A label-free electrochemical immunosensor for direct, signal-on and sensitive pesticide detection

Biosensors and Bioelectronics 31 (2012) 62–68

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A label-free electrochemical immunosensor for direct, signal-on and sensitive pesticide detection H.V. Tran a , R. Yougnia a , S. Reisberg a , B. Piro a , N. Serradji a , T.D. Nguyen b , L.D. Tran c , C.Z. Dong a , M.C. Pham a,∗ a

Univ. Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baïf, 75205 Paris Cedex 13, France Institute for Tropical Technology (ITT), Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam c Institute of Material Sciences (IMS), Vientamese Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam b

a r t i c l e

i n f o

Article history: Received 23 July 2011 Received in revised form 19 September 2011 Accepted 27 September 2011 Available online 2 October 2011 Keywords: Conjugated polymer Electrochemical immunosensor Square wave voltammetry Label-free detection Cross-reactivity Atrazine Pesticide

a b s t r a c t A new electropolymerizable monomer, [N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], has been designed for use in a label-free electrochemical immunosensor when polymerized on an electrode and coupled with a monoclonal anti-atrazine antibody. This monomer contains three functional groups: hydroxyl group for electropolymerization, quinone group for its transduction capability, and hydroxyatrazine as bioreceptor element. Square wave voltammetry shows that the polymer film, poly[N-(6-(4-hydroxy6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], presents negative current change following anti-atrazine antibody complexation and positive current change after atrazine addition in solution. This constitutes a direct, label-free and signal-on electrochemical immunosensor, with a very low detection limit of ca. 1 pM, i.e. 0.2 ng L−1 , one of the lowest reported for such immunosensors. This is far lower than the detection limit required by the European Union directives for drinkable water and food samples (0.1 ␮g L−1 ). The strategy described has great promise for the development of simple, cost-effective and reagentless on-site environmental monitors. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Due to the increasing use of pesticides worldwide, there is a perceived threat of environmental damage as well as health issues. Atrazine (6-chloro-N-ethyl-N-[1-methylethyl]-1,3,5-triazine-2,4diamine; ATZ) is a widely used pesticide that constitutes an excellent model for the study of small organic pollutants. It has been found to be a persistent environmental contaminant, occurring at trace levels in ground waters, and has been recognized as mutagenic, teratogenic besides having effects on the reproductive system.Many methods have been developed for atrazine analysis. The most conventional ones use GC–MS or HPLC (Dean et al., 1996; Ma et al., 2003). These techniques are very accurate but require sample pretreatment, expensive equipment and high-purity chemicals for the mobile phases. Moreover, chromatographic methods cannot be used for continuous, on site analysis (Cai et al., 2004). In the past twenty-five years, the application of immunoanalytical techniques (immunoassays and immunosensors) has increased significantly (Marinella et al., 2007). The binding properties of an

∗ Corresponding author. Tel.: +33 1 57277223. E-mail addresses: [email protected], [email protected] (M.C. Pham). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.09.035

antibody to an antigen have been used for the development of a broad variety of analytical techniques for high-speed environmental monitoring (Jiang et al., 2008). Enzyme-linked immunosorbent assays (ELISA) (Morozova and Levashova, 2005) are traditional solid-state methods (Lee and Kennedy, 2007). Immunosensors (Luppa et al., 2001) must be distinguished from immunoassays, where the transducer is not an integral part of the analytical system. They combine the sensitivity of the antibody–antigen interaction with fast, often direct, data acquisition possible with biosensor processes (Murphy, 2006). Immunosensors are potentially useful for the rapid detection of pesticides and now investigated with a view to environmental monitoring (Marty et al., 1995; Dennison and Turner, 1995). Depending on the transduction mechanism, they are generally classified as optical (fluorescence, chemiluminescence) (Rodriguez-Mozaz et al., 2004; Jain et al., 2004) or electrochemical (amperometric, potentiometric, conductimetric). (Yulaev et al., 2001; Grennan et al., 2003; Valera et al., 2010). These systems are sensitive, with very low detection limits (Zacco et al., 2007) (e.g. 30 pM), but need in general a label to detect the immune-reaction. However label-free transduction systems have obvious advantages. The most popular ones are optical systems based on Reflectometric Interference Spectroscopy (RIFS) (Brecht et al., 1995; Mouvet et al., 1996) and Surface Plasmon Resonance (SPR) (Bier and Schmid, 1994; Chegel et al., 1998) with two leading systems on the market:

