted aldehydegroups Cyclic voltammetry (CV) and EIS were employedto characterize the p-AP gold electrode (a) p-AP-GA goldelectrode (b) and p-AP-GA-Ab gold

Figure 3(a) shows the CV curves and Figure 3(b) Nyquistplots obtained with these surfaces

The Faradaic EIS measurements are in good agreementwith CV measurements the current density decreases (Fig-ure 3(a)) and the diameter of the semi-circles of the Nyquistdiagrams increases significantly after the different stages ofthe elaboration of the immunosensor (Figure 3(b)) Thisindicates that following the observed blocking effect of thep-AP film a further blocking effect was observed after thebinding of glutaraldehyde and the covalent immobilizationof the antibody

322 Number of Immobilized Antibodies (Binding Sites) Inorder to calculate the number of binding sites we calculate theantibody surface coverage 120579Ab using the following equation

1 minus 120579Ab =119877CTAP119877CTAb

(5)

119877CTAb Charge transfer resistance after antibody immobiliza-tion 119877CTAP Charge transfer resistance after grafting of theaminophenyl layer55 of the surface is found to be covered with antibodiesConsidering the surface density for a dense antibody layerdetermined in [47] the number of immobilized antibodieson the aminophenyl layer it should be 48 times 10

12 Abcm2

33 Detection of S aureus Cells Using ElectrochemicalImpedance Spectroscopy Having developed our sensor westudied its response towards different concentrations of theS aureus bacteria The electrodes were incubated in different

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 ISRN Electrochemistry

minus04 minus02 00 02 04 06minus8

minus6

minus4

minus2

0

2

4

6

A

B

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 400

4

8

12

16

A

B

00 05 10 15 20 25 30 35 40 45 5000051015202530

Z998400(120596) (kΩ)

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

minusZ998400998400(120596

) (kΩ

)

(b)

Cf

Rs

Rf RCT

CPE

Zw

(c)

Figure 2 (a) Cyclic voltammograms of 5mM Fe(CN)6

3minus4minus at scan rate of 100mVs (A) on bare electrode (B) on p-AP-Au electrode (b)Nyquist plots in 5mM Fe(CN)

6

3minus4minus at open circuit potential using frequency range of 10 kHz to 10mHz (A) on bare electrode (B) on p-AP-Au electrode (c) An electrical equivalent circuit for both a bare and a modified electrode which comprises a series resistance 119877

119878(resistance

of solution and of ohmic contacts) an interfacial film resistance 119877119891 an interfacial film capacity 119862

119891 a resistance related to the charge transfer

rate of redox reactions at the functionalized electrode 119877CT a constant phase element (CPE) related to the capacitance of the functionalizedAu electrodeelectrolyte interface

Once the p-aminophenyl layer had been formed theelectrochemical behavior of the modified gold electrode sur-face was investigated by cyclic voltammetry in the presenceof the Fe2+Fe3+ redox couple Figure 2(a) shows the cyclicvoltammogram for bare Au electrode and for aminophenyl-Au electrode in the presence of the Fe2+Fe3+ redox coupleThis voltammogram is highly affected by the deposited layerthe redox peaks have entirely disappeared as expected forcompletely blocked surfaces The aminophenyl (pAP) groupis assumed to be covalently grafted to the Au surface throughthe formation of an Au-C bond [43 44] From the literatureafter diazonium cation reduction the stability of a diazoniumcompound on gold electrodes is weaker than on glassy carbonelectrodes [45]

Electrochemical impedance spectroscopy measurementswere also performed to further characterize the modifiedsurfaces Impedance119885(120596) is a complex number according to(2)

119885 (120596) = 1198851015840(120596) + 119895 sdot 119885

10158401015840(120596) (2)

where 120596 is the angular frequency 119895 = radicminus1 is the imaginarynumber and120596 the angular frequency is equal to 2120587119891119891 beingac-frequency

