Development of a very sensitive electrochemical magneto immunosensor for the direct determination of ochratoxin A in red wine

Sensors and Actuators B 162 (2012) 327–333

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Development of a very sensitive electrochemical magneto immunosensor for the direct determination of ochratoxin A in red wine Patricio René Perrotta, Fernando Javier Arévalo, Nelio Roberto Vettorazzi, María Alicia Zón ∗ , Héctor Fernández ∗ Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Agencia Postal N◦ 3, 5800 Río Cuarto, Argentina

a r t i c l e

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Article history: Received 7 October 2011 Received in revised form 21 December 2011 Accepted 23 December 2011 Available online 29 December 2011 Keywords: Ochratoxin A Immunosensor Magnetic beads Screen printed-electrode Square wave voltammetry

a b s t r a c t An electrochemical immunosensor (EI) for the determination of ochratoxin A (OTA) in red wine samples was developed. This immunosensor was based on Protein G functionalized magnetic beads (MBs) as solid phase for affinity reaction between OTA and OTA monoclonal antibody (mAbOTA). A carbon screen printed-electrodes (CSPE) system was used as electrochemical transduction element. This immunosensor was based on a direct competitive assay between OTA in wine samples and OTA labeled with horseradish peroxidase (HRP) (OTA–HRP). The HRP, in the presence of hydrogen peroxide catalyzes the oxidation of pyrocatechol to benzoquinone, whose back electrochemical reduction was detected on a CSPE by square wave voltammetry (SWV). The experimental variables involved in the immunosensor response to OTA were evaluated. The performance obtained for the EI was an analytical range of 0.01–20 ppb, limit of detection (LOD) of 0.008 ppb, and IC50 = 0.272 ± 0.081 ppb. In addition, an acceptable accuracy with a relative standard deviation (RSD) of 5.56% and very good recoveries (92–110%) were found. This work shows the potential of our EI for the direct measurement of OTA in red wine samples combining SWV as electroanalytical technique with the MBs and CSPE. This EI has great advantages as direct measurement of red wine samples without a prior pretreatment, the small volume of sample, the short time consuming of experiences and LOD well below those established by the Regulatory Commission of the European Community (2 ␮g kg−1 ). © 2011 Elsevier B.V. All rights reserved.

1. Introduction Ochratoxin A (OTA), 7-(l-␤-phenylalanylcarbonyl)-carboxyl5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocumarin, is a secondary fungal metabolite produced mainly by several Aspergillus and Penicillium genera [1]. Mainly, OTA is found in improperly stored food such as cereals, dried fruit, nuts, and beverages such as beers and wines [2,3]. OTA is met in different organs and tissues of animals as well as in human blood and breast milk. This mycotoxin is a powerful nephrotoxic, teratogenic, immunosuppressive agent [4]. The International Agency for Research on Cancer (IARC) classified OTA in 2B Group (possibly carcinogenic agent) [3]. A level of 2 ␮g kg−1 of OTA in wines has been established by the Regulatory Commission of the European Community [5]. In recent years there has been a tremendous increase in reports on the determination of OTA immunosensors based on different platforms. This reflects the current interest in developing simple and rapid methods for the determination of OTA in different

∗ Corresponding authors. Tel.: +54 358 467 6440; fax: +54 358 467 6233. E-mail addresses: [email protected] (M.A. Zón), [email protected], [email protected] (H. Fernández). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.12.089

matrixes of the agroalimentary system and, in particular, wines. The occurrence of OTA in wines is of great concern; therefore, it is necessary accurate and sensitive analytical methods for its determination. Several procedures for determination of OTA in wine samples have been published [1]. One of the most used methods for OTA detection in wines is high pressure liquid chromatography (HPLC) with previous extraction methods and different detectors (fluorescence, UV, tandem mass spectrometric detection) [6–14]. While they are sensitive and have low limit of detection, have several disadvantages such as high costs of equipment, excessive uses of solvents which are harmful to the environment, the need for qualified staff, steps that involve cleaning and sample collection resulting in increased analysis times as well as higher costs [6,7,12–14]. In official method for OTA determination in wine samples, the extraction and cleaning steps involves the use of immunoaffinity columns (IAC), which are expensive and can be used for a single sample [7]. On the other hand, exist immunological methods such as enzyme linked immunosorbent assay (ELISA), which has the disadvantage of being inaccurate, little sensitive and have high detection limits [10,15,16]. They also require of highly training personnel, have a long tedious time of analysis and high cost equipment [17]. Various analytical methods have been described in the literature related to the constructions of

