Fabrication of an Electrochemical E. coli Biosensor in Biowells Using Bimetallic Nanoparticle-Labelled Antibodies

DOI: 10.1002/elan.201400370

Fabrication of an Electrochemical E. coli Biosensor inBiowells Using Bimetallic Nanoparticle-LabelledAntibodiesHaslet Eksi,*[a] Remziye G�zel,[b] Burcu G�ven,[c] Ismail Hakki Boyaci,[c] and Ali Osman Solak[a, d]

1 Introduction

Escherichia coli (E. coli), the most widely studied and ge-netically best-known microorganism in the enteric bacte-rial family [1] is commonly found in the intestinal trackof humans and other warm-blooded animals. Most of E.coli strains are harmless however; some kinds of E. colican cause severe human diseases such as diarrhea, urinarytrack infections. It is mainly transmitted to humansthrough the consumption of contaminated food or water.Therefore, E. coli is used as a biomarker to identify thefecal contamination. Although traditional microbiologicalmethods for detecting E. coli including multiple cure fer-mentation, membrane filter, and plate count method pro-vide high sensitivity and accuracy, they are time consum-ing and complicated in operation. Besides, they have pro-longed incubation time. Due to these reasons, it is verydifficult to determine E. coli immediately in the environ-mental monitoring, food analysis and clinical diagnostics.Researchers have employed several detection techniquesincluding optical [2–4], electrochemical [5–7], mass [8, 9]and thermal [10] methods, which depend on the type ofthe transducer to obtain high sensitivity and selectivity ina short analysis time. Among these methods, electro-chemical techniques provide great advantages like sim-plicity, miniaturization, speed and time. Besides these ad-vantages, it is a cost-efficient method when comparedwith the other detection techniques.

Immunoassays have been considered an important ana-lytical method that based on the specific recognition reac-tion of antigen-antibody interaction [11,12]. The combi-nation of immunoassays with electrochemical methods,called as electrochemical immunosensors, ensures an ac-curate and sensitive detection of analytes [13]. Nanoma-terials are usually employed in electrochemical immuno-sensors in order to enhance the signal amplification andimmobilization of biomolecules [14]. To obtain high elec-trochemical response in these immunosensors, variousnanomaterials such as nanoparticles [15] especially thecore-shell formations have been widely used as labels[16]. Gold and silver are the most preferred noble metals

Abstract : An electrochemical biosensor was developedfor the determination of Escherichia coli (E. coli) inwater. For this purpose, silver-gold core-shell (Ag@Au)bioconjugates and anti-E. coli modified PS-microwellswere designed in a sandwich-type format in order toobtain higher sensitivity and selectivity. Ag@Au bimetal-lic nanoparticles were synthesized by co-reductionmethod. The core-shell formation was analyzed by usingUV-Vis spectroscopy and transmission electron microsco-py. Biotin labeled anti-E. coli antibodies were coupledwith Ag@Au nanoparticles to form bioconjugates. Theelectrochemical immunosensor was prepared by immobi-lizing anti-E. coli on polystyrene (PS)-microwells viachemical bonding. These modified microwells were iden-tified with X-ray photoelectron spectroscopy and surface

enhanced Raman spectroscopy. E. coli was sandwichedbetween Ag@Au bioconjugates and anti-E. coli on PS-mi-crowells at different concentrations. The relationship be-tween the E. coli concentration and stripping current ofgold ions (Au3+) were investigated by square waveanodic stripping voltammetry at pencil graphite electrode.The proposed method can provide some advantages suchas lower detection limit and shorter detection time. Theelectrochemical response for the immunosensor waslinear with the concentration of the E. coli in the rangeof 101 and 105 cfu/mL with a limit of detection 3 cfu/mL.The procedure maintains good sensitivity and repeatabili-ty and also offers utility in the fields of environmentalmonitoring and clinical diagnosis.

