Impedimetric DNA Aptasensor for Sensitive Detection of Ochratoxin A in Food

Impedimetric DNA Aptasensor for Sensitive Detection ofOchratoxin A in Food

Gabriela Castillo,a Ilaria Lamberti,b Lucia Mosiello,c Tibor Hianik*a

a Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska dolina F1, 84248 Bratislava, Slovakiab University of Roma, Tre Department of Biology, Viale G. Marconi 446, 00149 Roma, Italyc ENEA, Italian National Agency for New Technologies, Energy and the Environment, Via Anguillarese 301, 00060 Roma, Italy*e-mail: [email protected]

Received: August 31, 2011;&Accepted: October 13, 2011

AbstractWe report a high sensitive biosensor based on DNA aptamers for detection of ochratoxin A (OTA). The thiolatedDNA aptamers specific to OTA of different configurations have been immobilized by chemisorption to the surfaceof a gold electrode. Electrochemical impedance spectroscopy (EIS) in the presence of a redox probe [Fe(CN)6]

�3/�4

has been used for the determination of the charge transfer resistance, Rct, followed by addition of OTA. The Rct in-creased with increasing OTA concentration in the range of 0.1–100 nM. The limit of detection (0.12–0.40 nM) de-pended on the configuration of the aptamers. The sensor was regenerable and validated in food samples with satis-factory recovery.

Keywords: DNA aptamer, Ochratoxin A, Electrochemical impedance spectroscopy, Biosensors

DOI: 10.1002/elan.201100485

1 Introduction

Electrochemical biosensors based on DNA aptamers (ap-tasensors) are of growing interest due to their high sensi-tivity and selectivity [1–5]. This is particularly due tounique properties of DNA or RNA aptamers – the singlestranded nucleic acids with high affinity to proteins or toother low and macromolecular compounds, which is com-parable with that of antibodies. In contrast to antibodies,aptamers are synthesized in vitro by the SELEX proce-dure [6,7]. Aptamers also present high versatility to beingchemically functionalized by biotin, thiol or aminogroups, allowing them to be immobilized on various solidsupports. In contrast with antibodies aptamers are morestable and the aptasensors can also be regenerated. Thesespecial characteristics open new routes in biosensor as-sembling for further practical applications, such as diag-nosis purposes in medicine or detection of toxicants infood or environment. Recently a DNA aptamer sensitiveto mycotoxin – ochratoxin A (OTA) – has been devel-oped [8]. OTA is a toxical fungal metabolite produced byAspergillus ochraceus and Penicillium verrucosum thathas been identified as a contaminant in cereals, coffee,cocoa, dried fruits, pork and occasionally in the field ofgrapes, during storage. It may also be present in bloodand kidneys of animals that have been fed contaminatedfoodstuff. Animal studies indicated that OTA is carcino-genic [9]. Already in 1991 the Joint FAO/WHO ExpertCommittee on Food Additives (JECFA) evaluated a pro-visional tolerable weekly intake (PTWI) of 112 ng/kg

body weight (b.w.) for OTA. This evaluation was basedon porcine nephropathies data [9]. Most recently, the Eu-ropean Commission has fixed the maximum concentra-tion of OTA in foodstuffs to: 3 mg/kg (7.4 nM) for cerealproducts, 5 mg/kg (12.4 nM) for roasted coffee and up to10 mg/kg (25 nM) for instant coffee. A similar contamina-tion limit was fixed for dry grapes (10 mg/kg) (EC No.466/2001, 1881/2006), but a contamination limit of 2 mg/L(5 nM) is valid for wine (EC No. 123/2005). An evenstronger limit of OTA contamination was fixed for allbaby food (0.5 mg/kg, EC No. 466/2001). Cereals, especial-ly in countries with hot climate have a strong tendency tobe contaminated by OTA. Many works also reported con-tamination of grape juices with approximately 7 mg/LOTA (see [9] and references herein). The development offast and efficient methods for OTA detection is thereforeof significant importance. Biosensors for OTA determina-tion developed so far were based particularly on the oxi-dation of OTA at glassy carbon electrodes (limit of detec-tion (LOD) 0.26 mM) [10], at cysteamine self-assembledmonolayers (LOD=10 pM) [11] or reduction of horse-radish peroxidase (LOD=0.25 nM) [12]. The indirectamperometric immunoassay utilized polyclonal antibodiesagainst OTA immobilized on screen-printed electrodes[13] or gold colloid was of similar LOD (0.2 nM) [14]. Anegligible matrix effect was observed for detection ofOTA in corn samples. Improved sensitivity has been ob-tained in indirect amperometric immunoassay in whichpolyclonal antibodies were immobilized on dextran modi-fied gold screen-printed electrodes (LOD=0.12 nM). The