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biosensors for OTA determination in wine samples [18–25] and red grapes tissues [26], which have different sensitivity and accuracy [18–21]. Also, very low levels of OTA have been detected in red wine samples on gold electrodes modified with cysteamine self-assembled monolayers [27]. In general terms, these devices have proven to be inexpensive, fast and can achieve a low limit of detection. A critical step in the construction of an immunosensor is the antibody immobilization. Adequate antigen–antibody interaction is found when the immobilized antibody is more exposed to the solution (better orientation of antibody for immune reaction) [28]. For OTA determinations in wine samples, several surfaces have been used for the antibodies immobilization in various electrochemical biosensors, such as self assembled monolayer (SAM) of different thiols on gold electrodes for generate a biosensor by surface plasmon resonance, where polyclonal antibodies against OTA were immobilized [19,21], and a surface of carbon screen-printed electrodes (CSPE) [19]. These electrodes have the great advantages to be used outside the laboratory [29]. An alternative is the use of a surface for detection (electrode) and other as support for the immune recognition (affinity reaction), for example, functionalized nano and microparticles of MBs. These are commonly used as support in separation processes of different molecules [30]. These consist of a core of magnetic material surrounded by an inert polymer. The inert polymer molecules can be functionalized by biological or chemical groups [31] with different applications such as: immobilization of amino acids [32], proteins [33,34] and different functional groups [35,36]. The use of microparticle of MBs

in separation process offers great advantages such as easy handling, reusability, homogeneous dispersion and a great surface area, which allows great improvement in the separation steps. The reactions between modified MBs with antibody and the analyte are characterized by its high reaction kinetics and sensitivity, a good reproducibility and the expansion of the working range of concentration for a given analyte due to the modulation of the number of MBs used [30,34,37]. The uses of the magnetic beads with recombinant Protein G covalently bonded to its surface confer a specific binding and orientation of the antibodies, because they have specific receptors for G-protein [28]. In this work, we develop an electrochemical magneto immunosensor for the determination of OTA in red wine samples using heterogeneous competitive immunoassays. Wine sample containing OTA with known concentration of enzyme-labeled antigen (OTA–HRP) was used. The antigen and the labeled antigen compete for a limited amount of OTA monoclonal antibody (mAbOTA), which was immobilized on MBs modified with Protein G (Fig. 1). The horseradish peroxidase (HRP) catalyzes the oxidation of pyrocatechol (H2 Q) to benzoquinone (Q) in the presence of hydrogen peroxide (H2 O2 ). Its back electrochemical reduction to pyrocatechol can be detected on the CSPE through SWV. The current obtained from the product of enzymatic reaction is proportional to the activity of the enzyme and inversely proportional to the amount of OTA in wine samples. Our immunosensor showed a very high sensitivity to determine trace levels of OTA in red wine samples, compared to other conventional techniques.

Fig. 1. Schematic representation of the ochratoxin A immunosensor based on electrochemical detection using competitive assays.

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2. Experimental 2.1. Chemicals All reagents used were of analytical reagent grade. OTA, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide (EDAC) and horseradish peroxidase (HRP) were obtained from Sigma Chemical Company, N-hydroxysuccinimide (NHS) was Fluka and OTA mouse monoclonal antibody IgG1 (mAbOTA) was purchased from Santa Cruz Biotechnology, Inc. Anhydrous dimethylformamide (DMF, Merck, HPLC grade) and 10 mM phosphate buffer solutions (PBS), 137 mM NaCl and 2.70 mM KCl, pH 7.00, as well as 50 mM citrate/50 mM phosphate buffer solution (CBS), pH 5.00, were prepared from their salts (Merck, p.a.). H2 O2 p.a. and H2 SO4 p.a. were purchased from Merck. Pyrocatechol (H2 Q) and o-phenyldiamine (OPD) were obtained from Sigma–Aldrich. Methanol and H2 O were Sintorgan (HPLC grade). Red wine samples of the Cabernet Sauvignon type (harvest 2006) Mendoza, Argentina and skimmed powdered milk were purchased in a local supermarket. 2.2. Materials and apparatus The CSPE based on working and counter electrodes of carbon and pseudo-reference electrodes of silver were purchased to Palm Sens. (The Netherlands). Before use, CSPE surface was electrochemically pretreated in 50 ␮L of 0.10 M KOH aqueous solution by a potential step of 1.2 V over 5 min according to a procedure previously described by Anjo et al. [38]. The MBs with recombinant Protein G covalently bonded to the surface (Dynabeads® Protein G) were obtained from Invitrogen Dynal. 96 well-microplates were purchased to Thermo scientific. Magnetic separator was Serono Diagnostic, Norwell, MA, USA. Cyclic (CV) and square wave (SWV) voltammetric measurements were performed with an AutoLab PGSTAT30 potentiostat, run with the GPES (version 4.9) electrochemical analysis software from Eco-chemie, Utrecht, The Netherlands. The CSPE was connected using a special type of homemade connector which makes the interface with the potentiostat. Colorimetric measurements were performed with ELISA reader, Multiskan EX. Absorbance measurements were performed using a Hewlett Packard spectrophotometer, Model 8452A, equipped with a temperature controller. The pH measurements were carried out with an HANNA instruments, Bench Meters, model pH 211, Romania. The buffer solutions were thermostatized to 37 ◦ C using a stove NEO LINE thermostat, Argentina. The samples with MBs were mixed with a vortex Vortemixer Speed Knob (China). For OTA determination by AOAC official method was use a high pressure liquid chromatography Hewlett Packard Series 1100 with a fluorescence detector Hewlett Packard 1046A. The step extraction was carried out using immunoaffinity columns, OchraTestTM , VICAM LP (Watertown, USA). 2.3. Conjugation of OTA with HRP OTA was bound to HRP by following a synthesis of activated ester to generate the OTA–HRP labeled [39]. Thus, 0.50 mg of OTA, 1.74 mg of NHS and 6.20 mg of EDAC were dissolved in 2.0 mL of methanol. The solution was stirred at 4 ◦ C in the darkness for 4 h to activate the carboxyl group of the mycotoxin. Then, 2.5 mL of 1.00 mg mL−1 HRP solution was added to mycotoxin activated solution, stored and shaken overnight at 4 ◦ C. After the reaction was completed, the solution (OTA–HRP labeled) was purified using a PD-10 column by means of gel filtration (packed with SephadexTM 25). Then, it was dialyzed in 10 mM PBS, pH 7.00 for 5 days by using cellulose dialysis membrane (cut-off molecular weight 10 KDa). The reaction product was confirmed by UV–vis spectrophotometry

