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Atrazine analysis using an impedimetric immunosensor based on mixed biotinylated self-assembled monolayer
Sensors and Actuators B 113 (2006) 711–717
Atrazine analysis using an impedimetric immunosensor based on mixed biotinylated self-assembled monolayer S. Hleli a,b , C. Martelet a , A. Abdelghani b , N. Burais a , N. Jaffrezic-Renault a,∗ b
a Centre de G´ enie Electrique de Lyon, CEGELY, ECL, 69134 Ecully Cedex, France Unit´e de Recherche de Physique des Semiconducteurs et Capteurs, IPEST, La Marsa, Tunisia
Available online 10 August 2005
Abstract Atrazine is one of the most commonly used pesticides in the world. Thus, it is regularly found in rain, surface, marine and ground water. For these reasons, analysis of this pollutant is of high importance for environmental protection during the forthcoming years. This work describes the development of an electrochemical immunosensor for the analysis of atrazine associated to biotinylated-Fab fragment K47 antibody. The sensors are based on mixed self-assembled monolayer consisting of 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamineN-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid (MHDA). The properties of mixed monolayer were characterized by cyclic voltammetry and impedance spectroscopy. The tethered neutravidin was used the biotin sites present in the mixed monolayer, with those associated to the biotinyl-Fab fragment K47 antibody. The electrical resistance, Rm , decreases gradually after each building step of the sensing membrane. These decreases could be attributed to rearrangements in the structure of the SAMs. The results show that immunosensor based on this method has a high sensitivity to atrazine antigen and a good linear response in the range 10–300 ng/ml with a detection limit of 20 ng/ml. © 2005 Elsevier B.V. All rights reserved. Keywords: Atrazine; Biotinyl-PE; Neutravidin; Mixed self-assembled monolayers; Impedance spectroscopy; Biosensors
1. Introduction Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3, 5-triazine 9) is a highly toxic compound and also among the most widely used pesticides. Their excessive use has lead to environmental contamination of water and soil ecosystems with severe consequences for plant, veterinary and human health [1,2]. The risk of long term exposition of photosynthetic microorganisms of the surface water to atrazine has been extensively studied [3–5]. Generally, chromatographic and ELISA techniques are used for the determination and detection of atrazine in soil and water [6,7]. Both methods need traditional sample preparation, though very efficient in extracting the target analyte, which is time consuming and produces large amount of solvent wastes. Although ∗
Corresponding author. Tel.: +33 472186243; fax: +33 478433717. E-mail address: [email protected] (N. Jaffrezic-Renault).
0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.07.023
conventional qualitative and quantitative assay methods are available alternative biosensor-based immunochemical methods are usually more convenient [8–10]. Antibodybased immunosensor technology allows quick and inexpensive analysis of pesticides in the laboratory or in the field [11]. In recent years, the generation of antibodies against small pesticide haptens has witnessed significant progress leading to the introduction of several immunoassays for environmentally sensitive small toxic molecules [12]. Among the various transduction techniques, electrochemical impedance spectroscopy (EIS) has previously been investigated to study the selective binding of antibody–antigen on gold [13–15]. Electric impedance spectroscopy is a sensitive technique, which monitors the electrical response of the system studied after application of a periodic small amplitude ac signal. Analysis of the system response provides information concerning the electrical behaviour of the interface and interaction occurring on it [16,17].
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In this contribution, an innovative way for sensitive detection of atrazine based on novel self-assembled monolayer is presented. Non-faradic impedance spectroscopy was applied to characterize the immunoreaction biotinyl-Fab fragment K47 and atrazine. The mixed self-assembled monolayer consisting of biotinyl-PE and MHDA was examined using electrochemical impedance spectroscopy and cyclic voltammetry. The neutravidin/biotin interaction and conformational effects induced within the SAM were detected using impedance spectroscopy.