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sensor was successfully validated in spiked wine samples[15]. A similar detection limit (0.14 nM) has been ob-tained by surface plasmon resonance (SPR) immunosen-sor. The high sensitivity has been obtained by signal am-plification using antibody-gold nanoparticle conjugates[16]. For comparison previous applications of SPR forOTA detection using polypyrrole films doped with chlo-ride (Ppy-Cl) resulted in substantially lower sensitivity(approximately 0.24 mM) [17]. Using molecularly imprint-ed polypyrrole (MIPPy) the sensitivity was improved bytwo fold (0.12 mM), which is, however not sufficient formost practical applications [18]. The best detection limithas been obtained by immunosensors using antibodiesimmobilized on carbon nanotubes (LOD=6.2 pM) [19].Direct, label-free detection of OTA using electrochemicalimpedance spectroscopy (EIS) based on immunosensorscomposed of OTA sensitive antibodies immobilized onvarious substrates has also been developed. As a matrixfor antibodies immobilization a gold surface was in par-ticularly used onto which OTA specific antibodies wereadhered using carbodiimide chemistry (LOD=1.2 nM)[20]. A chitosan–polyaniline hybrid conducting biopoly-mer film on an indium–tin oxide (ITO) electrode wasalso used for this purpose (LOD=2.4 nM) [21,22].Rather high sensitivity (LOD=6 pM) was obtained withan EIS immunosensor in which the specific antibodieswere immobilized on nanostructured zinc oxide depositedonto an ITO covered glass plate [23]. Most recently alsothe direct SPR and EIS immunosensors were reported[24]. In this work the monoclonal antibodies specific toOTA were immobilized at the surface using magneticbeads. Addition of OTA resulted in an increase of theSPR angle and of the charge transfer resistance in EISexperiments. The detection limit obtained (0.94 mg/L and0.01 mg/L�1 for SPR and EIS sensor, respectively) is suffi-cient for practical applications. Despite the fact that im-munosensors are rather popular and also ELISA kitassays are commercially available, the disadvantage ofthese methods consist in the necessity of using stable anti-bodies. The production of antibodies needs animals, is la-borious and expensive. Also the biosensors based on anti-bodies are difficult to regenerate. Aptamers can thereforebe a good alternative instead of antibodies.

According to WOS database the first aptasensor forOTA was reported most recently and utilized either elec-trochemiluminescence method of detection (LOD=17 pM) [25] or amperometric detection based on methyl-ene blue as a redox probe [26]. In the later case the effec-tive detection range of OTA was 2.4–48 nM. The sensitivi-ty of the chemiluminescence method for OTA detectionwas recently substantially improved by using Fe3O4 basedmagnetic nanoparticles (MNPs) and upconversion nano-particles (UCNPs) as labels [27]. The assay was based onimmobilizing aptamer DNA1 sequence onto the surfaceof MNPs, which allowed capturing and concentratingOTA from bulk samples. DNA1 sequence has been al-lowed to hybridize with UCNPs modified DNA2 se-quence, which could dissociate from DNA1 and results in

a decreased luminescent signal when the aptamer DNA1is recognized and bound to OTA. Under optimal condi-tions, the decreased luminescent intensity was proportion-al to the concentration of OTA in the range from 0.1 ng/L to 1 mg/L with a detection limit of 0.1 ng/L (0.25 pM)).In our recent work we reported a thickness shear modeacoustic aptasensor for OTA detection using biotinylatedaptamers that have been adsorbed on a thin gold layer ofa quartz crystal transducer covered by neutravidin. Thissensor allowed direct detection of OTA with LOD 30 nM[28]. Further an electrochemical aptasensor based on acompetitive assay (LOD=0.27 nM) has been developed[29]. The sensitivity of this sensor was validated in spikedwine. Slightly improved sensitivity has been obtained withan aptasensor based on magnetic beads technology(LOD=0.17 nM) [30]. However, in these works, OTAconjugated with enzymes such as alkaline phosphatasemust be used in a competitive ELISA like-assay. Thedirect, one step detection of OTA would be, however,more advantageous for practical applications. Most re-cently a simple colorimetric method for OTA recognitionhas been reported [31]. In this work gold nanoparticleswere modified by OTA sensitive aptamers. Addition ofOTA resulted in removal of the aptamers from the sur-face of nanoparticles and after addition of salts colourchanges have been observed due to nanoparticle aggrega-tion. This method allowed detection of OTA with anLOD of 20 nM. Another approach for direct detection ofOTA is based on EIS electrochemical aptasensors. Theresults in developing such aptasensors utilizing thiolatedaptamers specific to OTA chemisorbed on a gold surfacehave been presented by us at the AISEM conference inFebruary 2011 [32]. This biosensor revealed comparablesensitivity with the above-mentioned indirect assay(LOD=0.4 nM) and selectively detected OTA. Thesensor was regenerable in 1 mM HCl and validated incoffee and flour. Most recently a sensitive EIS biosensorbased on DNA aptamer specific to OTA covalently im-mobilized onto a mixed Langmuir–Blodgett monolayercomposed of polyanilyne-stearic acid and deposited onITO coated glass plates has been reported [33]. Thissensor revealed a similar detection limit (0.24 nM), how-ever the fabrication procedure has been more complicat-ed in comparison with simple chemisorption used in ourwork. The sensor discriminated between OTA and afla-toxin, however it has not been validated in food samples.We therefore extended our research in order to show ad-vantage of the EIS based aptasensor utilizing chemisorp-tion of aptamers. We have also shown that the presenceof calcium ions is important for the sensitivity of thesensor to OTA. We also analyzed the sensitivity of vari-ous aptamers configurations, including aptamer homo-dimers. The sensor discriminated between OTA andochratoxin B (OTB) and N-acetyl-l-phenylalanine(NAP). The aptasensor was regenerable in 1 mM HCland it was validated in coffee, flour and wine spiked byOTA. The detection limit was comparable to that of theHPLC method (0.12 nM) [34].

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Impedimetric DNA Aptasensor for Sensitive Detection of Ochratoxin A in Food

http://www.electroanalysis.wiley-vch.de

2 Materials and Methods

2.1 Reagents and Materials

The HPLC purified DNA aptamers have been purchasedfrom Thermo Fisher Scientific (Ulm, Germany). Theaptamer sequences are shown in Table 1. The binding siteof the aptamers is identical with that reported in [8](aptamer 1.12.2). However, the aptamer DNA1 is extend-ed by thymidine spacer dT15 terminated by a thiol group,DNA2 is identical to DNA1 but containing the hybrid-ized part of dT15-dA15 as a spacer. DNA3 is the homodi-mer of aptamer DNA1 containing two identical bindingsites and dT15-dA15 double helix spacer. DNA4 is theaptamer without spacer used in [33]. The sequence of thisaptamer is identical with that of DNA1 aptamer, but it isimmobilized on a gold surface at the 5’ end and does notcontain the dT15 spacer.