Keywords: Bimetallic nanoparticles · E. coli · Anodic stripping voltammetry · Pencil graphite electrode

[a] H. Eksi, A. O. SolakAnkara University, Faculty of Science, Department ofChemistry06560 Ankara, Turkey*e-mail: [email protected]

[b] R. G�zelDicle University, Faculty of Education21280 Diyarbakır, Turkey

[c] B. G�ven, I. H. BoyaciHacettepe University, Food Research CenterBeytepe 06800 Ankara, Turkey

[d] A. O. SolakKyrgyz-Turk Manas University, Faculty of Engineering,Department of Chemical Engineering720044 Bishkek, Kyrgyzstan

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in core-shell structures because of their interesting opticaland electronic properties in immunosensors [17]. Ag@Aucore-shell bimetallic nanoparticles are good examples fordetecting target molecule or analyte. These nanoparticlesare ideal for the applications in molecular sensing [18]and bio sensing because the Ag core improves the plas-monic enhancement while the Au shell provides chemicalstability and biological activity.

Electrochemical stripping analysis is a highly sensitivetechnique for determining trace analytes which includesanodic stripping voltammetry (ASV) [19,20], cathodicstripping voltammetry (CSV) [21] and adsorptive strip-ping voltammetry (AdSV) [22,23]. The most favorableone, ASV is a voltammetric method involves two stepssuch as preconcentration and stripping [24]. In the firststep, the metal ions in the sample are electrodepositedonto electrode surface at a negative potential. In thestripping step, the electrodeposited metal is oxidized fromthe electrode surface during the positive potential scan.The stripping current is proportional to the amount ofmetal ion in the sample and the peak potential is uniquefor each metal. Square wave anodic stripping voltamme-try (SWASV) is a type of ASV which combines thesquare wave and staircase wave in the stripping step. Thisprovides well-defined peaks for each analyte in an ex-tremely fast and sensitive way with low detection limits[25–28]. Nanoparticle labels coupled with DNA, enzyme,protein or microorganisms are detected with SWASV bydissolving the metal in acid solution because the concen-tration of metallic ions is proportional with the capturedanalyte. In this type of analysis, in order to obtain highaccuracy and sensitivity, a suitable electrode materialwith multiple features should be chosen. Pencil graphiteelectrodes (PGEs) are the best candidates for detectingbiological and chemical analytes with high electrical con-ductivity, low background signals, fast and easy usage,low-cost and wide availability [29–32].

The aim of this study was to develop a new kind ofelectrochemical immunosensor for E. coli by usingAg@Au bimetallic nanoparticle labeled antibodies. Weused PGEs as a working electrode which showed a highlysensitive square wave stripping signals for the detection ofE. coli. Studying with PGEs provided faster and simpleranalysis without long polishing procedures. This renewableelectrode offered a good reproducibility for individual sur-faces. For this purpose, we synthesized bimetallic nanopar-ticles by co-reduction method and coupled them with anti-E. coli antibodies to form bioconjugates. Sandwich typeimmunoassay was built on polystyrene (PS)-microwells assubstrate. Square wave anodic stripping voltammetry wasused for the rapid detection of E. coli at PGE. The strip-ping voltammograms were obtained for different concen-trations of E. coli. The relationship between the electro-chemical current and E. coli concentration was plotted. Fi-nally, we have applied the new biosensor for the determi-nation of E. coli in tap water. The proposed method pro-vides a fast, simple and economic approach for detectionof E. coli in water in a short period.

