Label free electrochemical immunoaffinity sensor based on impedimetric method for pesticide detection

Label-Free Electrochemical Immunoaffinity Sensor Based on Impedimetric Method for Pesticide Detection H. V. Tran, a S. Reisberg, a B. Piro,* a T. D. Nguyen, b M. C. Pham a a Univ. Paris Diderot, Sorbonne Paris CitØ, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baïf, 75205 Paris Cedex 13, France tel: + 33-1-57277224 b Institute for Tropical Technology (ITT), Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam *e-mail: piro@univ-paris-diderot.fr Received: July 4, 2012 Accepted: October 10, 2012 Published online: January 24, 2013 Abstract We present a new approach using a conducting polymer to combine immunoaffinity and electrochemical impedance spectroscopy for atrazine detection. The system is based on a competitive complexation between anti-atrazine anti- body and either atrazine present in the analyzed sample or hydroxyatrazine immobilized on the sensor surface. The process allows to detect atrazine at a very low detection limit (0.2 ngL À1 ) in a true label-free format (no redox probe added in solution) by following changes in the electrochemical impedance of the sensor. Keywords: Conducting polymers, Electrochemical impedance spectroscopy (EIS), Enzyme-linked immunosorbent assays (ELISA), Immunosensors, Label-free detection, Cross-reactivity, Atrazine, Pesticides DOI: 10.1002/elan.201200331 1 Introduction The increased use of pesticides and herbicides has led to serious problems of contamination of soil and water. The maximum value for atrazine in drinkable water set by the World Health Organization is 2 mgL À1 , and this limit is even lower in the European Union (0.1 mgL À1 ). Given this, atrazine has been often chosen as a model to develop analytical devices. Immunoassay technology with enzyme-linked immuno- sorbent assays (ELISA) is now seen as a gold-standard of immunoassays for pesticide analysis. The immunoassays kits are inexpensive, simple, adaptable to field use and constitute a rapid way to determine contaminants in envi- ronmental samples [1,2]. ELISA test is based on anti- body technology and involves the immobilization of a re- actant (an antibody, an antigen or a part of an antigen, called hapten) onto a solid surface with enzymes being used as markers for the presence of a specific antibody- antigen (Ab/Ag) or antibody-hapten (Ab/Hp) complex. However, ELISA tests are not efficient for simultaneous multiple analysis (multiplexing), continuous detection and transduction into an electronically processable signal. Conversely, label-free electrochemical immunosensors are efficient for these tasks [3,4]. Among them, impedimetric immunosensors are the subject of special attention [5–7]. Electrochemical Impedance Spectroscopy (EIS) consti- tutes an efficient way to follow the Ab/Ag or Ab/Hp in- teractions at electrode surfaces, by probing changes in ion diffusion and electrical capacitance [8]. In the last five years, the applications of EIS to the detection of small an- tigenic organic pollutants, as bisphenol A [9], organic toxins [10,11] or pesticides, mainly atrazine [7,12,13] have shown growing interest. Hleli et al. reported a detec- tion limit of 20 ngmL À1 atrazine using an impedimetric immunosensor based on mixed biotinylated self-assem- bled monolayer [14]. Later, Cosnier et al. reported the detection of extremely low atrazine concentration (10 pgmL À1 ) using a label-free impedimetric immunosen- sor based on a conducting polymer cleverly modified to bind the antibody probe using affinity interactions [7]. They showed that the immunoreaction of atrazine on the attached anti-atrazine antibody induces an increase in the charge transfer resistance proportional to the atrazine concentration. Among the key steps in the design of an immunosen- sor, the immobilization of the bioreceptor is decisive. The introduction of appropriate functionalities through chemi- cal modification of a monomer can provide polymer films with specific characteristics. Conducting polymers such as polypyrrole or polyaniline have been extensively studied for their great functionalization potentialities and advan- tageous electrochemical properties [15–18]. Polyquinone derivatives are less investigated but, nevertheless, present great and remarkable electrochemical properties as well as good