Impedance 119885(120596) is represented in the complex planeaccording to a Nyquist plot imaginary part (minus11988510158401015840(120596)) versusReal part (1198851015840(120596)) Figure 2(b) represents the Nyquist plots forbare Au electrode and for aminophenyl-Au electrode Indeedthe charge-transfer resistance estimated from the diameter ofthe semi-circle of the Nyquist plot is higher for the p-AP-Auelectrode than for the bare gold electrode This is due to thelower rate of electron transfer through the film showing highcoverage at the electrode

An electrical equivalent circuit for both a bare and amodified electrode shown in Figure 2(c) comprises a seriesresistance 119877

119878(resistance of solution and of ohmic contacts) a

resistance related to the charge transfer rate of redox reactionsat the functionalized electrode 119877CT a constant phase element(CPE) related to the capacitance of the functionalized Auelectrodeelectrolyte interface The capacitance of the inter-facial film 119862

119891in parallel with the resistance of the interfacial

film 119877119891are also included in the equivalent circuit

It is worth noting that the CPE reflects the nonide-ality of the double-layer at the functionalized gold elec-trodeelectrolyte interface due to the roughness and porosityof the interfacial film

ISRN Electrochemistry 5

minus04 minus02 00 02 04 06

minus3

minus2

minus1

0

1

2 A

B

C

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 40 50 60 700

8

16

24

32

C

B

A

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(b)

Figure 3 (a) Cyclic voltammograms of (A) p-AP-Au electrode (B) p-AP+GA-Au electrode and (C) antibody modified Au-p-AP+GA-Auelectrode recorded in the presence of Fe(CN

6)3minus4minus as a redox probe at scan rate of 100mVs (b) Nyquist plots of impedance spectra of (A)

Au-p-AP (B) with Au-p-AP+GA and (C) with antibody modified Au-p-AP+GA recorded in the presence of Fe(CN6)3minus4minus at a frequency

range between 01Hz and 100 kHz at open circuit potential

The CPE is defined as

119895 sdot 11988510158401015840(120596) =

1

119876

119895120596minus119899 (3)

where 119876 is the CPE constant and has the quality of asusceptance or 119876-capacitance 120596 is the angular frequency0 lt 119899 lt 1 is the factor describing deviation from idealcapacitor [46]

A specific electrochemical element of diffusion the War-burg element (119885

119908) is defined as

(119885119908) = 120590 (1 minus 119895) 120596

minus05 (4)

Here 120590 denotes the Warburg coefficient This equationrequires that 1198681198851015840(120596)119868 = 119868119885

10158401015840(120596)119868

32 Electrochemical Monitoring of Antibody Immobilization

321 Procedure of Covalent Grafting of the Antibody Follow-ing deposition of the p-aminophenyl (p-AP) film on the goldelectrode the terminal amine was activated by incubationfor 60min with glutaraldehyde (25) at pH 4 at roomtemperature [39] The value of pH was chosen close to pKaof aminophenyl group

After rinsing with PBS the electrode was incubated for 1hour in a 02mgmL solution of anti-S aureus antibodiesTheamino groups on the antibody molecule enable the covalentbonding with the glutaraldehyde activated amino functionson Au electrode The total time necessary for the wholeprocess of preparation of the immunosensor is 3 h

Finally after rinsing with PBS the electrodes were incu-bated for 20min in BSA (1) to block the unreacted aldehydegroups Cyclic voltammetry (CV) and EIS were employedto characterize the p-AP gold electrode (a) p-AP-GA goldelectrode (b) and p-AP-GA-Ab gold

Figure 3(a) shows the CV curves and Figure 3(b) Nyquistplots obtained with these surfaces

The Faradaic EIS measurements are in good agreementwith CV measurements the current density decreases (Fig-ure 3(a)) and the diameter of the semi-circles of the Nyquistdiagrams increases significantly after the different stages ofthe elaboration of the immunosensor (Figure 3(b)) Thisindicates that following the observed blocking effect of thep-AP film a further blocking effect was observed after thebinding of glutaraldehyde and the covalent immobilizationof the antibody