2 Experimental

2.1 Reagents and Chemicals

Chloroauric acid (99%, HAuCl4) and silver nitrate(AgNO3) were obtained from Merck (Darmstadt, Germa-ny). 11-mercaptoundecanoic acid (MUA), N-(3-dimethy-laminopropyl)-N’-ethylcarbodiimide hydrochloride(EDC), 2-morpholinoethanesulfonic acid monohydrate(MES), potassium hydrogen phosphate, dipotassium hy-drogen phosphate were purchased from Sigma-Aldrich(Taufkirchen, Germany). Trisodium citrate dehydrate, l-ascorbic acid and Tween 20 were obtained from Merck(Darmstadt, Germany). Ethanol absolute was obtainedfrom Riedel-de Hain (Seelze, Germany) and osmium tet-raoxide was purchased from JMC (London, UK). 5,5-Di-thiobis-(2-nitrobenzoic acid) (DTNB) was obtained fromAcros (Fair Lawn, NJ, USA). Biotin conjugated rabbitanti-E. coli polyclonal antibodies were obtained fromAbcam plc. (Cambridge, UK). Immunopure avidin andN-hydroxysuccinimide sodium salt (NHS) were obtainedfrom Pierce Biotechnology (Rockford, IL, USA). Allchemicals were used as received and all aqueous solutionswere prepared in double distilled water.

2.1.1 Solutions and Buffers

Millonig phosphate buffer was prepared by mixing207.5 mL of 2.26 % NaH2PO4.H2O solution and 42.5 mLof 2.52% NaOH solution and the pH was adjusted to 7.3.Glutaraldehyde solution (5 %) was prepared in Millonigphosphate buffer solution and stored at 4 8C. 1% osmiumtetroxide solution was prepared in Millonig phosphatebuffer solution and stored at 4 8C in an amber glass bottle[33]. 67 mM and 0.1 M phosphate buffer solutions wereprepared by using Na2HPO4, KH2PO4 and NaCl and pHwas adjusted with HCl or NaOH. PBST was prepared bymixing PBS with 0.05 % Tween 20 (v/v). 2-Morpholinoe-thanesulfonic acid buffer (MES; 0.05 M, pH 6.5) was ad-justed with 0.1 M NaOH and filtered from ChromafilCA-45/25 disposable syringe filter (D�ren, Germany)before using the solution. All solutions were preparedwith Milli-Q water (18 MW cm)

2.1.2 Microorganisms

E. coli was supplied from Refik Saydam National TypeCulture Collections, Ankara Turkey. The pure culture ofE. coli was grown in LB Broth at 37 8C for 18 h and oneml of pure culture was transferred to an Eppendorf tube.After centrifugation, the E. coli was washed with PBS.The culture was serially diluted between 10–107 cfu/mLwith PBS (67 mM, pH 7.4).

2.2 Instrumentation

Transmission electron microscopy (TEM) experimentswere performed with Technai G2 20 S-TWIN instrument



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(FEI Company, OR, USA) at accelerated voltage of200 kV for characterization of bimetallic nanoparticles. AFEI Tecnai G2 Spirit BioTwin (FEI Company, OR, USA)TEM was used to image E. coli samples at 120 kV. Bimet-allic nanoparticle labeled E. coli samples were preparedas follows. Firstly, E. coli bacteria were fixed in glutaral-dehyde solution (5 %) at 4 8C for 1 h. After removing theglutaraldehyde by centrifugation, E. coli was washed withbuffer solution at 4 8C for 20 min. Then E. coli was post-fixed in 1 % osmium tetroxide solution at 4 8C for 1 h.and washed with buffer solution for 20 min at 4 8C. Sam-ples were dehydrated in increasing concentrations of eth-anol at 4 8C for 10 min (25 %, 50 %, 70 %, 85%, 95%,100%). Finally, samples were washed two times in 100%ethanol. All samples were prepared by placing a drop offresh colloid on a carbon coated copper grid and then thesolvent was evaporated under vacuum.