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BIAcore (from BIAcore, Uppsala, Sweden) and IAsyse (from Fisons Applied Sensor Technology, Cambridge, UK). Nevertheless, electrochemical immunosensors (Murphy, 2006) could be competitive and revolutionize analysis, because of their simplicity and cheap technology. For pesticide detection, most of them use impedance (Hleli et al., 2006; Valera et al., 2007) or amperometry but mostly in label format (Besombes et al., 1995; Cosnier and Popescu, 1996). The use of electrogenerated polymers could avoid this drawback (Cosnier, 1999; Gerard et al., 2002). The introduction of appropriate functionalities through chemical modification of the monomer can provide polymer films with specific characteristics (Cosnier, 2003) without the help of a label. In this work, we describe an original electrogenerated polyquinone film functionalized by a hydroxyatrazine moiety for label-free electrochemical detection of atrazine. First, the synthesis and characterization of a new multifunctional monomer is described. [N-(6-(4-hydroxy-6-isopropylamino1,3,5-triazin-2-ylamino)hexyl)-5-hydroxy-1,4-naphthoquinone3-propionamide] (JUG-HATZ) contains three functional groups: the hydroxyl group for electropolymerization, the quinone group to be used as transducer, and hydroxyatrazine as bioreceptor element. Electropolymerization of JUG-HATZ leads to poly[N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2ylamino)hexyl)5-hydroxy-1,4-naphthoquinone-3-propionamide], poly(JUG-HATZ). By this method, the quinone and the hydroxyatrazine functions are preserved. The quinone function is well known to be particularly sensitive to its chemical environment, in terms of pH or ionic strength (Rubin et al., 2010) Therefore, the quinone group of poly(JUG-HATZ) can be used as a redox sensor for chemical or electrochemical modifications of its vicinity. These can be generated by heavy molecules, such as antibodies, or charged molecules immobilized at the vicinity of the film surface, influencing the diffusion layer by steric hindrance or electrostatic effects (Piro et al., 2007; Reisberg et al., 2008). The working principle of this sensor is illustrated in Fig. 1 and the polymer structure, poly(JUG-HATZ), is presented in Fig. 2. As shown, poly(JUG-HATZ) is able to bind to ␣-ATZ, i.e. the antibody directed towards unmodified atrazine, due to the cross-reactivity of ␣-ATZ for HATZ. After complex formation (HATZ/␣-ATZ), the faradic current of the quinone group should decrease (Fig. 1, step 2). The electrode modified by poly(JUG-HATZ) where HATZ is complexed by ␣-ATZ, poly(JUG-HATZ/␣-ATZ), is then utilized to detect ATZ in solution. Indeed, ␣-ATZ preferentially binds to ATZ, so a displacement equilibrium should occur (Fig. 1, step 3) between ATZ in solution and HATZ incorporated in the polymer. Addition of free atrazine removes the complexed antibodies from the electrode surface leading to an increase in the current. This signal-on system allows detection of one of the lowest atrazine concentrations (1 pM) recorded in the literature for this kind of electrochemical immunosensor.

2. Experimental 2.1. Chemicals and biological 5-Hydroxy-1,4-naphthoquinone (juglone), 1-naphtol (1NAP) and lithium perchlorate were purchased from Aldrich; silver nitrate (AgNO3 ) and succinic acid ((CH2 –COOH)2 ) from Fluka; cyanuric chloride (ClCN)3 ), isopropylamine (CH3 )2 CHNH2 ) and N-Boc-1,6-diaminohexane (C11 H24 N2 O2 ) from Alfa Aesar; N,N -dicyclohexylcarbodiimide (DCC) from Acros and 4(dimethylamino)pyridine (DMAP) from Aldrich. All other reagents used (NaOH, HCl) and solvents, acetonitrile (ACN), methanol (MeOH), dichloromethane (DCM), ethyl acetateand toluene, were PA (practical grade). Phosphate buffer saline (PBS, 0.137 M NaCl;

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0.0027 M KCl; 0.0081 M Na2 HPO4 ; 0.00147 M KH2 PO4 , pH 7.4) was provided by Sigma. Aqueous solutions were made with bi-distilled or ultrapure (MilliQ) water. Glassy carbon (GC) working electrodes (area, 0.07 cm2 ) were from BASInc, Tokyo, Japan; (␣-ATZ), monoclonal anti-atrazine antibody (Mw = 150 kDa) from Thermo Scientific, USA. Immune sera against OVA and ␣-NEF were obtained by immunizing mice of C57BL/6 strain (H-2b haplotype) with relevant protein antigens. 2 i.p. injections (a priming followed by a boost) were made with 300 ␮g of the antigen in 0.2 mL of PBS per mouse.