322 Number of Immobilized Antibodies (Binding Sites) Inorder to calculate the number of binding sites we calculate theantibody surface coverage 120579Ab using the following equation

1 minus 120579Ab =119877CTAP119877CTAb

(5)

119877CTAb Charge transfer resistance after antibody immobiliza-tion 119877CTAP Charge transfer resistance after grafting of theaminophenyl layer55 of the surface is found to be covered with antibodiesConsidering the surface density for a dense antibody layerdetermined in [47] the number of immobilized antibodieson the aminophenyl layer it should be 48 times 10

12 Abcm2

33 Detection of S aureus Cells Using ElectrochemicalImpedance Spectroscopy Having developed our sensor westudied its response towards different concentrations of theS aureus bacteria The electrodes were incubated in different

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Electrochemistry 5

minus04 minus02 00 02 04 06

minus3

minus2

minus1

0

1

2 A

B

C

EV (versus SCE)

J(m

Amiddotcm

minus2)

(a)

0 10 20 30 40 50 60 700

8

16

24

32

C

B

A

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(b)

Figure 3 (a) Cyclic voltammograms of (A) p-AP-Au electrode (B) p-AP+GA-Au electrode and (C) antibody modified Au-p-AP+GA-Auelectrode recorded in the presence of Fe(CN

6)3minus4minus as a redox probe at scan rate of 100mVs (b) Nyquist plots of impedance spectra of (A)

Au-p-AP (B) with Au-p-AP+GA and (C) with antibody modified Au-p-AP+GA recorded in the presence of Fe(CN6)3minus4minus at a frequency

range between 01Hz and 100 kHz at open circuit potential

The CPE is defined as

119895 sdot 11988510158401015840(120596) =

1

119876

119895120596minus119899 (3)

where 119876 is the CPE constant and has the quality of asusceptance or 119876-capacitance 120596 is the angular frequency0 lt 119899 lt 1 is the factor describing deviation from idealcapacitor [46]

A specific electrochemical element of diffusion the War-burg element (119885

119908) is defined as

(119885119908) = 120590 (1 minus 119895) 120596

minus05 (4)

Here 120590 denotes the Warburg coefficient This equationrequires that 1198681198851015840(120596)119868 = 119868119885

10158401015840(120596)119868

32 Electrochemical Monitoring of Antibody Immobilization

321 Procedure of Covalent Grafting of the Antibody Follow-ing deposition of the p-aminophenyl (p-AP) film on the goldelectrode the terminal amine was activated by incubationfor 60min with glutaraldehyde (25) at pH 4 at roomtemperature [39] The value of pH was chosen close to pKaof aminophenyl group

After rinsing with PBS the electrode was incubated for 1hour in a 02mgmL solution of anti-S aureus antibodiesTheamino groups on the antibody molecule enable the covalentbonding with the glutaraldehyde activated amino functionson Au electrode The total time necessary for the wholeprocess of preparation of the immunosensor is 3 h

Finally after rinsing with PBS the electrodes were incu-bated for 20min in BSA (1) to block the unreacted aldehydegroups Cyclic voltammetry (CV) and EIS were employedto characterize the p-AP gold electrode (a) p-AP-GA goldelectrode (b) and p-AP-GA-Ab gold

Figure 3(a) shows the CV curves and Figure 3(b) Nyquistplots obtained with these surfaces

The Faradaic EIS measurements are in good agreementwith CV measurements the current density decreases (Fig-ure 3(a)) and the diameter of the semi-circles of the Nyquistdiagrams increases significantly after the different stages ofthe elaboration of the immunosensor (Figure 3(b)) Thisindicates that following the observed blocking effect of thep-AP film a further blocking effect was observed after thebinding of glutaraldehyde and the covalent immobilizationof the antibody

322 Number of Immobilized Antibodies (Binding Sites) Inorder to calculate the number of binding sites we calculate theantibody surface coverage 120579Ab using the following equation

1 minus 120579Ab =119877CTAP119877CTAb

(5)