2. Experimental 2.1. Material Atrazine was purchased from SUPELCO. The antibody Fab fragment K47 was obtained from Technische Universitat Munchen, Germany and then biotinylated in Institut National de la Recherche Agronomique (INRA), Paris. Non-immune goat IgG was purchased from Sigma–Aldrich whilst neutravidin was obtained from Pierce. The affinity phospholipid dipalmitoyl-sn-glycero3-phospho-ethanolamine-N-(biotinyl) (biotinyl-PE) and 16mercaptohexadecanoic acid, for the formation of mixed monolayers were purchased from Avanti polar lipids and Sigma–Aldrich, respectively. The buffer solution used for all experiments was phosphate buffered saline (PBS) containing 140 mM NaCl, 2.7 mM KCl, 0.1 mM Na2 HPO4 , 1.8 mM KH2 PO4 , pH 7 and the redox couple Fe(CN)6 3− /Fe(CN)6 4− at a 5 mM concentration. All reagents were of analytical grade and ultrapure water (resistance 18.2 M cm−1 ) produced by a Millipore Milli-Q system was used throughout. 2.2. Fab fragment K47 antibody Genes encoding the variable heavy and light chain domains of the recombinant Fab fragment K47H were initially isolated from the hybridoma cell line K4E7 and cloned into the Fab expression vector pASK85 as described before [12]. This vector contains the murine heavy (CH 1) and light chain (CL ) constant regions of mouse IgG, thus complementing the variable antibody regions to a functional Fab fragment of native antibodies. Escherichia coli strain W3110 [18] was transformed with the recombinant vector for high density fermentation. Expression was performed in a 4 l bench top fermenter according to Schiweck and Skerra [19]. The Fab fragment was efficiently purified in a single step from the periplasmic fraction of E. coli by immobilized metal affinity chromatography (IMAC) as confirmed by SDS-PAGE analysis. The spectroscopically determined [20] yield of the purified Fab fragment was ca. 0.1 mg/l × OD550 for the fermenter culture (final OD550 ∼31). The specificity for striazine herbicides was verified for the Fab preparation by ELISA.
2.3. Preparation of mixed self-assembled monolayers Evaporated gold (∼300 nm thickness) was deposited on silicon, using a titanium baselayer (30 nm thickness) as substrate. Before modification, the gold surface was cleaned in an ultrasonic bath for 10 min in acetone, dried under a dry N2 flow and then dipped for 1 min into “piranha solution” 7:3 (v/v) 98% H2 SO4 /30% H2 O2 . The gold substrate was then rinsed two to three times with ultra-pure water and dried with a nitrogen flow. After cleaning, the gold electrodes were immediately immersed in an ethanol solution of 1 mM of MHDA and 0.1 mM of biotinyl-PE for 21 h at room temperature. After the formation of the mixed monolayer the substrate was rinsed four to five times with ethanol and dried under a nitrogen-flow. 2.4. Blocking and interaction biotinyl-PE with neutravidin Mixed self-assembled monolayer functionalized electrode was immersed in 10−7 M solution of non-specific IgG in PBS at pH 7, for 2 h in order to block the free space between the biotinylated species. The electrode was then throughly rinsed with PBS to remove excess of nonspecific IgG. Finally, the electrode was dipped into PBS containing 1 × 10−7 M neutravidin for 1 h 30 min, and then rinsed with PBS buffer to remove non-specifically adsorbed neutravidin. 2.5. Immunoassay of Fab fragment K47H Neutravidin functionalized electrode was immersed in 5 ml PBS at pH 7 with 2.6 × 10−7 M of biotinyl-Fab fragment K47H at room temperature for 1 h, and then thoroughly rinsed with PBS to remove weakly absorbed antibodies. Finally, electrode was exposed to various concentrations of antigen, and assayed in an electrochemical cell in order to monitor the immunoreaction in real time. 2.6. Impedance spectroscopy The measurement set-up for impedance consists of a classical three-electrode system, where the modified gold electrode (0.21 cm2 ) was used as working electrode, a platinum strip (0.54 cm2 ) as a counter electrode and a saturated calomel electrode (SCE) as reference electrode. The impedance analysis was performed with the Voltalab 80 impedance analyser in the frequency range 0.5 Hz–100 kHz, using a modulation voltage of 10 mV. During measurements the potential was kept at 0 V. The impedance measurements and cyclic voltametry measurements were performed in the presence of a 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as redox probe in PBS. The Zview modelling programme (Scribrer and Associates, Charlottesville, VA) was used to analyse impedance data. All electrochemical measurements were carried out at room temperature and in a Faraday cage.