UV-Vis absorption spectrums were measured with U-2800 spectrophotometer (Hitachi, Tokyo, Japan) usinga quartz cell with 10 mm of optical path length in a rangeof 300–700 nm. Surface enhanced Raman spectroscopy(SERS) was employed to identify the immunoassay pro-tocols by using a DeltaNu Examiner Raman Microscopysystem (DeltaNu Inc., Laramie, WY, USA) with a 785 nmlaser source, a motorized microscope stage sample holderand cooled charge-couple device (CCD, at 0 8C) detector.X-ray photoelectron spectroscopy (XPS) analysis was per-formed on PHI 5000 Versa-Probe X-ray photoelectronspectrometer (Berlin, Germany) with a monochromatizedAl Ka radiation (1486.6 eV) as an X-ray anode. The pres-sure inside the analyzer was arranged to 107 Pa. The max-imum of the C�H peak at 285.0 eV for C1s was taken asa baseline to form the band energy scale and a multipeaksoftware program was used to determine the atomic com-position of the modified surfaces. All electrochemicalmeasurements were performed using Gamry 300 electro-chemical analyzer system (Warminster, PA, USA)equipped with a BAS-C3 cell stand and the data were an-alyzed in the Gamry EChem Analyst software package(Gamry Instruments, PA, USA). A traditional three-elec-trode system was used in cyclic voltammetry (CV) andSWASV experiments. A PGE was employed as a workingelectrode, a silver wire as the reference electrode anda platinum wire as an auxiliary electrode. PGEs wereTombow (Tombow Pencils Co. Ltd., Tokyo, Japan) H0.7 mm in diameter and 6 cm in length purchased froma local bookstore. All electrochemical experiments werecarried out at room temperature in 96-well PS-micro-wells.

2.3 Preparation of Bimetallic Nanoparticles and TheirBioconjugates

Ag@Au core-shell bimetallic nanoparticles were preparedusing co-reduction method according to the literature[34,35]. The silver nanoparticles were prepared accordingto the Lei and Meisel�s method [36]. A volume of fivemL of the as prepared colloidal silver solution was added

to 30 mL of distilled water. One mL of 38.8 mM sodiumcitrate was added drop wise to this solution under vigo-rous stirring. After the solution was completely mixed up,1.2 mL of 10 mM HAuCl4.3H2O solution was added intothe solution. Finally, 0.4 mL of 100 mM ascorbic acid wasadded slowly to form the bimetallic core-shell structure.At this stage, the yellow color of the solution was turnedto reddish color, which shows that silver nanoparticleswere covered with gold shell. Ag@Au bimetallic nanopar-ticles were stored in the refrigerator for further use.

Ag@Au-antibody conjugates were prepared by usingself-assembled monolayer (SAM) technique. The surfacesof the bimetallic nanoparticles were modified with twodifferent molecules according to the characterizationmethod in the first step of the modification. MUA andDTNB were used for electrochemical experiments andSERS, respectively. Bimetallic nanoparticle-labeled anti-bodies were prepared by following procedure. Firstly, onemL of Ag@Au bimetallic nanoparticle in ethanol weretransferred into an Eppendorf tube and 150 mM MUAwas insert into this solution. The mixture was stirred forovernight to form MUA capped bimetallic nanoparticles(MUA-BNP). MUA-BNP solution was centrifuged toremove unbounded MUA in the solution. The superna-tant was carefully removed and precipitated nanoparticleswere washed twice with ethanol and 0.05 M MES buffersolution, respectively. MUA-Au@Ag nanoparticles weretreated with one mL of 50 mM/200 mM EDC/NHS solu-tion for 40 min to activate the carboxyl groups on MUA.The solution was centrifuged again and then activatednanoparticles were washed twice with MES buffer solu-tion. After the activation step, bimetallic nanoparticleswere incubated with 0.2 mg/mL avidin for 40 min to bindthe avidin to the carboxylate activated-Ag@Au nanoparti-cles. The formed avidin-BNP conjugates were then sepa-rated from the incubation solution via centrifugation andwashed two times with PBS to remove the unreactedavidin. Finally, the avidin-BNP conjugates were interactedwith the 0.5 mg/mL biotinylated E. coli antibody in PBSfor 40 min and washed with PBS buffer in order toremove unconjugated antibodies. Table 1 displays the pa-rameters used for proposed immunosensor.