119877CTAb Charge transfer resistance after antibody immobiliza-tion 119877CTAP Charge transfer resistance after grafting of theaminophenyl layer55 of the surface is found to be covered with antibodiesConsidering the surface density for a dense antibody layerdetermined in [47] the number of immobilized antibodieson the aminophenyl layer it should be 48 times 10

12 Abcm2

33 Detection of S aureus Cells Using ElectrochemicalImpedance Spectroscopy Having developed our sensor westudied its response towards different concentrations of theS aureus bacteria The electrodes were incubated in different

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 ISRN Electrochemistry

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

160

A

B

C

D

E

FG

Z998400(120596) (kΩ)

minusZ998400998400(120596

) (kΩ

)

(a)

24

2

16

12

08

04

0

1 2 3 4 5 6 7

B

A

ΔR

CTR

CTAb

Log (CFUmL)

(b)

Figure 4 (a) Nyquist plots of impedance spectra obtained for increasing concentrations of S aureus (ATCC 25923) in PBS and Fe(CN6)3minus4minus

(A) 101 CFU per mL (B) 102 CFU per mL (C) 103 CFU per mL (D) 104 CFU per mL (E) 105 CFU per mL (F) 106 CFU per mL and (G)107 CFU per mL (b) Relative variation of 119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) versus concentration of S aureus (ATCC 25923) inPBS and Fe(CN

6)3minus4minus (A) incubated on antibody modified aminophenyl-Au electrode and (B) incubated on aminophenyl-Au electrode

aliquots corresponding to different bacterial concentrationsin CFU per mL Figure 4(a) shows the Nyquist diagramsof the immunosensor obtained after incubation in increas-ing concentrations of the target bacterium S aureus Theimpedance spectra obtained in the presence of differentconcentrations of bacteria were modeled by the equivalentelectrical circuit presented in Figure 2(c)

331 Surface Coverage by Bacteria Thepercentage of surfacecoverage by bacteria 120579Bact was determined according to thefollowing equation

1 minus 120579Bact =119877CTAb119877CTBact

(6)

119877CTAb Charge transfer resistance after binding antibody119877CTBact Charge transfer resistance after coverage with bacte-riaAssuming that the surface area of one S aureus bacteriumis314 120583m2 [48] it follows that in presence of a concentrationof 10 CFU per mL the determined surface coverage of theimmunosensor is 29 corresponding to 9 times 10

6 bacteria percm2 on the Ab functionalized gold surface In the presence ofa concentration of 107 CFU per mL a surface coverage of theimmunosensor is 59 corresponding to 2 times 10

7 bacteria percm2 has been determined

332 Apparent Equilibrium Dissociation Constant The reac-tion flow between immobilized antibody and bacteria is asfollows

Ab + Bact 999448999471 Ab-Bact (7)

Mass action applied to the equilibrium gives

119870119889=

[Ab] [Bact][Ab-Bact]

(8)

where 119870119889represents the apparent equilibrium dissociation

constant [Ab] and [Bact] respectively are the concentrationsof antibody at electrode surface and of bacteria in solution(CFU per mL) [Ab-Bact] is the surface concentration ofAb-Bact complex

If 119891(Ab) = [Ab-Bact][Ab-Bact]max and 1 minus 119891(Ab) =

[Ab][Ab-Bact]maxThe expression of119870

119889becomes

119870119889= [Bact]

(1 minus 119891 (Ab))119891 (Ab)

(9)

The measured relative variation of 119877CT tΔ119877CT (Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is related tothe amount of Ab-Bact complex formed tΔ119877CTmax is themaximum variation obtained corresponding to saturation Itthen comes

119891 (Ab) =tΔ119877CT

tΔ119877CTmax (10)

The measured relative variation of 119877CT tΔ119877CT(Δ119877CT119877CTAb = (119877CTBact minus 119877CTAb)119877CTAb) after equilibrium withdifferent concentrations of bacteria [Bact] is presented inFigure 4(b)