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Fig. 1. Schematic showing the assembly of a mixed SAM based immunosensor.
3. Results and discussion 3.1. Electrochemical characteristics of mixed self-assembled monolayer Self-assembled monolayers consisting of long alkyl-thiols chains on gold have been shown to be stable in air, water and organic solvents at room temperature [21]. On the other hand, the biotin/neutravidin couple has a quite high binding affinity and can act as a bridge to anchor bioreceptor species. Therefore, a stable self-assembling system combined with a biotin/neutravidin couple has potential application for construction of biosensors. In this study, a mixed monolayer is chosen, which is composed of thiol acid, MHDA. It possess a thiol group allowing its immobilization on gold surfaces, and an hydrophilic terminal carboxyl group. The final stability of the mixed SAM layer is obtained through hydrophobic interaction between long alkyl (C16) chains. Fig. 1 shows the steps in the assembly of the mixed SAM based immunosensor, from initial mixed SAM formation, to the complete sensor which has bound the analyte (atrazine). In the last few years, impedance spectroscopy was used as an sensitive method to probe the interface properties of surface-modified electrodes [15–17]. Several authors demonstrated the possible use of this technique for a direct sensing of the interaction between immunospecies, and since then it has been expanding continuously. The complex impedance can be presented as a combination of the real, Zre , and imaginary, Zim , components originating mainly from the resistance and capacitance of the cell, respectively. A typical shape of a non-faradic impedance spectrum (presented in the form of a Nyquist plot) includes a semicircle region lying on the Zre -axis followed by a straight line. The semicircle portion, observed at higher frequencies, corresponds to the membrane process, whereas the linear part is characteristic of the lower frequencies range and represents the diffusion process. The semicircle diameter equals the membrane resistance, Rm . To fit much better the impedance spectrum, the constant phase element impedance (CPE, Eq. (1)) was introduced into the circuit instead of a capacitance: CPE = A−1 (jω)−n
parameter n in Eq. (1). The CPE becomes equal to capacitance when n = 1. The experimental non-faradic impedance spectra were fitted with computer simulated spectra using an electronic circuit shown in Fig. 2. This equivalent circuit includes the ohmic resistance of the electrolyte solution, Rs , the Warburg impedance, Zw , from the diffusion, the constant phase element, CPE, and membrane resistance, Rm . The latter three components, Zw , CPE and Rm , represent interfacial properties of the electrode, and they are affected by the surface modification. An excellent fitting between the simulated and experimental spectra was obtained for the bare Au-electrode and the mixed monolayer-modified Au-electrode Fig. 3. It can be seen that the diameter of semicircle at high frequency increases upon the stepwise formation of modifier on the electrode surface. The membrane resistance values, Rm , were extracted from the computer simulated spectra which are 410 and 1552 cm2 for bare Au-electrode and mixed modified Au-electrode, respectively. The values of the fractional coverage area of the mixed monolayer (θ) can be calculated from the impedance diagrams using equation (2) [30]: θ =1−
Rm R∗m
(2)
where Rm and R∗m are the values of the membrane resistance derived from the impedance diagram of the bare gold elec-
(1)
The constant phase element reflects an inhomogeneity and defect area of the layer [22–29]. The extent of the deviation from the Randles and Ershler model is controlled by the
Fig. 2. Equivalent circuit used to model impedance data in PBS solution.