biocompatibility, easy bio-functionalization and a very stable electroactivity in neutral aqueous medium [19]. These properties can be used to probe biomolecular interactions [20–24] due to the high sensitivity of the qui- none group to its local physico-chemical environment. TOPICAL CLUSTER 664  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2013, 25, No. 3, 664– 670 Full Paper In the present study, we describe an innovative strategy based on an original electrogenerated polyquinone film functionalized by a hydroxyatrazine moiety (HATZ) for sensitive and direct detection of atrazine (ATZ). The syn- thesized monomer [N-(6-(4-hydroxy-6-isopropylamino- 1,3,5-triazin-2-ylamino)hexyl)-5-hydroxy-1,4-naphthoqui- none-3-propionamide] (JUG-HATZ) contains three func- tional groups: the hydroxyl group for electropolymeriza- tion, the quinone group to be used as transducer, and hy- droxyatrazine (a structural analogue of ATZ) as biore- ceptor element (Scheme 1). Electropolymerization of JUG-HATZ leads to poly[N-(6-(4-hydroxy-6-isopropyla- mino-1,3,5-triazin-2-ylamino)hexyl)5-hydroxy-1,4-naph- thoquinone-3-propionamide], poly(JUG-HATZ) [25]. By this method, the quinone and the hydroxyatrazine func- tions are preserved. We have shown that poly(JUG- HATZ) is able to specifically bind a-ATZ, the antibody directed to atrazine, due to cross-reactivity of a-ATZ for HATZ. a-ATZ is eventually removed from the electrode surface if ATZ (the natural ligand of a-ATZ) is present in solution (see Figure 1). This architecture differs from other devices used in label-free impedimetric imunosen- sor, for which the antibody is immobilized on the surface. In the present work, electrochemical impedance spectros- copy (EIS) is used to identify the physico-chemical prop- erties of the electrode/electrolyte interface (such as ca- pacitances and charge transport resistances), which are dramatically influenced by the antibody binding/unbind- ing, Changes in these parameters are significant and can be used individually to determine ATZ in solution, for concentration as low as 0.2 pgmL À1 . 2 Experimental 2.1 Chemicals All reagents and solvents were PA type. Phosphate buffer saline (PBS, 137 mM NaCl; 2.7 mM KCl; 8.1 mM Na 2 HPO 4 ; 1.47 mM KH 2 PO 4 , pH 7.4) was provided by Sigma. Aqueous solutions were made with ultrapure (18 MW cm) water. Glassy carbon electrodes (GC, 3 mm in diameter, S= 0.07 cm 2 ) were purchased from BASInc. Anti-atrazine (a-ATZ) antibody (monoclonal) was pur- chased from Thermo Scientific, USA. Atrazine (ATZ) and atrazine-desethyl-2-hydroxy (ATD) were purchased from Supelco (USA). 5-hydroxy-1,4-naphthoquinone (JUG), 1-naphthol (1-NAP), lithium perchlorate (LiClO 4 ), acetonitrile (ACN) and ethanol (EtOH), practi- cal grade, were from Sigma-Aldrich. Dry Argon (Ar) was purchased from Air Liquide (France). N-(6-(4-hydroxy-6- isopropylamino-1,3,5-triazin-2-ylamino)hexyl)-5-hydroxy- 1,4-naphthoquinone-2(3)-propionamide (JUG-HATZ) and 5-hydroxy-3-thioacetic acid-1,4-naphthoquinone (JUGA) were synthesized in the lab [21]. 2.2 Preparation of the Poly(JUG-HATZ)-Modified Electrodes GC electrodes were polished by 1 mm alumina slurry then sequentially washed in an ultrasonic bath with distilled water, EtOH and ACN for 5 minutes. The electrochemi- cal synthesis of the polymer films was carried out by elec- trooxidation of 510 À3 M JUG-HATZ+10 À3 M 1-NAP+ 0.1 M LiClO 4 in ACN on GC electrodes, under Ar, at a constant potential of 1.25 V (vs. SCE) during 600 s. After that, electrodes were repeatedly washed with aceto- nitrile to remove residual monomers then put into an electrochemical cell containing PBS and scanned between À0.9 V and 0 V at a scan rate of 50 mVs À1 under Ar at- mosphere until complete stabilization of the voltammo- grams. After this treatment, electrodes were characterized by electrochemical impedance spectroscopy (EIS). TOPICAL CLUSTER Fig. 1. Schematic representation of the ion flux, before and after complexation and decomplexation. Electroanalysis 2013, 25, No. 3, 664 – 670  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 665 Electrochemical Immunoaffinity Sensor for Pesticide Detection 2.3 Complexation of a-ATZ on Poly(JUG-HATZ)- Modified Electrodes Poly(JUG-HATZ)-modified electrodes were immersed into PBS containing a-ATZ (the concentration of which depends on the experiment) overnight at 378C to achieve complexation between HATZ and a-ATZ. After that, the electrodes were rinsed thrice with ultrapure water and immersed into PBS for 1 h at 37 8C to remove nonspecifi- cally adsorbed a-ATZ. After this step, electrodes were characterized by EIS. Such electrodes are referred as poly(JUG-HATZ)/a-ATZ. The maximum surface concen- tration of a-ATZ is ca. 0.2 pmolcm À2 , i.e. 10 9 antibodies on an electrode area of 0.07 cm 2 . 