119870119889is equal to [Bact] when 119891 (Ab) = 05 The determined

value of119870119889 from Figure 4(b) is 1026 CFU per mL

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Electrochemistry 7

Table 1 A comparison of the analytical characteristics of theimmunosensor developed in this work with relevant immunosen-sors for S aureus bacteria detection based in the literature

Type oftransducer

Quantification limit(CFUmL)

Dynamic range(CFUmL) Reference

Impedance

10 10ndash106 [13]102 102ndash107 [12]10 10ndash106 [14]10 10ndash107 This work

34 Analytical Performance

341 Detection Limit Dynamic Range The charge transferresistance increases gradually as the bacteria concentra-tion increases after consecutive incubations from 10 up to107 CFU per mL The impedance immunosensor shows alinear relationship between the relative variation of chargetransfer tΔ119877ct and the decimal logarithmic value of S aureusconcentration [Bact] (Figure 4(b)) in a range of 10 to 107 CFUper mL with a slope of 04 and a correlation coefficient of0994 the limit of quantification being 10 CFU per mL

342 Reproducibility The reproducibility of the immun-osensor was investigated by repeating the experiments withthree different immunosensors prepared in the same condi-tions The response for three different immunosensors hasan average standard deviation of 20 which verifies thereproducibility of the system for the detection of S aureusThe drawback is that our immunosensor is not reusablebecause we were not able to regenerate our antibodies forother detections

343 Specificity To ensure that the response of our immun-osensor was not due to the adsorption of bacteria on theAu surface modified by p-aminophenyl different electrodeswere elaborated in the same conditions without the antibodyincubation step The results show that there is no significantvariation of the relative variation of the charge transferresistance tΔ119877ct of the modified electrode after incubationof the electrode in S aureus solutions (Figure 4(b))

The specificity of the impedimetric immunosensor sys-tem was determined for different bacteria strains at 106 CFUper mL The relation variation of charge transfer resistancetΔ119877ct of the immunosensor for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli increased less than 02whereas a marked increase by a factor 2 was found afterincubation with S aureus cells Therefore the specificity ofthe immunosensor is 10 for Staphylococcus saprophyticus(ATCC 15305) and Escherichia coli compared to 100 for Saureus The characteristics of previous relevant immunosen-sors for S aureus detection in terms of the limit of detection(LOQ) and of the dynamic range are given in Table 1 In thiswork the LOQ obtained for the developed immunosensor isthe lowest value and the largest dynamic range is obtained

4 Conclusion

An impedance-based immunosensor for the detection of Saureus has been developed Electrochemical grafting of thegold electrode surface by p-aminophenyl (p-AP) was demon-strated as a suitable simple and versatile platform for anti-body immobilization giving stable and robust biomolecularlayers It allows rapid inexpensive sensitive and selectivedetection of these bacteria This method permits antibodiesto be spatially addressed on a single gold chip in a microarrayand then allows simultaneous detection of different bacteriaFurther studies are in progress for the use on real samples

Acknowledgments

The authors would like to thank EGIDE for its supportthrough UTIQUE Program no 09G 1128 and through PHCMaghreb no 12 MAG 088 Amina Chrouda is grateful to theTunisian Ministry of Higher Education and Technology forthe ldquoMobility Grantrdquo

References

[1] B Kouidhi T Zmantar H Hentati and A Bakhrouf ldquoCellsurface hydrophobicity biofilm formation adhesives propertiesand molecular detection of adhesins genes in Staphylococcusaureus associated to dental cariesrdquo Microbial Pathogenesis vol49 no 1-2 pp 14ndash22 2010

[2] R Ananthanarayan and C K J Panikaran ldquoDiagnostic valueof mannitol for sugar fermentation in S aureusrdquo in Textbook ofMicrobiology S Kumar Ed vol 6 pp 178ndash186 2001

[3] I Hein A Lehner P Rieck K Klein E Brandl andMWagnerldquoComparison of different approaches to quantify Staphylococcusaureus cells by real-time quantitative PCR and applicationof this technique for examination of cheeserdquo Applied andEnvironmental Microbiology vol 67 no 7 pp 3122ndash3126 2001