2.4 Immunosensor Preparation

In the XPS and SERS experiments, polystyrene-modifiedindium tin oxide (ITO) surfaces were used for construct-ing immunoassay protocol for the determination of E.coli. Prior to modification, the ITO surfaces were cleanedin acetone, ethanol and water, respectively, with a sonica-tor in order to remove adsorbed substances on the elec-trode surface. ITO surfaces were dried under argon at-mosphere until they were ready to use. PS-modified ITOsurfaces were prepared as follows. Briefly, 0.025 mg/mLpolystyrene was dissolved in toluene at room temperatureand then 50 mL of this solution was dropped on the ITOsurface. The modified surfaces were dried at room tem-perature for overnight. The immunoassay protocol was



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built on PS-modified surfaces in the same way by using96-well micro plates which were prepared for the electro-chemical experiments. The preparation of Ag@Au bimet-allic nanoparticle-labeled antibodies and their interactionwith E. coli in microwells were shown in Scheme 1.

The immunoassay protocol for electrochemical experi-ments was constituted in polystyrene microwells. In thebeginning, avidin solution was added to PS-modified ITOsurfaces and incubated at 4 8C overnight. After removingthe solution, the avidin-modified surfaces were washedwith PBS buffer and PBST for three times. Then, biotinconjugated polyclonal antibody was coated on the avidin-modified surfaces. After multiple washing steps, E. colisolution was added on avidin-biotin modified surfacesand incubated at room temperature for 40 min. Next, thesurfaces were washed with PBS buffer for three times.Following this step, the E. coli modified surfaces wasrinsed into Ag@Au bimetallic nanoparticle-labeled anti-body solution for another 40 min to obtain sandwich im-munocomplex. After completing the immunoassay proce-dure, the microwells were washed with PBS buffer forthree times.

2.5 Electrochemical Experiments

Electrochemical experiments were performed in three-steps (activation, preconcentration and stripping) for eachelectrode in three different microwells of the 96-wellmicro plates. In the first well, the PGE was activated indiluted aqua regia in order to provide sensitivity and re-producibility of the electrode. PGE was dipped into dilut-ed acid solution and the surface of the electrode was acti-vated by chronoamperometry at 1.4 V for 6 min. Secondwas the accumulation microwell where Au3 + ions cathodi-cally electrodeposited onto PGE and the third well con-taining diluted acid solution was called as the strippingwell. CV was used to identify the electrochemical behav-ior of 1 mM HAuCl4 in diluted aqua regia at PGE. CVscans were performed between 1.4 and �0.1 V at a scanrate 200 mVs�1. SWASV measurements were performedin polystyrene microwells containing dilute acid solution.Ag@Au bimetallic nanoparticles were dissolved by theoxidation of metallic Au to Au3+ ions in acidic solution.The released Au3+ ions were determined by SWASV. TheAu3+ ions were deposited on PGE at �0.2 V for 10 min,then the electrode was put into another microwell con-taining 100 mL dilute acidic solution. The deposited met-allic gold was stripped from the PGE surface to Au3+

ions by scanning from 0 to 1.2 V by SWASV mode. Therelationship between the E. coli concentration and strip-ping current of gold were investigated.

2.6 Determination of E. coli in the Spiked Tap WaterSamples with Developed Immunoassay Procedure

100 mL of 102 cfu/mL was transferred an Eppendorf tubeand diluted to 1 mL with tap water. For conventionalplate-count method, 100 mL of this dilution was spread on

Table 1. Parameters for the preparation of bimetallic nanoparti-cle-labeled antibodies.

Parameters

Concentration of EDC/NHS 50 mM/200 mMConcentration of avidin (mg/mL) 0.2 mg/mLConcentration of antibody (mg/mL) 0.5 mg/mLIncubation temperature 25 8CIncubation time 40 min

Scheme 1. The preparation of Ag@Au bimetallic nanoparticle-labeled antibodies and their interaction with E. coli in microwells.



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SMAC-Agar plates and incubated over night at 37 8C.The numbers of colonies were counted on plates to deter-mine the colony-forming units per milliliter. For electro-chemical experiments, the same spiked tap water was in-jected to the PS-microwell and the cfu/mL was deter-mined with the developed method. The amount of the E.coli was detected from the stripping current of the Au3 +

and calculated with equation in the calibration graph.