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Fig. 3. Nyquist diagram (Zr vs. Zi ) for the non-faradic impedance measurements corresponding to (a) bare Au-electrode and (b) mixed self-assembled monolayer functionalized Au-electrode. All measurements were performed in a PBS pH 7.0 solution. Amplitude of alternating voltage 10 mV. Symbols show the experimental data in PBS solution. Solid curves show the computer fitting of the data using the equivalent circuits shown in Fig. 2.
trode and mixed self-assembled monolayer, respectively. In our system, the fractional coverage area was equal to 0.69. The Warburg impedance values, W, were extracted from computer simulated spectra which are 218.7 and 2043 cm2 for bare Au-electrode and mixed modified Au-electrode, respectively. As can be seen, the high value of the Warburg impedance of the gold electrode with the covered SAM shows that the layer is not acting as a blocking layer but as a diffusion layer because of the low percentage of area coverage. The experimental variations of impedance versus time show a stability in the range of 8%, which proves the mixed SAMs is quite stable. Such stability of the mixed monolayer offers us a very good basis for further construction. Moreover, cyclic voltammetry experiments further confirmed that the mixed SAM layer was successfully formed on the gold surface. When the electrode surface was modified by addition of material, the electron transfer kinetics of Fe(CN)6 4− /3− were perturbed. Fig. 4 shows the cyclic voltammograms of Fe(CN)6 4− /3− at the bare gold electrode (curve a) and mixed SAM covered electrode (curve b). As
Fig. 4. Cyclic voltammetric measurement with the presence of the 5 mM redox-probe K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]: (a) bare gold electrode; (b) mixed SAM/Au-electrode. All experiments were performed in PBS pH 7.0, scan rate 50 mV/s.
Fig. 5. Nyquist diagram (Zr vs. Zi ) for the non-faradic impedance measurements corresponding to: (a) mixed SAM/Au-electrode; (b) blocked with non-specific IgG/mixed SAM/Au-electrode; (c) neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode; (d) Fab fragment K47 antibody/neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode. Solid curves show the computer fitting of the data using the equivalent circuits shown in Fig. 2. Symbols show the experimental data.
shown in Fig. 4, the stepwise assembly of bare gold and mixed SAMs is accompanied by a decrease in the peak to peak separation between the cathodic and anodic waves of redox probe. This shows the formation of the mixed monolayer. 3.2. Detection of antibody and antigen–antibody interaction by impedance spectroscopy As previously indicated, the schematic diagram for sensor fabrication and antibody binding is shown in Fig. 1. Impedance measurements were performed in the frequency range 0.5 Hz–100 kHz. Complex impedance plots of the successive building-up of the sensing layers: mixed self-assembled monolayers/gold electrode, IgG/ mixed self-assembled monolayers/gold electrode, neutravidin/IgG/mixed self-assembled monolayers/gold electrode, Fab fragment K47/neutravidin/IgG/mixed self-assembled monolayers/gold electrode are shown in Fig. 5. Using the same equivalent circuit model described in Fig. 2, an excellent fitting between the simulation and experimental spectra was obtained. The parameter n was equal to 0.905, 0.91, 0.91 and 0.918 for the mixed modified electrode, successively blocked with IgG, neutravidin and Fab fragment K47 antibody, respectively. All these values are close to 1 and it means that CPE is essentially a capacitance. The membrane resistance value, Rm , were extracted from the respective computer simulated spectra as well. The formation of a new layer after the mixed self-assembled monolayer, induces decrease of membrane resistance. The membrane resistance was respectively equal to 1552, 1193, 1061 and 768.2 cm2 for the successive steps of modification electrode: mixed monolayer,
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Fig. 6. The constant phase element, CPE, at the electrode upon the stepwise assembly: (a) bare Au-electrode; (b) mixed SAM/Au-electrode; (c) blocked with non-specific IgG/mixed SAM/Au-electrode; (d) neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode; (e) Fab fragment K47 antibody/neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode.