All standard reagents as well as B-mercaptoethanol(MET) and N-acetyl-l-phenylalanine (NAP) were pur-chased from Sigma-Aldrich (USA). Ochratoxin A (OTA,M.W. 403.8) and ochratoxin B (OTB, M.W. 369.4) weresupplied by Romer Labs (Austria). The reagents usedwere of p.a. grade. For sensor preparation the gold elec-trode (diameter 2 mm) from CH Instruments Inc. (USA)was used.

2.2 Preparation of the Biosensor and the Samples

The thiolated aptamers were immobilized at the surfaceof the gold electrode by chemisorption. Prior to modifica-tion, the electrodes were polished using an electrode-pol-ishing kit consisting of 1.0, 0.3 and 0.05 mm aluminapowder (CH Instruments, USA) and then cleaned withfreshly prepared “piranha solution“ (3 :1 mixture of con-centrated H2SO4 and 30% H2O2) at 90 8C for 5 min andsubsequently rinsed thoroughly with Millipore water.Warning : Piranha solution is highly dangerous and mustbe handled with extreme caution. Then electrochemicalcleaning of 10 successive cycles in 0.5 M H2SO4 in the po-tential range from +0.2 to +1.5 V with a scan rate of100 mV/s was applied to gold electrodes. After that theelectrodes were properly rinsed again in deionised waterand dried under nitrogen.

Aptamer immobilization was done by self-assembling.The aptamers dissolved in TE buffer (10 mM TRIS+1 mM EDTA, pH 7.6) in a concentration of 5 mM wereadded at the surface of the gold electrode and kept in a

closed container preventing the solvent evaporation at4 8C for 16 h. Prior immobilization, aptamers solution wasincubated in a 90 8C water bath and then exposed to icecooling for providing proper folding of the aptamers.Once the immobilization was done, the gold electrodeswere rinsed in deionised water and incubated in 100 mMMET during 20 min for removing physically adsorbed ap-tamers and for blocking the bare electrode�s surface, thusavoiding nonspecific binding. The aptamers DNA2 andDNA3 containing complementary oligonucleotides wereprepared as follows: 0.5 mL of each aptamer solution in aconcentration of 5 mM were mixed and then immersed ina 95 8C water bath (volume approximately 50 mL) andsubsequently allowed to cool to room temperature (ap-proximately 25 8C). This ensures full DNA hybridizationas we approved earlier by circular dichroism [35].

The electrodes modified by aptamers and MET wererinsed several times in binding buffer (10 mM HEPES+120 mM NaCl+5 mM KCl+20 mM CaCl2, pH 7.0) fol-lowed by incubation for 30 min in different concentra-tions of OTA 0.1–100 nM. For this purpose a smallvolume (10 mL) of OTA dissolved in binding buffer wasadded at the surface of the sensor. Electrodes have thenbeen rinsed in distilled water and immediately used forelectrochemical experiments. In order to check the effectof calcium on OTA binding, we used binding buffer with-out and with 5, 10 and 20 mM Ca2+, respectively. For test-ing the sensor selectivity, 100 nM OTB dissolved in bind-ing buffer was incubated with the aptamer-MET modifiedelectrodes and the electrochemical impedance spectrawere recorded. We used also NAP as a possible interfer-ence. This residue is important part of OTA [9]. Thestock solutions of OTA, OTB and NAP were prepared bydissolving these reagents in a binding buffer in a concen-tration 248 nM.

The sensor has been validated in spiked coffee andflour extracts as well as in a red wine from Low Carpa-tian region (Slovakia) (Frankovka modra, Chateau Topol-cianky 2011, content of ethanol 12%). The coffee hasbeen diluted in hot water (approximately 95 8C) in a con-centration 10 % (w/v). After approximately 30 min extrac-tion the solution was filtered using Whatman No. 4 filterpaper. Similar procedure was applied for flour, howeverin this case demonized water (20 8C) was used for prepa-ration of 10% solution. The samples were spiked by 1, 5and 10 ppb (2.48, 12.4, 24.8 nM, respectively) of OTA.The detection of OTA has been performed as follows.The response of freshly prepared sensor in the presence

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Table 1. The oligonucleotide sequences of aptamers used, the value of dissociation constant, KD, and limit of detection (LOD) deter-mined for aptasensors in the presence of 20 mM Ca2+ in binding buffer (see Section 3).

Aptamer Sequence 5’ to 3’ KD (nM) LOD (nM)

DNA1 GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-dT15-SH 8.3�0.8 0.40�06DNA2 GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-dT15-SH+dA15 17.3�5.6 0.40�06DNA3 GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-dT15-SH

+dA15-ACAGGCTACGAGGGAAATGCGGTGGGTGTGGGCTAG3.3�1.0 0.25�0.04

DNA4 SH-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA 1.2�0.2 0.12�0.02

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of 1 mM [Fe(CN)6]�3/�4 (the redox probe) was measured

in a binding buffer. Then OTA was added in a desiredconcentration and the response has been measured. Afterthis the sensor was regenerated by immersing in 1 mMHCl for 10 s and then rinsed in demonized water and inbinding buffer. The matrix (coffee, flour or wine) withoutOTA has been added at the sensor surface and incubatedduring 30 min. The response was measured at the pres-ence of 1 mM redox probe. The sensor was then washedwith deionized water and in binding buffer and thespiked sample (coffee, flour or wine) was added at thesensor surface and incubated during 30 min. The sensorwas then washed in binding buffer and immersed in meas-uring cell containing 1 mM redox probe.