3 Results and Discussion

3.1 Characterization of Bimetallic Nanoparticles and E.coli Interaction

TEM was performed to investigate the size and shape ofsynthesized Ag@Au bimetallic nanoparticles. As shown inFigure 1c, Ag@Au nanoparticles are spherical in shapewith a smooth surface morphology. The diameter of nano-particles is found to be approximately 12 nm.

UV-Vis spectroscopy results were consistent with theTEM images. Figures 1a and 1b show that the UV-Visspectra of monometallic silver seed and Ag@Au bimetal-lic nanoparticles solution. Initially, silver colloids exhibitan absorbance band at 410 nm which was the typical sur-face plasmon resonance of silver nanoparticles. The ab-sorption bands of the AgNPs showed a red shift from

410 nm to 508 nm upon the Ag coverage, which is attrib-uted to AgNPs coated by gold ions to produce Au@Agbimetallic nanoparticles.

The interaction of bimetallic nanoparticle-labeled anti-body with E. coli is shown in Figure 2. From the TEMimages, the nanoparticle-labeled antibodies are linked toE. coli from the specific parts of the bacteria which arecalled epitopes.

3.3 Spectroscopic Analysis of Immunoassay Protocols

Schematic presentation of immunoassay protocol forSERS and XPS measurements was shown in Scheme 2.SERS characterization was utilized in order to correct thebinding procedure of sandwich-type immunoassay. Fig-ure 3a shows the bare PS-modified ITO surface. After thebiotinylated antibody conjugation, E. coli is attached onPS-modified ITO surface and some bands are observedbut due to intensity of CH2 vibration they are not charac-terized in details (Fig. 3b and, inset). According to theFigure 3c, when the bimetallic nanoparticle-labeled anti-bodies linked to E. coli, the SERS signals were strongerand distinguishable. It is understood that Ag@Au bimet-allic nanoparticles were exhibited good performance forsignal amplification. DTNB was used as a Raman labelfor E. coli immunoassays. The symmetric nitro band of

Fig. 1. TEM images of (a) Ag@Au bimetallic nanoparticles, (b) E. coli, (c)�(d) Ag@Au bimetallic nanoparticle labelled E. coli.



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DTNB was observed at 1368 cm�1 which indicates the bi-metallic nanoparticle-labeled antibody binds to E. coli. Inthe bare polystyrene modified ITO and E. coli modifiedsurface spectra, there is no nitro band observed. As it canbe seen from the SERS results, the Raman activity ofAg@Au labels used in the immunoassay increased theband signals. This result can be explained as the interac-tion of gold surface with sulphur containing linker mole-

cules. According to the studies in the literature, Ag@Aubimetallic nanoparticles exhibit higher SERS signalswhen the linker molecule contains a thiol group [37]. Thestrong SERS signals from the Raman label demonstratesthat the sandwich immunoassay protocol for the determi-nation of E. coli takes place as expected.

XPS analysis was performed in order to identify thecomposition of sandwich immunoassay protocol. Mainlytwo regions were investigated in the spectrum. The Auand Ag core spectrums of Ag@Au-E. coli-PS/ITO surfaceis shown in Figures 4a and 4b. Figure 4a shows the Ag3d

region for Ag@Au bimetallic nanoparticles. In the Agcore spectra 3d5/2 and 3d3/2 orbitals were appeared at368.5 eV and 374.6 eV, respectively, indicating the pres-ence of silver core. The Au 4f signal is shown in Figure 4bin which the Au 4f7/2 and 4f5/2 peak signals were observedat 87.5 eV and 83.7 eV. Au4f and Ag3d regions in the spec-trum were displayed that bimetallic nanoparticle-labeledantibodies were successfully joined to E. coli and thesandwich formation was formed. The molar ratios ofmetal atoms in the prepared bimetallic nanoparticles canbe identified from the spectrums. In Ag@Au bimetallicnanoparticles, the deposition of gold shell onto silvermetal reduces Ag 3d5/2 signal intensity in XPS. This indi-cates that the silver metal is found in the core and gold iscoated on the silver nanoparticles.