2.4 Detection of ATZ For ATZ detection, poly(JUG-HATZ)/a-ATZ electrodes were dipped 2 h at 378C into a 100 mL sample of water containing ATZ at concentrations between 0.1 pM and 0.1 mM, then washed with PBS at 378C for 30 min. After that, electrodes were characterized by EIS. 2.5 Methods EIS was performed using an Autolab PGSTAT30 equipped with the FRA module. Impedance spectra were recorded in PBS buffer at room temperature at a given potential within a frequency range from 10 kHz to 100 mHz with a perturbation amplitude of 10 mV. As a pretreatment before each experiment, a constant poten- tial corresponding to the one used for EIS was imposed, for 120 s. Solutions were systematically deaerated with Ar before and during experiments. 3 Results and Discussion 3.1 JUG-HATZ Electroactivity JUG, JUGA and JUG-HATZ were dissolved into PBS buffer at a concentration of 0.1 mM. Their electroactivity was investigated using cyclic voltammetry from À0.7 V to 0.1 V (vs. SCE) at a scan rate of 50 mVs À1 using GC elec- trodes. The results are showed on Figure 2. The redox peaks (E pa /E pc ) of JUG, JUGA and JUG-HATZ are situ- ated at À0.27/À0.31 V; À0.28/À0.33 V and À0.29/À0.36 V vs. SCE, respectively. Averaged peak potentials (E 1/2 ) are À0.29 V; À0.30 V and À0.33 V (vs. SCE), respectively. Changes in potentials for JUGA and JUG-HATZ com- pared to JUG reflect the presence of lateral chains of the substituents, which are slightly electrodonors (so that the reduction of the quinone moiety is slightly more difficult and occurs at lower potentials). Changes in currents, for the same concentration, reflect a decrease of the diffusion coefficient for JUGA and JUG-HATZ compared to JUG. 3.2 EIS on Poly(JUG-HATZ)-Modified Electrodes Electroactivity of poly(JUG-HATZ)-modified electrodes in PBS buffer is shown in Figure 3. Two main redox cou- ples are present, centered at around À0.44/À0.53 V and À0.72/À0.76 V vs. SCE. A third couple appears as should- ers at À0.27/À0.30 V vs. SCE. This redox system is com- parable to that of the monomer (Figure 2), with widened peaks due to electronic delocalisation within the polymer structure. In aqueous solution, quinones transfer two elec- trons and two protons in a concerted process. As previ- ously reported, we assume that the presence of three cou- ples of peaks in PBS is due to three different types of qui- TOPICAL CLUSTER Fig. 2. Electroactivities of (a) JUG; (b) JUGA and (c) JUG- HATZ. Concentration: 10 À4 M. Scan rate 50 mV s À1 . Potential range: À0.7 to 0.1 V (vs. SCE). Medium: PBS. GC electrode, S= 0.07 cm 2 . Fig. 3. CV for poly(JUG-HATZ)-modified GCE in argon-satu- rated solution of PBS (pH 7.4), scan rate 50 mVs À1 . E=À0.9 to 0 V (vs. SCE). 666 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2013, 25, No. 3, 664 – 670 Full Paper H. V. Tran et al. nones on the electrode surface: at the electrode/polymer interface, in the bulk of the polymer and at the polymer/ electrolyte interface [20–25]. The electrochemical stability of this modified electrode is excellent. It takes ca. 50 vol- tammetric cycles to stabilize the system in PBS [25] then it becomes perfectly stable (no measurable changes) for more than 250 cycles, or several days of storage in PBS. Electrochemical Impedance Spectroscopy (EIS) is an efficient method to probe the interfacial properties of sur- face-modified electrodes [26]. We performed EIS on poly(JUG-HATZ)-modified electrodes in the same po- tential domain than for cyclic voltammetry (Figure 4). To fit the experimental spectra, the equivalent circuit presented on Figure 5 was used. It includes R f , R ef and R s which are the resistances of the film, of the electrolyte- film interface and of the electrolyte, respectively. The constant phase elements represent the pseudocapacitan- ces of the electrolyte-film interface (CPE ef ) and of the film (CPE f ), respectively. In addition, we introduced an- other constant phase element (CPE d ) to