[4] RW Bennett ldquoStaphylococcal enterotoxin and its rapid identifi-cation in foods by enzyme-linked immunosorbent assay-basedmethodologyrdquo Journal of Food Protection vol 68 no 6 pp1264ndash1270 2005

[5] E Leoni G D Luca P P Legnani R Sacchetti S Stampiand F Zanetti ldquoLegionella waterline colonization detection ofLegionella species in domestic hotel and hospital hot watersystemsrdquo Journal of AppliedMicrobiology vol 98 no 2 pp 373ndash379 2005

[6] M Nayak A Kotian S Marathe and D ChakravorttyldquoDetection of microorganisms using biosensors-A smarter waytowards detection techniquesrdquo Biosensors and Bioelectronicsvol 25 no 4 pp 661ndash667 2009

[7] E Tully S P Higson and R OrsquoKennedy ldquoThe developmentof a ldquolabelessrdquo immunosensor for the detection of Listeriamonocytogenes cell surface protein Internalin BrdquoBiosensors andBioelectronics vol 23 no 6 pp 906ndash912 2008

[8] D Li Y Feng L Zhou et al ldquoLabel-free capacitive immunosen-sor based on quartz crystal Au electrode for rapid and sensitivedetection of Escherichia coli O157H7rdquo Analytica Chimica Actavol 687 no 1 pp 89ndash96 2011

[9] A Subramanian J Irudayaraj and T Ryan ldquoA mixed self-assembled monolayer-based surface plasmon immunosensorfor detection of E coli O157H7rdquo Biosensors and Bioelectronicsvol 21 no 7 pp 998ndash1006 2006

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 ISRN Electrochemistry

[10] O Laczka E Baldrich F X Munoz and F J Del CampoldquoDetection of Escherichia coli and Salmonella typhimuriumusing interdigitated microelectrode capacitive immunosensorsthe importance of transducer geometryrdquo Analytical Chemistryvol 80 no 19 pp 7239ndash7247 2008

[11] F Salam and I E Tothill ldquoDetection of Salmonella typhimuriumusing an electrochemical immunosensorrdquo Biosensors and Bio-electronics vol 24 no 8 pp 2630ndash2636 2009

[12] V Escamilla-Gomez S Campuzano M Pedrero and JM Pingarron ldquoElectrochemical immunosensor designs forthe determination of Staphylococcus aureus using 33-dithi-odipropionic acid di(N-succinimidyl ester)-modified gold elec-trodesrdquo Talanta vol 77 no 2 pp 876ndash881 2008

[13] F Tan P H M Leung Z-B Liu et al ldquoA PDMS microfluidicimpedance immunosensor for E coli O157H7 and Staphylo-coccus aureus detection via antibody-immobilized nanoporousmembranerdquo Sensors and Actuators B vol 159 no 1 pp 328ndash3352011

[14] M Braiek K Bekir Rokbani A Chrouda et al ldquoAn elec-trochemical immunosensor for detection of Staphylococcusaureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electroderdquo Biosen-sors vol 2 no 4 pp 417ndash426 2012

[15] M Delamar R Hitmi J Pinson and J M Saveant ldquoCovalentmodification of carbon surfaces by grafting of functionalizedaryl radicals produced from electrochemical reduction of dia-zonium saltsrdquo Journal of the American Chemical Society vol 114no 14 pp 5883ndash5884 1992

[16] C Saby B Ortiz G Y Champagne and D Belanger ldquoElec-trochemical modification of glassy carbon electrode usingaromatic diazonium salts blocking effect of 4-nitrophenyl and4-carboxyphenyl groupsrdquo Langmuir vol 13 no 25 pp 6805ndash6813 1997

[17] J Lyskawa and D Belanger ldquoDirect modification of a goldelectrode with aminophenyl groups by electrochemical reduc-tion of in situ generated aminophenylmonodiazonium cationsrdquoChemistry of Materials vol 18 no 20 pp 4755ndash4763 2006