2.2. Methods and apparatus For electrochemical experiments, a conventional onecompartment, three-electrode cell was used with an Autolab PGSTAT 30. The auxiliary electrode was a platinum grid and the reference electrode a commercial saturated calomel electrode (SCE, MetrOhm). Square wave voltammetry (SWV) was used to characterize ␣-ATZ complexation and ATZ detection. The following parameters were used: pulse height 50 mV, pulse width 50 ms, scan increment 2 mV, frequency 12.5 Hz. The electrolytic solution was PBS, bubbled with argon for 20 min before any experiment. The SWV scans were repeated until complete stabilization of the electrochemical signal (i.e. no difference observed between two successive responses). All electrochemical experiments were conducted at room temperature. All NMR spectra were recorded in DMSO at 298 K on a Bruker Avance III 400 MHz spectrometer. Data were processed using TOPSPIN 2.1 software (Bruker). FT-IR spectra were recorded on a NICOLET 860 Fourier transform spectrometer. Data were processed using OMNIC software (NICOLET).

2.3. Synthesis of JUG-HATZ To a vigorously stirred suspension of 3-(5-hydroxy-1,4-dioxo1,4-dihydronaphthalen-2(3)-yl) propanoic acid (JUG–COOH, 80 mg, 0.325 mmol) in a mixture of dry dichloromethane and dry N,N -dimethylformamide (DCM/DMF, 5:1) were added DCC (74 mg, 0.35 mmol), 4-(6-aminohexylamino)-6-(isopropylamino)1,3,5-triazin-2-ol hydrochloride (HATZ–NH2 ·2HCl, 172.6 mg, 0.70 mmol) and a catalytic amount of DMAP (2.0 mg, 0.015 mmol). The mixture was stirred for 40 min at 65 ◦ C after which, the white precipitate, N,N -dicyclohexylurea, was filtered from the lemon yellow solution. DCM was then evaporated at room temperature and distilled water added to precipitate the product. The crude product, a solid brown precipitate, was further purified by column chromatography on silica gel (DCM/MeOH, 0.05:1, v/v) to afford a mixture of the positional isomers (7/3) as a yellow solid (161 mg, 65%). FTIR/cm−1 : 3271 (O–H phenol); 3114 (N–H); 2928 (C–C, –CH2 –CH2 –); 2857 (C–H, weak, –CH2 –); 1727 (C O); 1663(C O, amide); 1643 (C O, strong, quinone); 1411 (C–N). 1 H NMR (400 MHz, DMSO): ı 1.10 (6H, d, CH ), 1.20 (4H, m, 3 i H , Hj ), 1.30–1.40 (4H, m, Hh , Hk ), 2.40 (3H, m, NHo , He ), 2.70 (3H, m, NHp –Hf ), 2.90 (2H, m, Hl ), 3.10 (2H, m, Hg ), 3.30 (1H, m, Hm ), 6.80 (1H, s, Hd juglone), 7.30 (1H, d, 8.0 Hz, Hc juglone), 7.50 (1H, d, 7.5 Hz, Ha juglone), 7.70 (1H, dt, 7.5 Hz, 8.0 Hz, Hb juglone), 7.90 (1H, s, NHq ), 9.90 (1H, s, OH , atrazine), and 11.90 (1H, s, OH , naphthol). See Supplementary Information, Fig. SI.1, for letters and colors. The TOF electrospray mass spectrum (ES+) gives the expected mass: Mw calc, 496.2434 g mol−1 , found 496.2522 g mol−1 .

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Fig. 1. Strategy for the electrochemical detection of atrazine based on the change in electroactivity of polymer film, poly(JUG-HATZ). SWV recorded with (1) poly(JUG-HATZ)modified electrode; (2) after complexation with ␣-ATZ, poly(JUG-HATZ/␣-ATZ)-modified electrode; (3) after addition of ATZ in solution.