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by appearance of new bands respect to OTA and HRP solutions. OTA–HRP showed absorption band with maxima at 330 nm, OTA at 250 nm and HRP at 402 nm in 10 mM PBS, pH 7.00 [40] (data not shown). On the other hand, conventional ELISA experiments were carried out for OTA–HRP labeled (data not shown). The results suggest that the covalent bond between OTA and HRP was carried out successfully. Finally, it was stored at 4 ◦ C (Fig. S1, Suppl. Material). 2.4. ELISA protocol Colorimetric checkerboards were performed in order to optimize the mAbOTA and OTA–HRP concentrations on a 96-well microtiter plate. The optimized concentrations were translated to electrochemical magneto immunosensor. 100 ␮L of different mAbOTA dilutions in 10 mM PBS, pH 7.00, were incubated into wells microtiter plates at 37 ◦ C for 1 h. After washing with 10 mM PBS, pH 7.00, 400 ␮L of a blocking solution of 3% (w/v) skimmed milk in 10 mM PBS, pH 7.00, was added to the wells and kept at 37 ◦ C for 30 min to avoid unspecific adsorptions. After washing with the same buffer solution, 100 ␮L different OTA–HRP dilutions in 10 mM PBS, pH 7.00, were added into each well and incubated for 1 h at 37 ◦ C, where competitions between OTA an OTA–HRP was carried out. Finally, after washing the wells with 10 mM PBS, pH 7.00, 100 ␮L of solution of 1 mM of OPD and H2 O2 in 100 mM CBS, pH 5.00, was used. The absorbance values were measured at 450 nm with ELISA reader. Assays were performed by duplicate. 2.5. Assay procedure for the electrochemical immunosensor Electrochemical magneto immunosensor was developed by direct competitive assay. MBs were used as the solid support to bind the mAbOTA to perform the immunoreaction. On the other hand, the surface of the CSPE was used to reduce the benzoquinone (Q) produced in the enzymatic reaction. Every immunoassay steps are shown in Fig. 1. Briefly, suspensions of 1.8 ␮L the MBs were transferred to EppendorfTM tubes and were washed with 10 mM PBS, pH 7.00, for three times to remove the NaN3 preservative, according to the manufacturer protocol. Then, 50 ␮L of optimized dilution of mAbOTA was added to MBs. The solution was stirred to 200 rpm at 37 ◦ C for 15 min to obtain mAbOTA–MBs complex. After incubation, each tube was positioned on the high magnetic field. Once MBs were deposited on the bottom of tube, the supernatant was removed. The mAbOTA–MBs were washed with 10 mM PBS, pH 7.00, for three times. This wash step allowed eliminates of unbound mAbOTA. The MBs were re-suspended in 50 ␮L of solution of OTA and OTA–HRP, and stirred to 200 rpm at 37 ◦ C for 15 min (competition step). Then, MBs were magnetically separated and the supernatant was removed, as noted above. Then, MBs were washed with 10 mM PBS, pH 7.00, for three times. Reactions catalyzed by enzymes have been largely used for analytical purposes in determinations of different substrates, inhibitors, etc. Gorton has explained the HRP catalytic mechanism [41]. Therefore, it is well known that in the presence of H2 O2 the enzyme catalyzes the oxidation of H2 Q to Q [42]. Therefore, the MBs were re-suspended in 20 ␮L of a solution of H2 O2 /H2 Q, both 10 ␮M, in 100 mM CBS, pH 5.00. After 5 min of incubation to 200 rpm at 37 ◦ C, the MBs were magnetically separated and 15 ␮L of the supernatant was transferred onto the surface of CSPE. The enzymatic product was detected by measuring the cathodic reduction of generated Q by SWV. All SWV measurements were performed in the potential range of 0.500–0.000 V, with square wave amplitude (ESW ) of 0.025 V, a staircase step height (ES ) of 0.005 V and a frequency (f) of 25 Hz. These values of ESW and ES are commonly used to heterogeneous electronic transfer of 2 e− [43] (see below).