blocking with IgG, neutravidin and Fab fragment K47 antibody. This decrease could be attributed to change in structure of mixed modified gold electrode, and inhomogeneity of the layer. Fig. 6 shows the constant phase element extracted from the computer fitting at the functionalized electrode upon the build-up of the blocking monolayer with IgG, neutravidin and association of biotinyl antibody. It is evident that the constant phase element at the electrode surface decreases. The electrode modified with Fab fragment K47 antibody was dipped into an electrochemical cell containing 5 ml of PBS at pH 7.0. Different volumes of 0.01 mg/ml of atrazine corresponding to different concentrations of atrazine in the cell were added into electrochemical cell at room temperature. Antigen–antibody interactions were monitored by impedance spectroscopy in PBS. The impedance measurement results of antigen in range of 0–1100 ng/ml are shown in Fig. 7. The semicircle diameter in the Nyquist plot seems to decrease with the antigen concentration, implying that more amount of antigen was linked to the interface and the mixed SAMs change its structure with different concentration adding of antigen as the antigen was not immobilized on the entire surface and thus do not act as a blocking layer. When the concentration of antigen was increased over 300 ng/ml, the change of impedance spectroscopy become gradually weak; showing that immobilization of the antibody on a gold electrode trends to saturation situation. In order to obtain the calibration data set, the values of membrane resistance differences Rm versus the added atrazine concentration were plotted in Fig. 8 (curve a). The change of membrane resistance was calculated according the equation: Rm = Rm(Ab) − Rm(Ab–Ag)
(3)
Fig. 7. Complex impedance plots of antigen–antibody/neutravidin/blocking with IgG/mixed SAM/gold electrode under various concentrations of antigen. The concentrations of antigen (ng/ml): (a) 0; (b) 10; (c) 30; (d) 50; (e) 80; (f) 120; (g) 200; (h) 600; (i) 1100. Applied frequency was from 0.5 Hz to 100 kHz.
Fig. 8. Calibration plots of the variation of membrane resistance Rm with the concentration of antigen.
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where Rm(Ab) is the value of membrane resistance as antibody immobilized on the electrode, Rm(Ab–Ag) the value of the membrane resistance after antigen binding to antibody. As can be seen in the figure, the plot is linear for high concentrations of atrazine and then reaches saturation. A linear relationship between the Rm values and the concentration of atrazine was established in the range from 10 to 300 ng/ml. The plot is represented by a linear regression equation, Rm = 1.23[atrazine](ng/ml) − 3.38, the minimum detectable concentration was around 20 ng/ml. To confirm that the above-observed impedance changes generated from the result of specific antibody–antigen interaction, the sensor was exposed to a solution of haemoglobin that are expected to bind non-specifically. Fig. 8 (curve b) shows the membrane resistance differences versus the added non-specific antigen concentration. As can be seen, the sensor was not sensitive to the non-specific binding and thus applicable for a selective determination of atrazine.
4. Conclusion Well controlled methods for the formation of sensitive membranes based on mixed self-assembled monolayers can lead to effective and highly sensitive molecular devices. The application of this innovative way for designing pesticide biosensors was proven to be quite efficient as associated with impedance spectroscopy measurement it allows a selective detection of atrazine up to the nano molar range. Furthermore impedance spectroscopy and cyclic voltammetry allow to characterize and to follow the building up of the successive layers, thus a coverage rate of 0.69 was found for the mixed self-assembled formed by MHDA and biotinyl-PE. Such a strategy can be transposed to other immunosensing couples and will be applied in the future for the GMOs detection.
Acknowledgment This work was financially supported by the European community under framework VI within project IMAGEMO (Contract: QLK3-2001-02141).