2.4. Electrochemical methods To prepare the modified electrode, the surface of the working electrode was polished with 0.3 ␮m alumina slurry on microcloth pads to a mirror-like surface. Residual alumina particles were removed by placing the electrode in an ultrasonic bath for 5–10 min. Then, the electrode was dried and washed with pure ACN before use. The medium was 5 × 10−3 M JUG-HATZ + 10−3 M 1-NAP + 0.1 M LiClO4 (Fluka, puriss electrochemical grade) in ACN (anhydrous, HPLC grade). Solutions were deoxygenated with pure dry argon for about 15–20 min prior to each experiment. Argon was continuously passed over the solution during the measurements. Potentials were scanned from 0.6 to 1.25 V (vs. SCE) at scan rate of 50 mV s−1 for 25 scans. The poly(JUG-HATZ) film was washed by pure ACN and pure water to remove adsorbed JUG-HATZ monomer. Then, it was put into a cell containing PBS and potential scanned from −0.95 V to 0.1 V at 50 mV s−1 until the last cyclic voltammogram curves almost overlap (i.e. complete stabilization). Then, the square wave voltammogram (SWV) was recorded. All electrochemical studies were performed at room temperature.

antibodies), under the same conditions as applied for ␣ATZ. The stability of the bioelectrode (poly(JUG-HATZ) film complexed with ␣-ATZ) was checked in PBS at room temperature for one week. Results are given in Supplementary Information, Fig. SI.3. 2.5.2. ATZ detection and selectivity For ATZ detection, the electrodes modified with poly(JUGHATZ/␣-ATZ) prepared as described in Section 2.5.1 were used. They were dipped into ATZ solution (concentration varying between 0.1 pM and 10 ␮M) for 2 h at 37 ◦ C, then washed three times with PBS at room temperature and once with PBS at 37 ◦ C for 30 min. After that, the SWV was recorded. The selectivity of the bioelectrode was also tested with a structural analogue of atrazine, 2-amino-4-chloro-6-isopropylamino-1,3,5triazine, (desethylatrazine-ATD), following the same procedure as for ATZ detection. 3. Results and discussion 3.1. Synthesis of JUG-HATZ

2.5. Bioelectrode preparation 2.5.1. ˛-ATZ complexation For complexation between ␣-ATZ and HATZ incorporated in the electrode surface, the electrode modified with poly(JUGHATZ) was immersed into PBS containing ␣-ATZ overnight at 37 ◦ C, for ␣-ATZ between 1 fM and 10 nM. Then, the electrode was rinsed three times with distilled water and immersed in PBS for 1 h at 37 ◦ C to remove physisorbed ␣-ATZ. Finally, square wave voltammetry was performed to record the quinone electroactivity after this complexation step, as a control. In order to check the complexation specificity, we used also two control antibodies, ␣-OVA (anti-ovalbumin IgG antibodies) and ␣-NEF (anti-negative regulatory factor IgG

JUG-HATZ is a monomer with three functional groups: the hydroxyl group for electropolymerization, the quinone group for its redox properties, and the HATZ as a receptor for complexation with ␣-ATZ. It was synthesized in four steps. The coupling reaction is schematized in Supplementary Information (Fig. SI.1). First, the quinone ring was substituted by a carboxyethyl group via an already described substitution reaction (Salmon-Chelin et al., 2001) to give JUG–COOH. In a second step, a hexanamine-modified atrazine was synthesized from N-Boc1,6-hexanediamine and Atrazine. During this processing step, the chlorine group is replaced by the hydroxyl giving HATZ–NHBoc. Then, in the third step, the protecting group (Boc) is hydrolysed in acidic medium to give HATZ–NH2 . Finally, in the fourth

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Fig. 2. Poly(JUG-HATZ) structure.

step, HATZ–NH2 was coupled to the JUG–COOH via a peptide bond using the acid group of JUG–COOH and the amine group of the HATZ–NH2 . The synthesis of JUG–COOH, HATZ–NHBoc, and HATZ–NH2 are described in Supplementary Information (Fig. SI.7).

3.2. Electrochemistry of poly(JUG-HATZ)-modified electrodes 3.2.1. Poly(JUG-HATZ) electrosynthesis The electrochemical synthesis of the polymer films was carried out by electrooxidation of 5 × 10−3 M JUG-HATZ + 10−3 M 1-NAP + 0.1 M LiClO4 in ACN on GC electrodes, under Ar, by 25 potential scans at 50 mV s−1 between 0.6 and 1.25 V vs. SCE. The irreversible electrooxidation peak of the monomer occurs above ca. 1.03 V during the first scan, then gradually shifts to higher potentials and current increases upon cycling (Fig. SI.2). FT-IR spectra of the resulting polymer layer were recorded; the observed bands are compared to those of JUG–COOH and JUG-HATZ, and their assignments are given in Supplementary Information, Table SI.3. The structure of poly(JUG-HATZ) is presented in Fig. 2.