2.3 Electrochemical Measurements and Determination ofthe Surface Density of Aptamers

Electrochemical experiments were performed with a po-tentiostat/galvanostat AUTOLAB PGSTAT 12 (EcoChemie, The Netherlands) in a Teflon cell of a 5 mLvolume using standard three electrode configuration:working gold electrode (diameter 2 mm) a Pt wire thatserved as auxiliary electrode and Ag/AgCl/1 M KCl refer-ence electrode (CH Instruments or BAS, USA). Electro-chemical impedance spectroscopy (EIS) was performedwith FRA module of AUTOLAB in a frequency range of0.1 Hz–100 kHz and AC voltage of 5 mV. Simultaneouslyalso a DC voltage of 0.165 mV amplitude has been ap-plied. This voltage corresponds to the formal potential of1 mM [Fe(CN)6]

�3/�4.For determination of the aptamer density on a gold sur-

face we used the method described in [36]. First we deter-mined the working area, Aw, of the bare gold electrode(prior to immobilization of DNA aptamers). Accordingto Randles–Sevcik equation the peak current, ip, for a re-versible couple such as [Fe(CN)6]

�3/�4 at 25 8C can be ex-pressed as [37]:

ip ¼ ð2:69� 105Þ n3=2AwCD1=2v1=2 ð1Þ

where n is the number of electrons, Aw is the workingarea (in cm2), C is the concentration in (mol/cm3), D isthe diffusion coefficient (in cm2/s) and v is the potentialscan rate (in V/s). Thus, from the plot of ip as a functionof the scan rate, which is linear for a reversible redoxcouple, the Aw can be determined from the slope of thisdependence. In the experiment we determined ip as func-tion of the scan rate using CV in the presence of 1 mM[Fe(CN)6]

�3/�4 in a binding buffer (pH 7.0) and in a poten-tial range from �0.2 V to +0.6 V vs. Ag/AgCl at scanrates in the range of 0.1–0.5 V/s.

The surface density of DNA aptamers has been deter-mined by CV method in the presence of 50 mM[Ru(NH3)6]

3+ in a binding buffer at pH 7.0 [36]. Briefly,the aptamer modified electrode has been pretreatedduring 5 s at +150 mVvs. Ag/AgCl in a binding buffer

containing 50 mM [Ru(NH3)6]3+. Then the CV has been

applied in a potential range from �500 mV to +150 mVat a scan rate of 0.5 V/s. The integration of the cathodicpeak at the first scan yields the charge Q correspondingto [Ru(NH3)6]

3+ ions adsorbed at the sensor surface. Thecorresponding surface concentration of DNA aptamers(in cm�2), GApta, can be determined by the equation:

GApta ¼ QzNA=ðnmFAwÞ, ð2Þ

where z=3 is the charge of redox molecules, NA =6.022 �1023 mol�1 is Avogadro�s number, n=3 is the number ofelectrons in the redox reaction, m is the number of nucle-otides in DNA aptamers, F=9.6485� 104 C mol�1 is theFaraday constant. All experiments were performed at25 8C.

3 Results and Discussion

Using mfold program [38], which is available on the inter-net, we analyzed the aptamer oligonucleotide sequence(DNA1). We found two structures containing loops thatslightly differ by Gibbs energy. These structures are pre-sented in Figure 1. Energetically the more favourablestructure (DG=�1.2 kcal/mol) contains a loop betweennucleotides 16 and 28 (Figure 1 A). A larger loop wasfound for the second structure (Figure 1 B) (DG=�0.88 kcal/mol). Further we analyzed the aptamer struc-ture using the QGRS Mapper program [39], that allowsto predict the existence of guanine quadruplexes. Ouranalysis shows that the aptamer contains one guaninequadruplex connected by loops as it is shown in Figure 2.The existence of a quadruplex is also supported by ourrecent data on the study of thermodynamic properties ofOTA aptamers [28]. The phase transition temperature formost stable aptamers in the presence of 20 mM Ca2+ hasbeen 48.3�0.5 8C, which is close to the melting tempera-ture of DNA aptamers sensitive to the fibrinogen bindingsite of thrombin that also contains one guanine quadru-plex. For this aptamers the quadruplex structure has beenwell established using various methods including circulardichroism (CD) [35]. Most recently the existence of aquadruplex in OTA aptamers has been approved by CDmethod [31]. The changes in Gibbs energy for OTA ap-tamers determined from melting data was 3.8 kcal/mol,which is higher in comparison with that obtained frommfold program. However, this program does not takeinto account the quadruplexes, which are rather stablealso thanks to the stabilizing role of K+ ions.

The binding site for OTA in the aptamers is not knownyet. However, as it has been shown in the original workby Cruz-Aguado and Penner [8] this aptamer has ratherhigh affinity to OTA. This affinity substantially increasesin the presence of 20 mM Ca2+ (dissociation constantKD =49 nM). At the same time, no binding of OTA wasobserved without calcium or magnesium ions. It is there-fore surprising that in the paper by Prabhakar et al. [33]

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the binding of OTA to aptamers has been observed in abinding buffer that did not contain calcium (binding con-stant KA =1.21�107 M�1, which correspond to a dissocia-tion constant KD =82.6 nM). In one series of the experi-ments we therefore examined the effect of calcium on theimpedance spectra of the aptasensor.