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Atrazine analysis using an impedimetric immunosensor based on mixed biotinylated self-assembled monolayer S. Hleli a,b , C. Martelet a , A. Abdelghani b , N. Burais a , N. Jaffrezic-Renault a,∗ b
a Centre de G´ enie Electrique de Lyon, CEGELY, ECL, 69134 Ecully Cedex, France Unit´e de Recherche de Physique des Semiconducteurs et Capteurs, IPEST, La Marsa, Tunisia
Available online 10 August 2005
Abstract Atrazine is one of the most commonly used pesticides in the world. Thus, it is regularly found in rain, surface, marine and ground water. For these reasons, analysis of this pollutant is of high importance for environmental protection during the forthcoming years. This work describes the development of an electrochemical immunosensor for the analysis of atrazine associated to biotinylated-Fab fragment K47 antibody. The sensors are based on mixed self-assembled monolayer consisting of 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamineN-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid (MHDA). The properties of mixed monolayer were characterized by cyclic voltammetry and impedance spectroscopy. The tethered neutravidin was used the biotin sites present in the mixed monolayer, with those associated to the biotinyl-Fab fragment K47 antibody. The electrical resistance, Rm , decreases gradually after each building step of the sensing membrane. These decreases could be attributed to rearrangements in the structure of the SAMs. The results show that immunosensor based on this method has a high sensitivity to atrazine antigen and a good linear response in the range 10–300 ng/ml with a detection limit of 20 ng/ml. © 2005 Elsevier B.V. All rights reserved. Keywords: Atrazine; Biotinyl-PE; Neutravidin; Mixed self-assembled monolayers; Impedance spectroscopy; Biosensors
1. Introduction Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3, 5-triazine 9) is a highly toxic compound and also among the most widely used pesticides. Their excessive use has lead to environmental contamination of water and soil ecosystems with severe consequences for plant, veterinary and human health [1,2]. The risk of long term exposition of photosynthetic microorganisms of the surface water to atrazine has been extensively studied [3–5]. Generally, chromatographic and ELISA techniques are used for the determination and detection of atrazine in soil and water [6,7]. Both methods need traditional sample preparation, though very efficient in extracting the target analyte, which is time consuming and produces large amount of solvent wastes. Although ∗
Corresponding author. Tel.: +33 472186243; fax: +33 478433717. E-mail address: [email protected] (N. Jaffrezic-Renault).
0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.07.023
conventional qualitative and quantitative assay methods are available alternative biosensor-based immunochemical methods are usually more convenient [8–10]. Antibodybased immunosensor technology allows quick and inexpensive analysis of pesticides in the laboratory or in the field [11]. In recent years, the generation of antibodies against small pesticide haptens has witnessed significant progress leading to the introduction of several immunoassays for environmentally sensitive small toxic molecules [12]. Among the various transduction techniques, electrochemical impedance spectroscopy (EIS) has previously been investigated to study the selective binding of antibody–antigen on gold [13–15]. Electric impedance spectroscopy is a sensitive technique, which monitors the electrical response of the system studied after application of a periodic small amplitude ac signal. Analysis of the system response provides information concerning the electrical behaviour of the interface and interaction occurring on it [16,17].
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S. Hleli et al. / Sensors and Actuators B 113 (2006) 711–717
In this contribution, an innovative way for sensitive detection of atrazine based on novel self-assembled monolayer is presented. Non-faradic impedance spectroscopy was applied to characterize the immunoreaction biotinyl-Fab fragment K47 and atrazine. The mixed self-assembled monolayer consisting of biotinyl-PE and MHDA was examined using electrochemical impedance spectroscopy and cyclic voltammetry. The neutravidin/biotin interaction and conformational effects induced within the SAM were detected using impedance spectroscopy.
2. Experimental 2.1. Material Atrazine was purchased from SUPELCO. The antibody Fab fragment K47 was obtained from Technische Universitat Munchen, Germany and then biotinylated in Institut National de la Recherche Agronomique (INRA), Paris. Non-immune goat IgG was purchased from Sigma–Aldrich whilst neutravidin was obtained from Pierce. The affinity phospholipid dipalmitoyl-sn-glycero3-phospho-ethanolamine-N-(biotinyl) (biotinyl-PE) and 16mercaptohexadecanoic acid, for the formation of mixed monolayers were purchased from Avanti polar lipids and Sigma–Aldrich, respectively. The buffer solution used for all experiments was phosphate buffered saline (PBS) containing 140 mM NaCl, 2.7 mM KCl, 0.1 mM Na2 HPO4 , 1.8 mM KH2 PO4 , pH 7 and the redox couple Fe(CN)6 3− /Fe(CN)6 4− at a 5 mM concentration. All reagents were of analytical grade and ultrapure water (resistance 18.2 M cm−1 ) produced by a Millipore Milli-Q system was used throughout. 2.2. Fab fragment K47 antibody Genes encoding the variable heavy and light chain domains of the recombinant Fab fragment K47H were initially isolated from the hybridoma cell line K4E7 and cloned into the Fab expression vector pASK85 as described before [12]. This vector contains the murine heavy (CH 1) and light chain (CL ) constant regions of mouse IgG, thus complementing the variable antibody regions to a functional Fab fragment of native antibodies. Escherichia coli strain W3110 [18] was transformed with the recombinant vector for high density fermentation. Expression was performed in a 4 l bench top fermenter according to Schiweck and Skerra [19]. The Fab fragment was efficiently purified in a single step from the periplasmic fraction of E. coli by immobilized metal affinity chromatography (IMAC) as confirmed by SDS-PAGE analysis. The spectroscopically determined [20] yield of the purified Fab fragment was ca. 0.1 mg/l × OD550 for the fermenter culture (final OD550 ∼31). The specificity for striazine herbicides was verified for the Fab preparation by ELISA.