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3. Results and discussion 3.1. Optimization of mAbOTA and OTA–HRP concentrations by ELISA The concentration of commercial reagent of mAbOTA is not provided by Santa Cruz Biotechnology. The synthesized OTA–HRP concentration is also unknown, so different dilutions were prepared for both. Therefore, the optimal dilutions of mAbOTA and OTA–HRP were determined by ELISA experiments. For mAbOTA solution, two dilutions of 1/1000 and 1/2000 of mAbOTA were prepared in 10 mM PBS, pH 7.00. These dilutions were prepared from the solution of the commercial reagent. The ELISA assays were carried out using solutions of OTA–HRP in the range of 1–1/2000. When the dilution factor of mAbOTA was 1/1000, the absorbance values were higher for all OTA–HRP dilutions in comparison to a 1/2000 dilution factor of mAbOTA (Fig. 2). This is due to the greater amount of mAbOTA immobilized on the wells. Lower dilution factors were not studies because higher concentration of mAbOTA can cause formation of antibody layers one above another, therefore, the interaction with antigen occurs with the outer layer, which can be loose during the washing steps, yielding a wrong reading [44]. On the other hand, higher dilution factors (<1/2000) imply smaller quantities of antibody adsorbed, resulting in a decrease of the absorbance. The chosen value for the dilution factor was 1/1000 in order to achieve the best sensitivity. On the other hand, the optimal dilution factor for OTA–HRP was obtained varying its concentration for a constant dilution factor of mAbOTA. For lower concentration of OTA–HRP, the absorbance was also smaller, due to a lower enzyme concentration. Dilution factors higher than 1/5 (higher concentrations), produces the maximum binding capacity to the immobilized mAbOTA, since constant values of absorbance were observed (see Fig. 2). Therefore, the OTA–HRP dilution which gave the value of 50% of the maximum signal (dilution 1/64) was chosen for all experiments, because it allows a competition in a wide range of OTA concentration and a very good sensitivity. These values of dilution factor for mAbOTA (1/1000) and OTA–HRP (1/64) were used with the MBs in the EI for all experiments. 3.2. Electrochemical magneto immunosensor Different parameters were optimized with the objective of increasing the performance of the EI. These parameters are related

Fig. 2. Optimization of OTA–HRP labeled and mAbOTA concentrations. ELISA assays carried out for different mAbOTA factor dilution: () 1/1000 and (䊉) 1/2000. OTA–HRP dilution factors were 1 to 1/2000. Each point is an average of two replicated measurements.

Fig. 3. Effect of MBs loading onto current response. Dilution factors of OTA–HRP labeled and mAbOTA were 1/64 and 1/1000, respectively.

to the activation of CSPE, MBs amount, and volume and concentration of redox mediator and enzymatic substrate solutions. 3.2.1. CSPE optimization To remove organic ink constituents or contaminants at CSPE and to increase surface roughness and functional group activation [45,46], a pre-treatment was applied to CSPE. Cyclic voltammograms obtained for the CSPE in 1 mM H2 Q in CBS, pH 5.00, after the electrochemical activation treatment by the method of Anjo et al. (see Section 2.2), showed a better definition of the redox couple of H2 Q as compared to those obtained on non-activated electrodes (Fig. S2, Suppl. Material). The cyclic voltammogram obtained for activated CSPE showed a well-defined anodic peak (Ep,a = 0.364 V) and its corresponding cathodic peak (Ep,c = 0.110 V) when the scan was reversed. As it is well known, the oxidation of H2 Q to Q and the reduction of Q back to H2 Q is a quasi-reversible two-electron redox process [47]. On the other hand, after activation, a reproducibility test was performed using 5 different CSPE. These experiments were carried out by SWV measurements of a 100 ␮M H2 Q + 100 ␮M H2 O2 in CBS solution. The relative standard deviation (RSD) was determined from measurements of net peak current (Ip,n ) obtained for each CSPE, giving a value of RSD = 7.62%, which indicate the good reproducibility of the electrodes after activation. On the other hand, an increase in current intensity of 1.5 times was observed after pretreatment. 3.2.2. Optimization of the magnetic beads amount The optimization on the amount of MBs, which act as support for affinity reaction, was carried out using OTA–HRP and mAbOTA concentrations optimized by ELISA tests. The MBs amount was varied in the range of 15–120 ␮g, which corresponds to 0.5–4.0 ␮L of MBs solution (Fig. 3). This figure shows that an increasing of the amount of MBs produces an increase in Ip,n , reaching a maximum value at 60 ␮g of MBs. Higher amounts produced a slight but continued Ip,n decrease. Homogeneous dispersions in 10 mM PBS, pH 7.00, were found for amounts ≤60 ␮g of MBs. Higher amounts of MBs produce non homogeneous dispersions at the stirring rate used, forming a precipitate in the bottom of the tube. Therefore, the interaction between mAbOTA and MBs is not complete. For this reason, 54 ␮g (1.8 ␮L) was chosen for all experiments. For a given MBs amount, the amount of mAbOTA (1/1000) added was sufficient for saturate all MBs. Higher mAbOTA concentrations were added for this MBs amount. However, any current increase for OTA–HRP constant concentration (1/64) was observed (results not shown).

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Fig. 4. Calibration curve for OTA in red wine extracts obtained by the use of electrochemical magneto immunosensor. B/Bo is the binding percentage. Curve parameters were as follows: IC50 = 0.272 ± 0.081 ppb, Hill Slope = −0.45 ± 0.07, correlation coefficient for the adjusted equation, r = 0.994. Amperometric measurements were performed at 37 ◦ C in a 10 mM phosphate–citrate buffer (pH 5.00) solution containing 10 ␮M H2 O2 and 10 ␮M H2 Q. Each point is an average of three replicated measurements.