It is well known that electrochemical impedance spec-troscopy (EIS) is highly sensitive to surface modifications.Using the redox probe [Fe(CN)6]

�3/�4 it is possible to am-plify the detection of affinity interactions at surfaces. Thishas been demonstrated for detection of proteins by DNAaptamers as receptors. Because a DNA aptamer is nega-tively charged the anionic redox probe will repeal fromthe sensor surface causing an increase of the charge trans-fer resistance (Rct). Binding of protein resulted in a de-crease (lysozyme) [40] or increase (thrombin) [41] of thenegative charge at the surface, causing decrease or in-crease of Rct, respectively. This approach has been used

so far mostly for the detection of proteins. However, bothin our work [32] and in the work by Prabhakar et al. [33]it has been shown that this method can be applied alsofor the determination of small molecules like OTA.

The surface density of aptamers was determined ac-cording to the method described in Section 2.3. First, theworking area of the electrode was calculated from theplot of ip vs. v1/2. This dependence was linear. For examplefor the anodic current we obtained ip =9.5 v1/2 (R2 =0.98)From the slope of this dependence the Aw value was de-termined according to Equation 1. This value was typical-ly 0.026�0.001 cm2. CV of the aptasensor in the presenceof 50 mM [Ru(NH3)6]

3+ has been characterized by redoxpeaks with a formal potential of 0.213 V and a peak sepa-ration of 0.036 V. The integration of the cathodic peaktypically yields a charge Q=1.31� 10�6 C for DNA 1 andQ=2.22� 10�6 C for DNA3, respectively. Using Q and Aw

values and Equation 2 the surface density of aptamerswas determined. For the DNA1 aptamer we obtained asurface density of 1.85 �1013 cm�2. This value is in goodagreement with the one previously reported for immobili-zation of single stranded DNA [36]. The surface densityof aptamer dimers (DNA3) was slightly lower, 1.48 �1013 cm�2, probably reflecting a larger part of binding sitedue to the presence of two guanine quadruplexes. TheEIS response was analyzed by means of the Randlesequivalent circuit (Figure 3 Inset) and fitted using the Au-tolab FRA software. The Nyquist plot of the sensinglayer composed of DNA1 aptamers at various concentra-tions of OTA and in the presence of 20 mM Ca2+ is pre-sented in Figure 3. The plot consists of a semicircle athigher frequencies and of a linear part at lower frequen-cies. The diameter of the semicircle is proportional to theRct value while the linear part corresponds to the diffu-

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Fig. 1. 2D structures of OTA aptamers generated by mfold program.

Fig. 2. The guanine quadruplex in the OTA aptamer structuredetermined according to QGRS Mapper program.




sion of the redox probe, which is represented by the War-burg impedance. It can be seen that with increased con-centration of OTA the diameter of the semicircle increas-es which is evidence of increased Rct values. At neutralpH (pH 7.0) in water, OTA is negatively charged. This isdue to ionization of carboxyl (pKa =4.2–4.4) and phenolicmoieties (pKa =7.0–7.3) [42,43]. Therefore both mono-(OTA�) and dianions of OTA (OTA2�) are present atphysiological pH. This means that under the experimentalconditions used (pH 7) OTA is negatively charged. Thus,binding of OTA to aptamers should increase the negativecharge of the surface and induce repealing of the redoxprobe. As a result the Rct value should increase. Thiseffect has been certainly observed. This is also in agree-ment with EIS data published in [24] on OTA imunosen-sor in which addition of OTA also resulted in an increaseof Rct in the presence of a 2.5 mM [Fe(CN)6]

�3/�4 redoxprobe. However, opposite Rct changes were reported in[33], in which a 5 mM [Fe(CN)6]

�3/�4 redox probe wasused. It should, however, be noted that EIS experimentsusing redox probes require special care, in particularly alow concentration of the redox probe is required (see[44]). At the same time other parameters of the circuitsuch as capacitance and Warburg impedance changedonly slightly (Table 2). The changes in Rct can serve as ananalytical signal useful for analysis of the aptamer�s sensi-tivity and selectivity, in particular in the presence of calci-um ions. Considering different results obtained in ourwork and in [33], we studied the properties of an aptasen-sor based on an identical DNA4 aptamer. However thedifferences lay in the method of aptamers immobilization.While we used chemisorption for aptamers immobiliza-tion, in [33] aptamers modified by amino groups havebeen immobilized on polyaniline-stearic acid films bycrosslinking with glutaraldehyde. The Nyquist plot of theaptasensor composed of DNA4 was similar to that forDNA1. With increased OTA concentration Rct also in-creases. The presence of Ca2+ affects significantly thebinding of OTA to aptamers as it is evident from Figure 4in which the plot of Rct for the aptasensor as a function ofcalcium concentration without OTA and in the presenceof 10 mM OTA is presented. It can be seen that for [Ca2+

]>5 mM, the Rct value slightly increases for aptasensorwithout OTA, but substantial increase took place in thepresence of 10 nM OTA. It is likely that the presence ofcalcium enhances the binding of OTA to the aptasensor.It is interesting that calcium does not result in a decreasein Rct for sensors without OTA as it has been expectedconsidering the positive charge of calcium ions that canpartly neutralize the negative charge of DNA aptamersbackbone. Such an effect was observed for dsDNA immo-bilized on a gold surface in the presence of Zn2+ or Mg2+

ions [45]. We can speculate that Ca2 + probably affectsthe aptamer�s conformation and thus influences also theEIS properties of the sensing layer. Similarly to our work,the enhanced binding of OTA in the presence of calciumions was reported in [30]. This has been attributed tocomplexation of Ca2+ with negatively charged carboxyl

or hydroxyl groups of OTA and negatively charged phos-phate backbone of aptamers.