Fig. 2. UV-Vis spectra of (a) Ag nanoparticles, (b) Ag@Au bimetallic nanoparticles.

Scheme 2. Schematic presentation of immunoassay protocol for SERS and XPS measurements.

Fig. 3. SERS spectra of (a) bare PS-ITO, (b) E. coli modifiedPS-ITO surface (inset), (c) Ag@Au labelled immunoassay proto-col.



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3.4 Determination of Electrochemical Parameters

In the first part of the electrochemical experiments, thebehavior of gold was identified by CV at PGE in the 96-well polystyrene microwells. Figure 5a shows the CV be-havior of 1 mM HAuCl4 in diluted aqua regia in order todetermine the oxidation and reduction peak potentials ofgold at PGE. CV scans were performed between 1.4 and

�0.1 V at a scan rate 200 mV/s. Under these conditions,the CV curve shows that the cathodic reduction of Au3+

takes place at �0.1 V at PGE. During the reversal scan,a well-defined anodic peak was located at 0.97 V which isthe characteristic oxidation peak of electrodepositedgold. From the CV curve, �0.2 V was chosen as the accu-mulation potential in diluted aqua regia for further stud-ies.

Fig. 4. XPS core spectra of Ag@Au labelled immunoassay protocol (a) Ag, (b) Au.

Fig. 5. (a) Cyclic voltammogram of 1 mM HAuCl4 in dilute aqua regia at PGE, scan rate 0.2 V/s. (b) Square wave anodic strippingvoltammogram of Au3+ at PGE. (c) Response of Ag wire in supporting electrolyte at PGE



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In order to optimize the analytical procedure, the strip-ping potential of Au at PGE was determined by SWASV.Gold nanoparticles were dissolved in diluted acidic solu-tion to oxidize gold nanoparticles (Au0) into Au3 +. Au3+

was preconcentrated at �0.2 V for 120 s and then it wasstripped from the PGE surface between �0.2 and 1.4 V.Figure 5b displays the ASV voltammogram of Au/Au3 + indiluted aqua regia. The voltammogram shows that thegold nanoparticles were dissolved successfully in theworking media, preconcentrated and stripped at the de-sired potential. The SWASV parameters for stripping ex-periments were used as follows; frequency 15 Hz, pulsewidth 50 mV and step width 2 mV.

In this experimental conditions to understand whetherAg reference electrode interfere the analysis at this po-tential range, same experimental parameters were appliedto PGE in supporting electrolyte. �0.2 V accumulationpotential was utilized to PGE in the supporting electro-lyte and stripping analysis were performed in the samepotential range used for Au3+ . As shown in the Figure 5c,there is no current response in the supporting electrolytesolution in the same conditions. In this way, it is clearlyunderstood that Ag reference electrode has no interfer-ence effects in the potential range where the accumulatedgold metal stripped out of the surface of PGE.

3.5 Analytical Performance

The electrochemical immunoassay was utilized to deter-mine different concentrations of E. coli. After the immu-nocomplex was formed on the surface of the polystyrenemicrowells, the acid solution was added to the microwelland then the Ag@Au bimetallic nanoparticles were dis-solved in the acidic solution. To detect E. coli by SWASVin the final step of the immunoassay, the gold metal mustbe released from the immobilized substrate.

Figure 6a showed the SWASV voltammograms of theelectrochemical immunosensor after the interaction withdifferent concentrations of E. coli. It can be observed

from ASV curve that the stripping current of gold wasproportional to the increasing concentration of E. coli.When the concentration of E. coli was increased, theamount of the bimetallic nanoparticle-labeled antibodyon the polystyrene microwells increased correspondingly.The calibration curve shows a linear relationship betweenthe stripping current of Au/Au3+ conversion and the loga-rithmic value of E. coli concentration from 101 to 105 cfu/mL, with a LOD of 3 cfu/mL. The linear regression equa-tion was Ip(mA)=0.6589 log [E. coli (cfu/mL)] – 0.0303(mA), with a correlation coefficient of 0.990.