3.2.3. Optimization of enzymatic substrate and redox mediator volumes and concentrations In order to optimize the Ip,n values, H2 Q and H2 O2 concentrations were evaluated. Small H2 Q concentrations permitted to use a minor H2 O2 concentration and avoid the HRP denaturalization [48]. Both concentrations were varied in a range from 10 to 1000 ␮M. An increase in Ip,n is obtained for H2 O2 and H2 Q concentrations up to 1000 ␮M. In order to ensure that the enzyme reaction rate depends only on HRP concentration, an identical concentration of 10 ␮M was chosen for both, H2 O2 and H2 Q. This concentration allows to obtain a good response (Fig. S3, Suppl. Material). Volume ranges of H2 O2 and H2 Q solution added on CSPE were from 20 to 100 ␮L. Final concentrations were the same for each experiment. When the volume of solution decreases, the Ip,n increases, due to an increment of concentration of Q enzymatically produced. Therefore, the chosen volume was 20 ␮L. Since the minimum volume required to cover CSPE is 15 ␮L, volumes below 20 mL were not used. 3.3. Direct detection of OTA in red wine samples Red wine samples were spiked with OTA for generate the calibration curve. On the other hand, OTA–HRP was added to obtain a 1/64 dilution factor. Firstly, the red wine solutions were analyzed with the developed immunosensor without immobilization of mAbOTA on MBs in order to check the absence of possible unspecific adsorptions. After incubation step, no signal appears after addition of H2 Q + H2 O2 solution which would indicate the absence of unspecific adsorptions on functionalized MBs. This fact is very important for the EI, considering that the wine is the more complex fluid after the blood [49]. 3.3.1. Calibration curve An OTA free red wine was used as matrix for the generation of a calibration curve. The wine was previously analyzed by AOAC official method using immunoaffinity columns coupled to a HPLC with fluorimetric detector, showing the absence of OTA. Each solution for the calibration curve was prepared spiking different amounts of pure commercial reagent of OTA in order to achieve the desired concentrations. Also, the OTA–HRP was added to each solution in optimized dilution factor (1/64). Using the optimized parameters, a dose–response curve for OTA was carried out with the solutions above mentioned from 0.01 to 20 ppb (Fig. 4). The calibration curve was constructed as binding percentage (B/Bo) vs logarithm of

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Fig. 5. Correlation between the OTA concentration values obtained from the electrochemical immunosensor (EI) and those obtained from the HPLC official method. Slope = 0.95 ± 0.04; intercept = 0.46 ± 0.22; r = 0.992. CV = 6.24%. ∗ the OTA concentration (log cOTA ). Bo is the maximum Ip,n obtained without competition and B is the Ip,n obtained for the competition between OTA–HRP and different concentrations of free OTA. The calibration curve was fitted to a four-parameter logistic equation according to the following formula:

y=D+

A−D [1 + 10 exp((log IC50 − log x)(Hill Slope))]

where A and D are the maximum and minimum B/Bo values, respectively, while IC50 is the concentration of OTA which produces 50% of A, x is the OTA concentration and Hill Slope is the slope at the linear portion of the sigmoid curve. Higher OTA concentrations showed responses related to saturation zone of mAbOTA immobilized. The relative standard deviations (RSD) for OTA solutions were from 3.0% to 8.5% for three repetitive measurements. The limit of detection (LOD), calculated as 85% of A value [31], was 0.008 ppb. This LOD value is much lower than the value set by the Regulatory Commission of the European Community, allowing us to ensure that this immunosensor is suitable for the determination of OTA in red wine samples. The limit of detection determined in this work for OTA in red wines is lower than values in previous reports, i.e. HPLC [6–14], ELISA [10,15,16], capillary electrophoresis [11] and several biosensors [18–26]. Recently, a similar limit of detection (0.004 ppb) for OTA in red wines using a gold electrode modified with a cysteamine self-assembled monolayer has been reported [27]. However, in spite of a very low limit of detection, an extraction procedure has to be done prior to the detection step in such system [27]. 3.3.2. Determination of OTA by HPLC In order to achieve another criterion to test the accuracy of the electrochemical immunosensor developed for OTA quantification in red wines, the results were compared with those obtained by the AOAC 2001.01 official method [7]. Same standard solutions used for calibration curve were used to perform this analysis. The curve obtained by Visconti method could be represented by the following linear regression equation: ∗ H(A.U) = (0.607 ± 0.156) [A.U] + (0.324 ± 0.024) [A.U ppb−1 ] cOTA

where H is the peak height of the liquid chromatogram in arbitrary ∗ units (A.U) and cOTA is the OTA concentration in ng mL−1 . The linear regression coefficient was r = 0.989. The concentrations determined by our immunosensor were plotted vs those obtained from HPLC-immunoaffinity column cleanup with fluorimetric detection [7]. An identity line was found

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Table 1 Statistical analysis of immunosensor responses for three red wine samples with different amounts of spiked OTA. Each point is an average of three replicated measurements. ∗ cOTA (ppb)a

∗ cOTA (ppb)b

CV (%)c

0.10 1.0 10

0.11 0.92 9.68

7.10 5.26 4.31

a b c

Recovery (%) 110 92 97

OTA concentrations in red wine samples. Average value of OTA concentration determined by our immunosensor. Percentage variation coefficient.

with the following parameters (Fig. 5): intercept = 0.46 ± 0.22 ppb; slope = 0.95 ± 0.04. The linear regression coefficient was r = 0.992.