In order to obtain further insight in the effect of Ca2+

we also plotted the normalized relative changes of Rct

(DRct/Rct0) as a function of OTA for the aptasensor com-posed of DNA4 aptamer without and with 20 mM Ca2 +

in binding buffer. This plot is presented in Figure 5. It canbe seen that at relatively low OTA concentrations of 0.1–3 nM a sharp increase of OTA took place with subse-quent saturation at higher toxin concentrations. As ex-pected (see Figure 4) the larger values of Rct were ob-

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Fig. 3. Nyquist plot of the gold electrode modified by DNA1aptamer/mercaptoethanol with different OTA concentrations inthe presence of 1 mM redox probe [Fe(CN)6]

�3/�4. AC impedancerecorded at a DC potential of 0.165 V and at 5 mV of AC voltagefrom 0.1 Hz to 100 kHz. Inset (A) components of the Randlesequivalent circuit: RS is the electrolyte resistance, C is the inter-facial capacity, Rct the charge transfer resistance and ZW is theWarburg element.

Fig. 4. Plot of Rct as a function of the calcium concentration inbinding buffer for the aptasensor composed of DNA4 aptamerwithout (open circles) and in the presence of 10 nM OTA (solidcircles).




served in the presence of calcium ions. In both cases theplot has the shape of Langmuir adsorption isotherm [37]and can be described by the following equation:

ðDRct=Rct0Þ ¼ ðDRct=Rct0Þmax ½c=ðKD þ cÞ� ð3Þ

where (DRct/Rct0)max is the maximum charge transfer rela-tive resistance variation and c is the OTA concentration.The KD value is a measure of the affinity of the analyteto the receptors on the sensor surface. A lower dissocia-tion constant corresponds to higher affinity, or higher sta-bility of the analyte–receptor complex. Using Equation 3and the least square method one can obtain KD =8.4�1.1 nM for an aptasensor without Ca2+ and KD =1.2�0.2 nM in the presence of 20 mM Ca2+ . Thus the affinityof OTA is approximately 7 times higher in the presenceof calcium ions in comparison with an aptasensor withoutcalcium in binding buffer. For comparison the KD valuecalculated on association constant reported in [33] wasmuch higher (82.6 nM). The later value is even two foldhigher in comparison with that reported in [8] for aptam-ers in a volume. It is likely that the polyaniline surfaceand aptamer crosslinking by glutaraldehyde could affectthe aptamers conformation and as a result the affinity ofaptamers to OTA. The KD value obtained in our work ismuch lower in comparison with that determined by fluo-rescence method for free aptamers in a volume [8]. Thismay be evidence of improved affinity of aptamers immo-bilized by thio-linker at the gold surface to OTA. TheLangmuir binding curve also suggests that OTA binds tothe aptamers independently. The limit of detection deter-mined for signal to noise level S/N=3 was 0.12�0.02 nM,which is below the recommended content of OTA in vari-

ous foods and thus, the sensor is adequate for practicaluse.

The aptamers configuration had significant effect onthe sensor properties. Figure 6 shows the plot of normal-ized relative changes of Rct as a function of OTA concen-trations for aptasensors composed of OTA sensitive ap-tamers of various configurations listed in Table 1.

It can bee seen that for all aptasensors the bindingcurves are rather similar with highest response for DNA4aptamer. For these aptasensors we also calculated the KD

values (see Table 1). The highest affinity to OTA as re-vealed from comparison of KD values, was obtained foraptasensor composed of DNA4 aptamer. The comparisonof the binding properties of conventional aptamers withaptamers dimer (DNA 1 and DNA 3, respectively), re-vealed improved binding properties for aptamer dimer.This is a consequence of the second binding site at aptam-ers dimer. The binding affinity of a biosensor composedof DNA2 aptamer with rigid supporting part does not sig-nificantly differ from DNA1 based biosensor. The LODfor the aptasensors studied was in the range from 0.12 to0.40 nM (Table 1). The differences between DNA1 andDNA4 aptamer based biosensors are not clear and re-quire special study. The values of Rct for these biosensorswithout OTA (10.4�2.3 kW and 12.4�3.5 kW, respective-ly) do not differ significantly. We can only conclude thatdT15 spacer is not necessary for proper functioning of theOTA aptasensor.

The aptasensors were selective to OTA. No significantchanges in Rct were observed when ochratoxin B (OTB)or N-Acetyl-l-phenylalanine (NAP) in a concentration of100 nM were added to the sensor surface (Figure 7). Itwas also possible to regenerate the sensor by immersingit in 1 mM HCl for 30 s and subsequently by washing it indeionised water according to the method published in

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Fig. 5. Plot of the normalized relative changes of the chargetransfer resistance, DRct/Rct0, as a function of OTA concentrationfor the aptasensor composed of DNA4 without (open circles)and in the presence of 20 mM Ca2+ (solid circles) in bindingbuffer. DRct =Rct�Rct0, Rct0 is the charge transfer resistance with-out OTA and Rct that at certain OTA concentration. The fulllines are fits according to Equation 3. Results represent mean�SD obtained at 3 independently prepared biosensors.

Fig. 6. Plot of relative changes of charge transfer resistance,DRct/Rct0, as a function of OTA concentration for aptasensorswith immobilized aptamers of various configurations (see legendand Table 1). The full line is the fit according to Equation 3. Re-sults represent mean�SD obtained at 3 independently preparedbiosensors.