3.2.2. Electrochemical characterizations The electroactivity of poly(JUG-HATZ) films was investigated in PBS in the negative potential range corresponding to the quinone redox reaction. As shown in Fig. 3, two main couples are detected, centered on −0.42/−0.51 V, and −0.72/−0.78 V vs. SCE, together with shoulders at −0.26 V and −0.28 V vs. SCE. The redox system is highly stable with respect to cycling (several hundreds of cycles without significant current decrease). Of interest is the fact that the potential range is sufficiently low to avoid interference from other electroactive species that may be present, including ATZ, which is electrooxidized at much higher potential. The effect of the scan rate on the CV peaks was investigated. The results are shown in Supplementary Information, Fig. SI.4a. The anodic and cathodic peak currents, ipa and ipc , of poly(JUGHATZ) film are linearly proportional to the scan rate, , with equations: ipa (␮A) = 5.118 + 3.917v (mV s−1 ) (R2 = 0.99913) and ipc (␮A) = −1.170 − 4.160v (mV s−1 ) (R2 = 0.9961) (Supplementary Information, Fig. SI.4b). This indicates that the current is limited by electron transfer and not by diffusion. By charge integration, 2.8 × 10−9 mol cm−2 of quinone are estimated to be electroactive.

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decreases by ca. 55%. However, it is only ca. 15% for ␣-OVA, and 10% for ␣-NEF (see Supplementary Information, Fig. SI.5). Therefore, poly(JUG-HATZ) is more specific for ␣-ATZ than for other antibodies, i.e. the HATZ moiety is available for complexation and remains specific on the polymer backbone.

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3.2.3. ˛-ATZ immobilization SWV was used to characterize complexation between ␣-ATZ in solution and HATZ present on the polymer film. SWVs of poly(JUGHATZ)-modified electrode are shown on Fig. 4. The potential was swept from −0.85 V to 0 V vs. SCE (oxidation scan). Two oxidation peaks appear in this range. The main peak presents a maximum around −0.42 V/SCE, a smaller peak appears around −0.72 V/SCE, and a shoulder at −0.26 V/SCE. These peaks correspond to those observed on CV in Fig. 3. As expected, formation of the ␣-ATZ/HATZ complex causes the current to fall: This is high for the poly(JUGHATZ)-modified film (before complexation, plain squares, curve 1), and diminishes after ␣-ATZ addition (open circles, curve 2). An optimal ␣-ATZ concentration was determined by using ␣-ATZ concentrations between 1 fM and 10 nM (between 0.15 pg mL−1 and 1.5 ␮g mL−1 ). As expected, the drop increases with the ␣ATZ concentration and is largest and reproducible for 10 nM or 1.5 ␮g mL−1 .However, besides the specific complexation, antibodies can also bind by non-specific interactions. To confirm that the current decrease is mostly due to antibody complexation and not to physisorption, we used two control antibodies, ␣-OVA and ␣-NEF. Results were compared to ␣-ATZ. In these experiments, an antibody concentration of 10 nM was used. With ␣-ATZ, the current -5

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E / V vs. SCE Fig. 4. SWV of poly(JUG-HATZ)-modified GC electrode (plain squares , curve 1); , curve 2), and after complexation with ␣-ATZ, [␣-ATZ] = 10 nM (open circles, after addition of ATZ, [ATZ] = 0.1 nM (plain up-triangles , curve 3). SWV conditions detailed in Section 2. S = 0.07 cm2 . Medium: PBS.