3.3.3. Recovery test In order to study the performance of the immunosensor developed, recovery tests were carried out. Samples of red wine naturally contaminated with OTA were not found in the market. For the purpose of recovery studies, an OTA free red wine sample was spiked with different amounts of pure commercial reagent. The chosen concentrations were 0.1 ppb, 1.0 ppb and 10 ppb. The results obtained from measurements with the electrochemical immunosensor are shown in Table 1. The values obtained were very good, with recoveries between 92–110%, showing the big performance of the immunosensor described.

4. Conclusions The electrochemical magneto immunosensor developed combines magnetic beads as solid phase for affinity reaction and carbon screen printed-electrode for electrochemical measurement step. This sensor was able to determine OTA at trace levels in red wine samples. The parameters of affinity reaction and electrochemical measurement were optimized. The immunosensor showed a high analytical performance in terms of excellent limit of detection (0.008 ppb), sensitivity (IC50 = 0.272 ± 0.081 ppb), high specificity, analytical range of interest in red wine samples (0.01–20 ppb), reproducibility and an acceptable accuracy. This device has several advantages over other methods of determination of OTA in wine, such as direct measurement without any pre-treatment, using small volumes (harmful solvents are avoided), and the possibility of introducing this type of immunosensor in field measurements because of to its small size and easy handling. All of these features make this device a high potential tool for the measurement of OTA in wine samples.

Acknowledgements Financial supports from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaría de Ciencia y Técnica (SECyT) from the Universidad Nacional de Río Cuarto are gratefully acknowledge. P.R. Perrotta acknowledges to CONICET for a doctoral research fellowship. We thank to Dra. L. Ponzone and Dra. S. N. Chulze, from the Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Fisico-Químicas y Naturales, Universidad Nacional de Río Cuarto, for their help in HPLC measurements.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2011.12.089.

References [1] N.W. Turner, S. Subrahmanyam, S.A. Piletsky, Analytical methods for determination of mycotoxins: a review, Anal. Chim. Acta 632 (2009) 168–180. [2] S. Bräse, A. Encinas, J. Keck, C.F. Nising, Chemistry and biology of mycotoxins and related fungal metabolites, Chem. Rev. 109 (2009) 3903–3990. [3] IARC, 1993 International Agency for Research on Cancer (IARC), 1993, Some naturally occurring substances: food items and constituents, (IARC) Monograph Evaluation of Carcinogenic Risk to Humans No 56. [4] D. Ringot, A. Chango, Y.J. Schneider, Y. Larondelle, Toxicokinetics and toxicodynamics of ochratoxin A, an update, Chem. Biol. Interact. 159 (2006) 18–46. [5] Commission Regulation (EC) No. 1881/2006 of 19th December 2006, http:// eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:364:0005:0024: en:pdf. [6] A. Visconti, M. Pascale, G. Centonze, Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography, J. Chromatogr. A 864 (1999) 89–101. [7] A. Visconti, M. Pascale, G. Centonze, Determination of ochratoxin A in wine and beer by immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study, J. AOAC Int. 84 (2001) 1818–1827. [8] A. Leitner, P. Zöllner, A. Paolillo, J. Stroka, A. Papadopoulou-Bouraoui, S. Jaborek, E. Anklamb, W. Lindner, Comparison of methods for the determination of ochratoxin A in wine, Anal. Chim. Acta 453 (2002) 33–41. ˜ C. Leache, M. Viscarret, A. Pérez de Obanos, C. Araguás, A. [9] E. González-Penas, López de Cerain, Determination of ochratoxin A in wine using liquid-phase microextraction combined with liquid chromatography with fluorescence detection, J. Chromatogr. A 1025 (2004) 163–168. [10] Z. Zheng, J. Hanneken, D. Houchins, R.S. King, P. Lee, J.L. Richard, Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC, Mycopathologia 159 (2005) 265–272. ˜ C. Leache, A. López de Cerain, E. Lizarraga, Comparison [11] E. González-Penas, between capillary electrophoresis and HPLC–FL for ochratoxin A quantification in wine, Food Chem. 97 (2006) 349–354. [12] A. Aresta, R. Vatinno, F. Palmisano, C.G. Zambonin, Determination of ochratoxin A in wine at sub ng/mL levels by solid-phase microextraction coupled to liquid chromatography with fluorescence detection, J. Chromatogr. A 1115 (2006) 196–201. [13] M. Solfrizzo, G. Panzarini, A. Visconti, Determination of ochratoxin A in grapes, dried vine fruits, and winery byproducts by high-performance liquid chromatography with fluorometric detection (HPLC–FLD) and immunoaffinity cleanup, J. Agric. Food Chem. 56 (2008) 11081–11086. [14] A. Fabiani, C. Corzani, G. Arfelli, Correlation between different clean-up methods and analytical techniques performances to detect ochratoxin A in wine, Talanta 83 (2010) 281–285. [15] D. Flajs, A.M. Domijan, D. Ivicˇı, B. Cvjetkovicˇı, M. Peraica, ELISA and HPLC analysis of ochratoxin A in red wines of Croatia, Food Control 20 (2009) 590–592. [16] T.Y. Rusanova, N.V. Beloglazova, I.Y. Goryacheva, M. Lobeau, C. Van eteghem, S. De Saeger, Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine, Anal. Chim. Acta 653 (2009) 97–102. [17] E. Mannaert, P. Daenens, Development of a radioimmunoassay for the determination of N-desmethylzopiclone in urine, Analyst 119 (1994) 2221–2226. [18] S.H. Alarcon, L. Micheli, G. Palleschi, D. Compagnone, Development of an electrochemical immunosensor for ochratoxin A, Anal. Lett. 37 (2004) 1545–1558. [19] M.M. Ngundi, L.C. Shiriver-Lake, M.H. Moore, M.E. Lassman, F.S. Ligher, C.R. Taitt, Array biosensor for detection of ochratoxin A in cereals and beverages, Anal. Chem. 77 (2005) 148–154. [20] B. Prieto-Simón, M. Campás, J.L. Marty, T. Noguer, Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples, Biosens. Bioelectron. 23 (2008) 995–1002. [21] X.H. Wang, S. Wang, Sensors and biosensors for the determination of small molecule biological toxins, Sensors 8 (2008) 6045–6054. [22] J. Yuan, D. Deng, D.R. Lauren, M.I. Aguilar, Y. Wu, Surface plasmon resonance biosensor for the detection of ochratoxin A in cereals and beverages, Anal. Chim. Acta 656 (2009) 63–71. [23] H. Kuang, W. Chen, D. Xu, L. Xu, Y. Zhu, L. Liu, H. Chu, C. Peng, C. Xu, S. Zhu, Fabricated aptamer-based electrochemical “signal-off” sensor of ochratoxin A, Biosens. Bioelectron. 26 (2010) 710–716. [24] M. Heurich, M.K. Abdul Kadir, I.E. Tothill, An electrochemical sensor based on carboxymethylated dextran modified gold surface for ochratoxin A analysis, Sens. Actuators B 156 (2011) 162–168. [25] L.-G. Zamfir, I. Geana, S. Bourigua, L. Rotariu, C. Bala, A. Errachid, N. Jaffrezic-Renault, Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection, Sens. Actuators B 159 (2011) 178–184. [26] M.A. Fernández-Baldo, F.A. Bertolino, G.A. Messina, M.I. Sanz, J. Raba, Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues, Talanta 83 (2010) 651–657. [27] P.R. Perrotta, N.R. Vettorazzi, F.J. Arévalo, A.M. Granero, S.N. Chulze, M.A. Zon, H. Fernández, Electrochemical studies of ochratoxin A mycotoxin at gold electrodes modified with cysteamine self-assembled monolayers. Its ultrasensitive quantification in red wine samples, Electroanalysis 23 (2011) 1585–1592. [28] A. Margni, Inmunología e Inmunoquímica. Fundamentos, 5th ed., Panamericana, Buenos Aires, 1996. [29] K.C. Honeychurch, J.P. Hart, Screen-printed electrochemical sensors for monitoring metal pollutants, Trends Anal. Chem. 22 (2003) 456–469.