[18] G Z Liu T Bocking and J J Gooding ldquoDiazonium salts stablemonolayers on gold electrodes for sensing applicationsrdquo Journalof Electroanalytical Chemistry vol 600 no 2 pp 335ndash344 2007

[19] A Adenier M-C Bernard M M Chehimi et al ldquoCovalentmodification of iron surfaces by electrochemical reduction ofaryldiazonium saltsrdquo Journal of the American Chemical Societyvol 123 no 19 pp 4541ndash4549 2001

[20] A Chausse M M Chehimi N Karsi J Pinson F Podvoricaand C Vautrin-Ul ldquoThe electrochemical reduction of diazo-nium salts on iron electrodes The formation of covalentlybonded organic layers and their effect on corrosionrdquo Chemistryof Materials vol 14 no 1 pp 392ndash400 2002

[21] J J Gooding ldquoAdvances in interfacial design for electrochemi-cal biosensors and sensors aryl diazonium salts for modifyingcarbon and metal electrodesrdquo Electroanalysis vol 20 no 6 pp573ndash582 2008

[22] R Polsky J C Harper D R Wheeler and S M BrozikldquoMultifunctional electrode arrays towards a universal detectionplatformrdquo Electroanalysis vol 20 no 6 pp 671ndash679 2008

[23] A Hayat L Barthelmebs A Sassolas and J-L Marty ldquoAnelectrochemical immunosensor based on covalent immobiliza-tion of okadaic acid onto screen printed carbon electrode viadiazotization-coupling reactionrdquo Talanta vol 85 no 1 pp 513ndash518 2011

[24] C M A Brett S Kresak T Hianik and A M O Brett ldquoStudieson self-assembled alkanethiol monolayers formed at appliedpotential on polyerystalline gold electrodesrdquo Electroanalysisvol 15 no 5-6 pp 557ndash565 2003

[25] P Allongue M Delamar B Desbat et al ldquoCovalent modifi-cation of carbon surfaces by aryl radicals generated from theelectrochemical reduction of diazonium saltsrdquo Journal of theAmerican Chemical Society vol 119 no 1 pp 201ndash207 1997

[26] G Liu E Luais and J J Gooding ldquoThe fabrication of stablegold nanoparticle-modified interfaces for electrochemistryrdquoLangmuir vol 27 no 7 pp 4176ndash4183 2011

[27] B P Corgier A Laurent P Perriat L J Blum and C AMarquette ldquoA versatile method for direct and covalent immobi-lization of DNA and proteins on biochipsrdquo Angewandte ChemieInternational Edition vol 46 no 22 pp 4108ndash4110 2007

[28] A-E Radi X Munoz-Berbel M Cortina-Puig and J-LMarty ldquoA third-generation hydrogen peroxide biosensor basedon horseradish peroxidase covalently immobilized on elec-trografted organic film on screen-printed carbon electroderdquoElectroanalysis vol 21 no 14 pp 1624ndash1629 2009

[29] J L Bahr and J M Tour ldquoHighly functionalized carbonnanotubes using in situ generated diazonium compoundsrdquoChemistry of Materials vol 13 no 11 pp 3823ndash3824 2001

[30] O A de Fuentes T Ferri M Frasconi V Paolini and R San-tucci ldquoHighly-ordered covalent anchoring of carbon nanotubeson electrode surfaces by diazonium salt reactionsrdquo AngewandteChemie vol 50 no 15 pp 3457ndash3461 2011

[31] B Chen A K Flatt H Jian J L Hudson and J M TourldquoMolecular grafting to silicon surfaces in air using organictriazenes as stable diazonium sources and HF as a constanthydride-passivation sourcerdquo Chemistry of Materials vol 17 no19 pp 4832ndash4836 2005

[32] A J Downard andM J Prince ldquoA simple approach to patternedprotein immobilization on silicon via electrografting fromdiazonium salt solutionsrdquoLangmuir vol 17 pp 5581ndash5586 2001

[33] A-E Radi X Munoz-Berbel V Lates and J-L Marty ldquoLabel-free impedimetric immunosensor for sensitive detection ofochratoxin Ardquo Biosensors and Bioelectronics vol 24 no 7 pp1888ndash1892 2009