To detect atrazine, the poly(JUG-HATZ/␣-ATZ) modified electrode is immersed for 2 h in a solution containing atrazine, then the electrode is washed and SWV is performed in PBS. Results are shown on Fig. 4 (plain up-triangles, curve 3). The addition of ATZ causes the current to increase, suggesting that ␣-ATZ is removed from the film surface, due to the competitive binding between free ATZ in solution and HATZ incorporated in the film. It is proposed that the dissociation of the HATZ/␣-ATZ complex strongly enhances the ionic flux through the interface and leads to this current increase. It is noteworthy to recall the ability of the combining site of an antibody to react with more than one antigenic determinant. This is known as cross-reactivity (Berzofsky and Schechter, 1981) and arises because the cross-reacting antigen shares a structure similar to that of the immunizing antigen (here ATZ) used to generate antibodies. The phenomenon of cross-reactivity is an intrinsic characteristic of all antibodies but depends also on the relative concentrations of cross-reactant (Dankwardt, 2001). In our case, the antibody ␣-ATZ generated from ATZ (chlorinated s-triazine) can also bind to the hydroxy s-triazine, but with lower affinity (Schlaeppi et al., 1989; Wortberg et al., 1996; Charlton et al., 2001). We took advantage of this cross-reactivity to design our sensor. In Section 3.2 we showed that ␣-ATZ binds to poly(JUG-HATZ). Nevertheless, the lower affinity of ␣-ATZ for hydroxyatrazine (HATZ) than for ATZ, combined with the higher concentration of ATZ in solution (compared to hydroxyatrazine on the surface), will promote competitive poly(JUG-HATZ/␣-ATZ) decomplexation. Therefore, addition of free atrazine to the solution removes ␣-ATZ from the electrode, leading to a current increase in SWV. 3.3.1. Selectivity To demonstrate the selectivity of the bioelectrode, we performed the decomplexation with a structural analogue of ATZ, desethylatrazine (ATD) (see structure (C) in Scheme 1). SWV was performed on a poly(JUG-HATZ/␣-ATZ) electrode; after addition of ATD (1 nM) a very slight current change is detected (+10%), whereas the addition of atrazine (same concentration, 1 nM) markedly increases the current (+90%) (see Supplementary Information, Fig. SI.6). These results show that the immunosensor is selective under these conditions. 3.3.2. Sensitivity To determine the detection threshold of the system, the SWV response was measured as a function of the ATZ concentration. Results are shown in Fig. 5 for concentrations between 0.1 pM and 10 ␮M and are expressed as I/I × 100 with I = Ipoly(JUG-HATZ) − Ipoly(JUG-HATZ/␣-ATZ) and I = Ipoly(JUG-HATZ/␣-ATZ) . The relative current increases to reach a maximum at ∼10 nM. The relationship between I/I and ATZ concentration is linear. The slight current decrease observed for higher concentrations might be due to ATZ adsorption on the electrode surface. This detection limit of 1 pM (i.e. 0.2 pg mL−1 ) can be compared to literature results on label-free immunosensors using conducting polymers as transducers. Ionescu et al. (2010) have reported that it was possible to detect 10 pg mL−1 atrazine by Electrochemical Impedance Spectroscopy (EIS) using immunosensor based on polypyrrole film N-substituted by nitrilotriacetic acid electrogenerated on gold electrode.

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(A)

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Scheme 1. Chemical structures of atrazine (A, ATZ), its 2-hydroxy analogue (B, HATZ), its dealkylated derivative (C, ATD), and JUG-HATZ monomer (D, JUG-HATZ). 100

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Fig. 5. Percentage of I/I measured at −450 mV/SCE, after addition of ATZ for concentrations from 1 pM up to 10 ␮M. I = Ipoly(JUG-HATZ) − Ipoly(JUG-HATZ/␣-ATZ) and I = Ipoly(JUG-HATZ/␣-ATZ) . SWV conditions described in Section 2.

It is shown in this work that atrazine can be electrochemically detected by a “signal-on” process using square wave voltammetry, based on a novel multifunctional conducting polymer, poly(JUGHATZ). A poly(JUG-HATZ)-modified electrode acts both as the complexation and the transduction element. Quinone and HATZ groups are preserved in the polymer; quinone is the transducer and HATZ is available for complexation. It keeps its specificity when covalently attached to the polymer backbone and presents higher specificity for ␣-ATZ than for other antibodies like ␣-OVA or ␣-NEF. It is proposed, for the transduction scheme, that the presence of ␣-ATZ/HATZ complex is detected via change in signal due to steric hindrance which modifies the cation transport rate at the polymersolution interface and, therefore, modifies the redox kinetics of the quinone group. A special feature of this immunosensor is that it takes advantage of cross-reactivity, in that the addition of atrazine displaces the equilibrium and removes the formerly complexed antibody, and the poly (JUG-HATZ) electroactivity is enhanced. The current falls following anti-atrazine antibody complexation and

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