P.R. Perrotta et al. / Sensors and Actuators B 162 (2012) 327–333 [30] K. Aguilar-Arteaga, J.A. Rodriguez, E. Barrado, Magnetic solids in analytical chemistry: a review, Anal. Chim. Acta 674 (2010) 157–165. [31] M. Hervás, M.A. López, A. Escarpa, Simplified calibration and analysis on screen-printed disposable platforms for electrochemical magnetic bead-based immunosensing of zearalenone in baby food samples, Biosens. Bioelectron. 25 (2010) 1755–1760. [32] E. Van Leemputten, M. Horisberger, Immobilisation of enzymes on magnetic particles, Biotechnol. Bioeng. 16 (1974) 385–396. [33] Y.Y. Lin, G. Liu, C.M. Wai, Y. Lin, Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons, Electrochem. Commun. 9 (2007) 1547–1552. [34] A. Lermo, S. Fabiano, S. Hernández, R. Galve, M.P. Marco, S. Alegret, M.I. Pividori, Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors, Biosens. Bioelectron. 24 (2009) 2057–2063. [35] M. Yang, L.H. Li, Determination of trace hydrazine by differential pulse voltammetry using magnetic microspheres, Talanta 55 (2001) 479–484. ˇ [36] J. Prod˘elalová, B. Rittich, A. Spanová, K. Petrová, M.J. Ben˘e, Isolation of genomic DNA using magnetic cobalt ferrite and silica particles, J. Chromatogr. A 1056 (2004) 43–48. [37] H. Font, J. Adrian, R. Galve, M.C. Estévez, M. Castellari, M. Gratacós-Cubarsí, F. Sánchez-Baeza, M.P. Marco, Immunochemical assays for direct sulfonamide antibiotic detection in milk and hair samples using antibody derivatized magnetic nanoparticles, J. Agric. Food Chem. 56 (2008) 736–743. [38] D.M. Anjo, M. Kahr, M.M. Khodabakhsh, S. Nowinski, M. Wanger, Electrochemical activation of carbon electrodes in base: minimization of dopamine adsorption and electrode capacitance, Anal. Chem. 61 (1989) 2603–2608. [39] F.S. Chu, F.C. Chang, R.D. Hinsdill, Production of antibody against ochratoxin A, Appl. Environ. Microbiol. 31 (1976) 831–835. [40] E. Märtlbauer, G. Terplan, Ein enzymimmunologisches Verfahren zum Nachweis, Arch. Lebensmittelhyg. 39 (1988) 133–156. [41] L. Gorton, Carbon paste electrodes modified with enzymes, tissues and cells, Electroanalysis 7 (1995) 23–45. [42] C. Ruan, Y. Li, Detection of zeptomolar concentrations of alkaline phosphatase based on a tyrosinase and horse-radish peroxidase bienzyme biosensor, Talanta 54 (2001) 1095–1103. [43] J.J. O’ Dea, J. Osteryoung, R.A. Osteryoung, Theory of square wave voltammetry for kinetic systems, Anal. Chem. 53 (1981) 695–701. [44] S.S. Deshpande, Enzyme Immunoassays, From Concept to Product Development, Chapman & Hall, New York, 1996. [45] H. Wei, J.J. Sun, Y. Xie, C.G. Lin, Y.M. Wang, W.H. Yin, G.N. Chen, Enhanced electrochemical performance at screen-printed carbon electrodes by a new pretreating procedure, Anal. Chim. Acta 588 (2007) 297–303. [46] P. Fanjul-Bolado, D. Hernández-Santos, P.J. Lamas-Ardisana, A. Martín-Pernía, A. Costa-García, Electrochemical characterization of screen-printed and conventional carbon paste electrodes, Electrochim. Acta 53 (2008) 3635–3642. [47] E.S. Forzani, G.A. Rivas, V.M. Solís, Amperometric determination of dopamine on vegetal-tissue enzymatic electrodes. Analysis of interferents and enzymatic selectivity, J. Electroanal. Chem. 435 (1997) 77–84. [48] Y. Xiao, H.X. Ju, H.Y. Chen, Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine monolayer, Anal. Chim. Acta 391 (1999) 73–82. [49] A.M. Gurban, B. Prieto-Simón, J.L. Marty, T. Noguer, Malate biosensors for the monitoring of malolactic fermentation: different approaches, Anal. Lett. 39 (2006) 1543–1558.