[46]. The sensor�s regeneration at least up to 10 times hadno significant effect on the sensor response and OTA de-termination was practically identical with that of a freshlyprepared biosensor. Thus, the biosensor is sufficientlysensitive and selective for practical purposes. We also va-lidated the biosensor for determination of OTA in spikedcoffee and flour extracts as well as in red wine. For thispurpose we used 10 % (w/v) of coffee and flour solutionin water and red wine from Low Carpatian wine region.The samples were spiked with 1, 5 and 10 ppb of OTA

and compared with that measured just in buffer. We haveshown that after incubation of the sensor in a spikedsample during 30 min and after thoroughly washing thesensor surface in a binding buffer the matrix effect wassuccessfully eliminated. The sensor response to OTA forall samples was similar to that in buffer (Table 3). Weshould however note that the rinsing procedure is impor-tant to eliminate any matrix effect that was remarkablefor coffee and wine, but rather small for flour. Regardingto this, the results agree well with that reported for OTAdetermination in spiked wine [20].

4 Conclusions

The aptasensor based on thiolated aptamers chemisorbedon gold can detect ochratoxin A (OTA) with a high sensi-tivity depending on the aptamer�s configuration (LOD=0.12–0.40 nM), which is sufficient for determination ofOTA in food samples. Easy regeneration and relativelylow matrix effects make this sensor an effective tool fordirect detection of OTA in food analysis. We validatedthe sensor in a limited number of food samples. Thereforeextension of this validation is required in further studiesincluding optimization of the assay. DNA aptamers re-vealed good selectivity to OTA, however additional stud-ies are required to examine the binding of other OTA an-alogs to the sensor. Differences in sensor response ob-tained in our work and in [33] indicate that the surfacemodification and the method of aptamer immobilizationare crucial for behaviour and response of the sensor. Atthe same time, supplementary analysis is necessary inorder to understand the mechanisms of binding smallmolecules such as OTA to DNA aptamers. From ourwork we can conclude that the presence of calcium is es-sential for OTA binding to aptamers since calcium ionsfavour the quadruplex formation on aptamers and im-prove its binding affinity to the analyte of interest. Onthe other hand, EIS as a sensitive detection method pro-vides satisfactory results when working in recognition oflow molecular compounds by using non-labelled probes,as aptamers. It permits a simple and relatively rapid de-tection of the analytes. These two properties in the case

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Fig. 7. Plot of Rct for the aptasensor composed of DNA1 apta-mer in the presence of 100 nM NAP, OTB or OTA (gray col-umns) or without these compounds in a binding buffer (whitecolumns). Results represent mean�SD obtained at 3 independ-ently prepared biosensors.

Table 2. The parameters of Randles circuit for aptasensor(DNA1 apatmer) in the presence of various concentrations ofOTA.

[OTA] (nM) Rs (kW) Rct (kW) C (nF) W� 10�5

0 14.87 10.35 352 0.1210.1 15.03 16.21 347 0.0980.3 15.01 18.43 343 0.0971.0 14.95 23.30 315 0.0953.0 14.93 25.57 310 0.09310.0 14.98 30.86 280 0.09130.0 15.02 39.80 256 0.090100.0 15.04 57.02 241 0.089

Table 3. The determination of OTA by impedimetric aptasensor (DNA1 aptamer) in spiked coffee, flour and wine. Results representmean�SD obtained from three independent experiments. RSD (relative standard deviation)= standard deviation/mean�100 %; RE(relative error)= [(true value-measured value)/true value] �100 %; n=3.

Sample OTA added, ppb OTA found, ppb RSD (%) RE (%) Recovery (%)

Coffee 1 0.85�0.03 3.5 15.0 855 4.4�0.04 0.9 12.0 87

10 8.8�0.03 0.3 12.0 88Flour 1 1.07�0.02 1.8 7.0 107

5 5.4�0.03 0.6 8.0 10810 10.4�0.07 0.6 4.0 104

Wine 1 0.78�0.01 1.3 22.0 785 4.04�0.06 1.5 19.2 81

10 9.13�0.02 0.2 8.7 91




of OTA, or more generally talking in food analysis, are ofvital importance for biosensor assembling.

Acknowledgments

This work was supported by the Slovak Research and De-velopment Agency (Contract No. APVV-0410-10, LPP-0341-09), by the MNT-ERA.NET Project IntelliTip andIRSES 7 FP program of EU, Materials Enhancement forTechnological Applications (META), Grant agreementNo. 269182.

References

[1] Y. Xiao, B. D. Piorek, K. W. Plaxco, A. J. Heeger, J. Am.Chem. Soc. 2005, 127, 17990.

[2] T. Hianik, J. Wang, Electroanalysis 2009, 21, 1223.[3] A. K. H. Cheng, D. Sen, H-Z. Yu, Bioelectrochemistry 2009,

77, 1.[4] E. E. Ferapontova, K. V. Gothelf, Current Org. Chem. 2011,

15, 498.[5] F. Wei, C.-M. Ho, Anal. Bioanal. Chem. 2009, 393, 1943.[6] C. Tuerk, L. Gold, Science 1990, 249, 505.[7] A. D. Elington, J. W. Szostak, Nature (London) 1990, 346,

818.[8] J. A. Cruz-Aguado, G. Penner, J. Agric. Food Chem. 2008,

56, 10456.[9] A. El Khoury, A. Atoui, Toxins 2010, 2, 461.

[10] S. C. B. Oliveira, V. C. Diculescu, G. Palleschi, D. Compag-none, A. M. Oliveira-Brett Anal. Chim. Acta 2007, 588, 283.