3.6 Real Sample Analysis

The developed immunoassay procedure was applied todetect the E. coli in real water samples. For this purpose,the electrochemical immunosensor was tested in tapwater and E. coli inoculated tap water, and the resultswere compared with the traditional counting method. Intap water, E. coli was not determined with both methods.E. coli concentration was detected 33 cfu/mL for inoculat-ed tap water with developed electrochemical methodwhich was consisted to the result in plate countingmethod (20 cfu/mL). These results showed that our ap-proach is appropriate in detecting E. coli in real watersamples.

When we overview the literature, there are numerousmethods for the determination E. coli in water samples.Most researchers employed modified electrodes to detectE. coli. For example, K. Abu-Rabeah and co-workers de-veloped a highly sensitive amperometric E. coli biosensorusing a polypyrrole-amine modified glassy carbon elec-trode [38] and H. Tang and co-workers used a self-assem-bled monolayer-based bienzyme biosensor to determineE. coli density [39]. However, cleaning and modifyingsteps for the electrodes were causing loss of time. In ourelectrochemical biosensor, we used PGEs without modifi-cation on their surface and no cleaning steps were appliedto the electrode surface. The other advantage of our bio-

Fig. 6. Square wave anodic stripping voltammograms (a) and the calibration curve (b) corresponding to the increase of the strippingcurrent of Au3+ with the concentration of E. coli.



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sensor, we prefer to use the wells of PS-microplate as anelectrochemical cell in order to study in small volumes.SWASV combined with sandwich-type immunoassaysprovide a better LOD value compared with the othermethods in the literature. Li and co-workers developedan impidimetric biosensor for the rapid detection of E.coli with a detection limit of 106 cfu/mL [40]. X. Zhangetal. reported an electrochemical immunoassay based onCu@Au bimetallic nanoparticles with a detection limit30 cfu/mL [41]. The selection of labels has an importantrole on the sensitivity of the immunoassay. Thus, we se-lected Ag@Au bimetallic nanoparticles to enhance thesignal amplitude. These nanoparticles improve the perfor-mance of the electrochemical immunoassay and increasethe sensitivity of the E. coli biosensor.

4 Conclusions

In this research, we have reported a sandwich type-elec-trochemical immunosensor for the rapid detection of E.coli in water using PEGs in 96-well micro plates. Thecombination of sandwich-type immunoassay and SWASVwas improved the sensitivity of the proposed immunosen-sor. Compared with the traditional methods, this kind ofcombination provided higher specificity and sensitivityfor the developed technique in 96 min. Furthermore,PGE displayed excellent performance such as low back-ground signals and high electrical conductivity withoutany modification on its surface. Besides, the proposedPGE-based biosensor offered a disposable and economi-cal procedure in a less time-consuming way. A linear rela-tionship between the stripping current of Au3+ and E.coli concentration was found in the range of 101 to105 cfu/mL, with a LOD of 3 cfu/mL. This detection limitis better than many other electrochemically based E. colisensors in the literature. Additionally, the proposed bio-sensor was tested for the detection of E. coli in real watersamples and the results display that the electrochemicalimmunosensor is available for the determination of E.coli. It can be expected that the proposed method holdpromise for the rapid detection of different microorgan-isms with disposable electrodes in the field of environ-mental analysis and clinical diagnosis.

Acknowledgement

This work was supported by The Scientific and Technolog-ical Research Council of Turkey, Project Number:109T633.

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Received: July 22, 2014Accepted: September 25, 2014

Published online: && &&, 2014



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H. Eksi,* R. G�zel, B. G�ven,I. H. Boyaci, A. O. Solak

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Fabrication of an Electrochemical E.coli Biosensor in Biowells UsingBimetallic Nanoparticle-LabelledAntibodies



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Fabrication of an Electrochemical E. coli Biosensor in Biowells Using Bimetallic Nanoparticle-Labelled Antibodies