characterize dif- fusion of ions from the electrolyte bulk to the electrode, which better fits experimental data than the classical War- burg element [27]. The impedance of a CPE is expressed as Z CPE ¼ Q À1 0 w Àn with n a corrective term between 1 (perfect capacitor) and 0, and Q 0 = 1/jZj for n = 1andw=1 rad s À1 . For our fitting, values of n were kept between 0.8 and 1. Values extracted from fittings (given on Figure 6) are in good agreement with cyclic voltammetry (Figure 3): diffusion (CPE d ) appears to be maximal at around À0.4 V, where the slope of the film capacitance (CPE f )is the highest. The electrolyte-film capacitance (CPE ef )is the lowest at À0.5 V, as well as the film resistance (R f ). These last three parameters are the most informative about changes which can occur on the electrode surface and can be used to characterize, at a given potential, anti- body complexation or decomplexation. 3.3 ATZ Detection It is noteworthy to recall the ability of the combining site of an antibody to react with more than one antigenic de- terminant. This is known as cross-reactivity and arises be- cause 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. In our case, the antibody a-ATZ generated from ATZ (chlorinated s-triazine) can also bind to HATZ (the hy- droxylated s-triazine), but with lower affinity [28–30]. We took advantage of this cross-reactivity to design our sens- ing strategy. As explained, poly(JUG-HATZ) is able to bind to a-ATZ, which covers the electrolyte-film inter- face. However, a-ATZ recognizes HATZ with less affini- ty than for ATZ (which is the natural ligand of a-ATZ) so that, when put into contact with ATZ, a-ATZ is re- moved from the electrode surface (this is illustrated on Figure 1). We have demonstrated in previous studies that the electroactivity of polyquinone-modified electrodes is sen- sitive to changes in cation diffusion, sodium in particular [19]. This can be generated by heavy molecules at the vi- cinity of the film surface, influencing the diffusion layer by steric hindrance. This is what occurs when a-ATZ is present at the electrolyte-film interface. a-ATZ has a mo- lecular weight of ca. 150 kDa and an hydrodynamic volume of about 25 nm 3 , which makes a projected surface of 25 2/3 =8.5 nm 2 , so that a-ATZ can strongly inhibits ion exchange. Upon ATZ detection, the antibody is released and the electrolyte-film interface is cleared, so that the TOPICAL CLUSTER Fig. 4. Nyquist plot obtained for a poly(JUG-HATZ)-modified electrode at different formal DC potentials vs. SCE in 100 mM PBS buffer solution, pH 7.4. Spectra are presented between 100 kHz to 1.265 Hz. Symbols correspond to experimental data and lines correspond to fittings. Fig. 5. Equivalent circuit used to fit experimental spectra. R s is the solution resistance, R ef and CPE ef are the electrolyte-film in- terface resistance and pseudocapacitance (CPE is a constant phase element), R f and CPE ef are the film resistance and pseudo- capacitance. For the CPE ef , values of n were kept between 0.9 and 1. Electroanalysis 2013, 25, No. 3, 664 – 670  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 667 Electrochemical Immunoaffinity Sensor for Pesticide Detection film resistance R f associated to ion diffusion is expected to decrease. For the same reasons, a-ATZ decomplexation should influence the film pseudocapacitance, CPE f, considering that this pseudo-capacitance comes from ion diffusion. The electrolyte-film interface pseudocapacitance CPE ef should also change when a-ATZ is removed, for a differ- ent reason. CPE ef is given by: CPE ef ¼ ee 0 =d with e the relative permittivity of the medium, e 0 the per- mittivity of vacuum and d the double layer thickness. The antibody at the electrolyte-film interface does not change the permittivity of the medium but due to steric hin- drance, it significantly increases the double layer thick- ness d. On the contrary, when the antibody is removed, this double layer thickness decreases, so that CPE ef should increase. R f , CPE f and CPE ef are given as a func- tion of ATZ concentration, on Figure 7. Values for R f , CPE f and CPE ef before addition of ATZ are 1306 W, 1.05 mF and 0.12 mF, respectively (these values appear as dashed lines on Figure 7). As shown, at an ATZ concen- tration of 10 À13 M, changes for R f , CPE f and CPE ef are not significant, due to the standard deviation. However, for 10 À12 M, ca. 0.2 ngL À1 , variations become significant. This value can be considered as the limit of detection. These variations are relatively linear (in a log scale) up to 10 À7 M (ca. 20 mgL À1 ), which covers practical concentra- tions. Considering the size of a-ATZ, the maximum sur- face concentration of a-ATZ is ca. 10 9 antibodies on an electrode area of 0.07 cm 2 . This value is in accordance with the lowest detectable quantity of ATZ which is 6 10 8 molecules (10 À12 M in 100 mL). 