[11] P. R. Perrotta; N. R. Vettorazzi, F. J. Arevalo, A. M. Gra-nero, S. N. Chulze, M. A. Zon, H. Fernandez, Electroanaly-sis 2011, 23, 1585.

[12] M. A. Alonso-Lomillo, O. Dom�nguez-Renedo, L. Ferreira-Goncalves, M. J. Arcos-Mart�nez, Biosens. Bioelectron. 2010,25, 1333.

[13] S. H. Alarcon, G. Palleschi, D. Compagnone, M. Pascaled,A. Visconti, I. Barna-Vetro, Talanta 2006, 69, 1031.

[14] X-P. Liu, Y-L. Deng, X-Y. Jin, L-G. Chen, J-H. Jiang, G-L.Shen, R-Q. Yu, Anal. Biochem. 2009, 389, 63.

[15] M. Meike Heurich, M. K. A. Kadir, E. Ibtisam, I. E. Tothill,Sens. Actuators B 2011, 156, 162.

[16] A. E. Urusov, S. N. Kostenko, P. G. Sveshnikov, A. V. Zher-dev, B. B. Dzantiev, Sens. Actuators B 2011, 156, 343.

[17] C. C. Jorn, E. Yu, P. C. Lai, Synth. Met. 2004, 143, 253.[18] C. C. Jorn, E. Yu, P. C. Lai, Reactive and Functional Poly-

mers 2005, 63, 171.[19] A. Kaushik, P. R. Solanki, M. K. Pandey, K. Kaneto, S.

Ahmad, B. D. Malhotra, Thin Solid Films 2010, 519, 1160.

[20] A-E. Radi, X. Munoz-Berbel, V. Lates, J-L. Marty, Biosens.Bioelectron. 2009, 24, 1888.

[21] R. Khan, M. Dhayal, Electrochem. Commun. 2008, 10, 492.[22] R. Khan, M. Dhayal, Biosens. Bioelectron. 2009, 24, 1700.[23] A. A. Ansari, A. Kaushik, P. R. Solanki, B. D. Malhotra, Bi-

oelectrochemistry 2010, 77, 75.[24] L-G. Zamfir, I. Geana, S. Bourigua, L. Rotariu, C. Bala, A.

Errachid, N. Jaffrezic-Renault, Sens. Actuators B 2011, 159,178.

[25] Z. P. Wang, N. Duan, X. Hun, S. J. Wu, Anal. Bioanal.Chem. 2010, 398, 2125.

[26] H. Kuang, W. Chen, D. Xu, L. Xu, Y. Zhu, L. Liu, H. Chu,C. Peng, C. Xu, S. Zhu, Biosens. Bioelectron. 2010, 26, 710.

[27] S. Wu, N. Duan, Z. Wang. H. Wang, Analyst 2011, 136,2306.

[28] I. Lamberti, L. Mosiello, T. Hianik, Chem. Sens. 2011, 1,11.[29] L. Barthelmebs, A. Hayat, A. W. Limiadi, J-L. Marty, T.

Noguer, Sens. Actuators B 2011, 156, 932.[30] L. Bonel, J. C. Vidal, P. Duato, J. R. Castillo, Biosens. Bio-

electron. 2011, 26, 3254.[31] C. Yang, Y. Wang, J-L. Marty, X. Yang, Biosens. Bioelec-

tron. 2011, 26, 2724.[32] G. Castillo, I. Lamberti, L. Mosiello, T. Hianik, in Sensors

and Microsystems. Lecture Notes in Electrical Engineering(Ed: C. Di Natale), Springer, Heilderberg 2012,DOI:10.1007/978-1-4614-0935-9_6

[33] P. Prabhakar, Z. Matharu, B. D. Malhotra, Biosens. Bioelec-tron. 2011, 26, 4006.

[34] P. Burdaspal, T. M. Legarda, J. Gilbert, J. Assoc. Offic.Anal. Chem. Int. 2001, 84, 1445.

[35] S. Ponikov�, K. Tluckov�, M. Antal�k, V. V�glasky, T.Hianik, Biophys. Chem. 2011, 155, 29.

[36] B. Ge B, Y-C. Huang, D. Sen D, H-Z. Yu, J. Electroanal.Chem. 2007, 602, 156.

[37] J. Wang, Analytical Electrochemistry, Wiley-VCH, NewYork 2006.

[38] M. Zuker, Nucleic Acids Res. 2003, 31, 3406.[39] O. Kikin, L. D’Antonio, P. S. Bagga, Nucleic Acids Res.

2006, 34, W676.[40] M. C. Rodriguez, A-N. Kawde, J. Wang, Chem. Commun.

2005, 4267.[41] J. A. Lee, S. Hwang, J. Kwak, S. I. Park, S. S. Lee, Sens. Ac-

tuators B 2008, 129, 372.[42] S. Uchiyama, Y. Saito, J. Food. Hyg. Soc. Jpn. 1987, 28, 453.[43] R. D. Whitley, R. Wachter, F. Liu, N. H. Wang, J. Chroma-

togr. 1989, 465, 137.[44] A. Bogomolova, E. Komarova, K. Reber, T. Gerasimov, O.

Yavuz, S. Bhatt, M. Aldissi, Anal. Chem. 2009, 81, 3944.[45] Y-T. Long, C-Z. Li, H-B. Kraatz, J. S. Lee, Biophys. J. 2003,

84, 3218.[46] S. Tombeli, M. Minunni, M. Mascini, Methods 2005, 37, 48.

TO

PIC

AL

CLU

STE

R




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Impedimetric DNA Aptasensor for Sensitive Detection of Ochratoxin A in Food