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Biographies Patricio R. Perrotta obtained his degree of biochemist (2005) from Cordoba National University (Córdoba, Argentina). At present he is doing the PhD at the Río Cuarto National University with a fellowship from Argentine Research Council (CONICET). His doctoral thesis focuses on the development and characterization of electrochemical sensors and immunosensors based on the use of self-assembled monolayers, biomolecules, magnetic nanoparticles and screen-printed electrodes. Fernando J. Arévalo obtained his PhD in chemistry (2009) from Río Cuarto National University (Río Cuarto, Argentina). He is actually doing a postdoctoral training in the group of Electroanalysis at the Chemistry Department, Faculty of Exact, Physicochemical and Natural Sciences (Río Cuarto National University). At present, he also is an assistant professor at the same department. Dr. Arévalo is an active member of the Electroanalysis Group at the Chemistry Department, and his research interests focus on the development and characterization of electrochemical (bio)sensors based on the use of nanostructured materials. Nelio R. Vettorazzi obtained his PhD in chemistry (1983) from Río Cuarto National University (Río Cuarto, Argentina). He is full professor at the same University in Applied Analytical Chemistry. He is a member of the Chemistry Department Council. His research now is focusing in the development of electrochemical (bio)sensors by using nanomaterials for different applications, mainly in the agroalimentary area. María A. Zon obtained her PhD in chemistry (1985) from Río Cuarto National University (Río Cuarto, Argentina). She did the postdoctoral training at Cordoba ˜ University (Córdoba, Espana) between 1990 and 1992. She is full professor at Río Cuarto National University and Independent Researcher at Argentine Research Council (CONICET). She has been the secretary of the Analytical Chemist Argentina Association (2007–2009). Her research now is focusing in the development of electrochemical (bio)sensors by using nanomaterials for the determination of different analites such as mycotoxins, antioxidants and hormones. She has over 40 peer-reviewed papers and three book chapter. She has been co-editor of an electroanalytical book. Héctor Fernández obtained his PhD in chemistry (1978) from Río Cuarto National University (UNRC) (Río Cuarto, Argentina). He did the postdoctoral training (1980–1982) at University of New York at Buffalo, Buffalo (USA). Currently, he is full professor at UNRC and principal researcher at Argentine Research Council (CONICET). He was dean of the Faculty of Exact, Physico-Chemical and Natural Sciences (UNRC, 1992–1999) and head of the Department of Chemistry at the Faculty of Exact, Physico-Chemical and Natural Sciences (2001–2004). He was president of the Argentinean Society of Analytical Chemists (2007–2009). His research interest focus on several subjects, such as electrochemistry of mycotoxins, hormones and synthetic and natural antioxidants, studies on ultramicroelectrodes and electrodes modified by self-assembled monolayers of thiols, carbon nanotubes, antibodies, etc., and their use for electroanalytical applications. Development of electroanalytical techniques for the determination of antioxidants, mycotoxins and hormones in real matrixes (plants, cereal, foods, sera of animal origin, etc., respectively). Design and characterization of chemical sensors, electrochemical (bio)sensors and immunoelectrodes based on nanostructured materials. He has over sixty peer-reviewed papers and three book chapters and has been the editor of a book. He belongs to the editorial board of Journal of Biosensors and Bioelectronics and he is a AAQA, AAIFQ and SIBAE fellow.