3.4 Selectivity The transduction mechanism is based on the cross-reac- tivity of a-ATZ towards HATZ. That is why it is impor- tant to evaluate the selectivity of this sensor towards an- other structural analogue of atrazine, for example 2- TOPICAL CLUSTER Fig. 6. Effect of the offset potential on the characteristics of poly(JUG-HATZ) modified electrode: (a) diffusion peudo-capacitance, (b) film pseudo-capacitance, (c) electrolyte-film interfacial pseudo-capacitance, and (d) film resistance. Results obtained for offset po- tentials between À0.8 V and 0 V vs. SCE, in PBS, fitted from the equivalent circuit of Figure 5 in the range of 100 kHz–1.265 Hz. For the CPEs, values of n were kept between 0.8 and 1. 668 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2013, 25, No. 3, 664 – 670 Full Paper H. V. Tran et al. amino-4-chloro-6-isopropylamino-1,3,5-triazine (desethy- latrazine ATD, see Scheme 1). Experiments were performed as described in Section 3.3, with ATD then with ATZ, at two different concentra- tions: 1 nmolL À1 and 10 nmolL À1 . Relative changes in re- sistance (R f ), film capacitance (CPE f ) and electrolyte-film capacitance (CPE ef ) are summarized and compared in Table 1. It appears that changes related to ATD are systemati- cally and significantly lower than those related to ATZ. For CPE ef , changes to ATD represents only 34% of the one obtained for ATZ, at 10 nM ATD. As expected, the selectivity is lower for high concentrations than for low concentrations. TOPICAL CLUSTER Scheme 1. Chemical structures of atrazine (ATZ), its 2-hydroxy analog (HATZ), its dealkylated derivative (ATD) and JUG- HATZ. Table 1. Relative changes in resistance (DR f ), film capacitance (DCPE f ) and electrolyte-film capacitance (DCPE ef ) as a function of the target. DR f (%)=100 (R a-ATZ ÀR a-ATZ/antigen )/R a-ATZ ; DCPE f (%)=100(CPE fa-ATZ ÀCPE fa-ATZ/antigen )/CPE fa-ATZ ; DCPE ef (%)=100(CPE efa-ATZ ÀCPE efa-ATZ/antigen )/CPE efa-ATZ . % DR f % DCPE f % DCPE ef ATZ ATD ATZ ATD ATZ ATD 1nM À24 À5 + 300 À70 + 170 +15 10 nM À26 À7 +320 + 100 + 230 + 90 Fig. 7. Changes in films resistance (R f ); capacitance of conduc- tive film (CPE f ) and capacitance of film-electrolyte interface (CPE ef ) as function of the concentration of atrazine in solution in the decomplexation step. Results obtained for an offset poten- tial of À0.5 V vs.SCE, fitted from the the equivalent circuit of Figure 5, in the range of 100 kHz–1.265 Hz. For the CPEs, values of n were kept between 0.8 and 1. Values for R f , CPE f and CPE ef before addition of ATZ appear as dashed lines. Electroanalysis 2013, 25, No. 3, 664 – 670  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.electroanalysis.wiley-vch.de 669 Electrochemical Immunoaffinity Sensor for Pesticide Detection 4 Conclusions We have shown that atrazine can be directly detected by electrochemical impedance spectroscopy using a novel multifunctional conducting polymer, poly(JUG-HATZ). It acts both as the complexation and the transduction ele- ment for a label-free, reagentless electrochemical immu- nosensor. 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TOPICAL CLUSTER 670 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Electroanalysis 2013, 25, No. 3, 664 – 670 Full Paper H. V. Tran et al. . Label-Free Electrochemical Immunoaffinity Sensor Based on Impedimetric Method for Pesticide Detection H. V. Tran, a S. Reisberg, a B. Piro,* a T continuous detection and transduction into an electronically processable signal. Conversely, label-free electrochemical immunosensors are efficient for these tasks [3,4]. Among them, impedimetric immunosensors. 667 Electrochemical Immunoaffinity Sensor for Pesticide Detection film resistance R f associated to ion diffusion is expected to decrease. For the same reasons, a-ATZ decomplexation should influence