[34] S Mahouche-Chergui S Gam-Derouich C Mangeney andMM Chehimi ldquoAryl diazonium salts a new class of couplingagents for bonding polymers biomacromolecules and nanopar-ticles to surfacesrdquo Chemical Society Reviews vol 40 no 7 pp4143ndash4166 2011

[35] D Belanger and J Pinson ldquoElectrografting a powerful methodfor surface modificationrdquo Chemical Society Reviews vol 40 no7 pp 3995ndash4048 2011

[36] A Adenier E Cabet-Deliry A Chausse et al ldquoGrafting of arylgroups on carbon or metallic surfaces without electrochemicalinductionrdquo Chemistry of Materials vol 17 pp 491ndash501 2005

[37] A Ramanavicius A Finkelsteinas H Cesiulis and ARamanaviciene ldquoElectrochemical impedance spectroscopyof polypyrrole based electrochemical immunosensorrdquoBioelectrochemistry vol 79 no 1 pp 11ndash16 2010

[38] P Geng X ZhangWMeng et al ldquoSelf-assembledmonolayers-based immunosensor for detection of Escherichia coli usingelectrochemical impedance spectroscopyrdquo Electrochimica Actavol 53 no 14 pp 4663ndash4668 2008

[39] A-E Radi XMunoz-Berbel M Cortina-Puig and J-L MartyldquoAn electrochemical immunosensor for ochratoxin A basedon immobilization of antibodies on diazonium-functionalized

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Electrochemistry 9

gold electroderdquo Electrochimica Acta vol 54 no 8 pp 2180ndash2184 2009

[40] A Mesnage X Lefevre P Jegou G Deniau and S PalacinldquoSpontaneous grafting of diazonium salts chemical mechanismon metallic surfacesrdquo Langmuir vol 28 pp 11767ndash11778 2012

[41] P A Brooksby and A J Downard ldquoElectrochemical and atomicforce microscopy study of carbon surface modification viadiazonium reduction in aqueous and acetonitrile solutionsrdquoLangmuir vol 20 no 12 pp 5038ndash5045 2004

[42] P A Brooksby and A J Downard ldquoMultilayer nitroazobenzenefilms covalently attached to carbon An AFM and electrochem-ical studyrdquo Journal of Physical Chemistry B vol 109 no 18 pp8791ndash8798 2005

[43] M Kullapere M Marandi L Matisen et al ldquoBlocking prop-erties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groupsrdquo Journal of Solid State Electrochemistry vol16 no 2 pp 569ndash578 2012

[44] A L Gui G Liu M Chockalingam et al ldquoA comparativestudy of electrochemical reduction of 4-nitrophenyl covalentlygrafted on gold and carbonrdquo Electroanalysis vol 22 no 16 pp1824ndash1830 2010

[45] A Chira O-I Covaci and G L Radu ldquoA comparative studyof gold electrodes modification methods with aromatic com-pounds based on diazoniumand thiol chemistryrdquoUPBScientificBulletin B vol 74 no 1 pp 183ndash192 2012

[46] J Bisquert G Garcia-Belmonte P Bueno E Longo and LO S Bulhoes ldquoImpedance of constant phase element (CPE)-blocked diffusion in film electrodesrdquo Journal of ElectroanalyticalChemistry vol 452 no 2 pp 229ndash234 1998

[47] J T La Belle K Bhavsar A Fairchild et al ldquoA cytokineimmunosensor for multiple sclerosis detection based uponlabel-free electrochemical impedance spectroscopyrdquo Biosensorsand Bioelectronics vol 23 no 3 pp 428ndash431 2007

[48] L G Harris S J Foster R G Richards P Lambert D Sticklerand A Eley ldquoAn introduction to Staphylococcus aureus andtechniques for identifyingand quantifying S aureus adhesins inrelation to adhesion to biomaterials reviewrdquo European Cells ampMaterials vol 4 pp 39ndash60 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of