2.3. Preparation of mixed self-assembled monolayers Evaporated gold (∼300 nm thickness) was deposited on silicon, using a titanium baselayer (30 nm thickness) as substrate. Before modification, the gold surface was cleaned in an ultrasonic bath for 10 min in acetone, dried under a dry N2 flow and then dipped for 1 min into “piranha solution” 7:3 (v/v) 98% H2 SO4 /30% H2 O2 . The gold substrate was then rinsed two to three times with ultra-pure water and dried with a nitrogen flow. After cleaning, the gold electrodes were immediately immersed in an ethanol solution of 1 mM of MHDA and 0.1 mM of biotinyl-PE for 21 h at room temperature. After the formation of the mixed monolayer the substrate was rinsed four to five times with ethanol and dried under a nitrogen-flow. 2.4. Blocking and interaction biotinyl-PE with neutravidin Mixed self-assembled monolayer functionalized electrode was immersed in 10−7 M solution of non-specific IgG in PBS at pH 7, for 2 h in order to block the free space between the biotinylated species. The electrode was then throughly rinsed with PBS to remove excess of nonspecific IgG. Finally, the electrode was dipped into PBS containing 1 × 10−7 M neutravidin for 1 h 30 min, and then rinsed with PBS buffer to remove non-specifically adsorbed neutravidin. 2.5. Immunoassay of Fab fragment K47H Neutravidin functionalized electrode was immersed in 5 ml PBS at pH 7 with 2.6 × 10−7 M of biotinyl-Fab fragment K47H at room temperature for 1 h, and then thoroughly rinsed with PBS to remove weakly absorbed antibodies. Finally, electrode was exposed to various concentrations of antigen, and assayed in an electrochemical cell in order to monitor the immunoreaction in real time. 2.6. Impedance spectroscopy The measurement set-up for impedance consists of a classical three-electrode system, where the modified gold electrode (0.21 cm2 ) was used as working electrode, a platinum strip (0.54 cm2 ) as a counter electrode and a saturated calomel electrode (SCE) as reference electrode. The impedance analysis was performed with the Voltalab 80 impedance analyser in the frequency range 0.5 Hz–100 kHz, using a modulation voltage of 10 mV. During measurements the potential was kept at 0 V. The impedance measurements and cyclic voltametry measurements were performed in the presence of a 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as redox probe in PBS. The Zview modelling programme (Scribrer and Associates, Charlottesville, VA) was used to analyse impedance data. All electrochemical measurements were carried out at room temperature and in a Faraday cage.
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Fig. 1. Schematic showing the assembly of a mixed SAM based immunosensor.
3. Results and discussion 3.1. Electrochemical characteristics of mixed self-assembled monolayer Self-assembled monolayers consisting of long alkyl-thiols chains on gold have been shown to be stable in air, water and organic solvents at room temperature [21]. On the other hand, the biotin/neutravidin couple has a quite high binding affinity and can act as a bridge to anchor bioreceptor species. Therefore, a stable self-assembling system combined with a biotin/neutravidin couple has potential application for construction of biosensors. In this study, a mixed monolayer is chosen, which is composed of thiol acid, MHDA. It possess a thiol group allowing its immobilization on gold surfaces, and an hydrophilic terminal carboxyl group. The final stability of the mixed SAM layer is obtained through hydrophobic interaction between long alkyl (C16) chains. Fig. 1 shows the steps in the assembly of the mixed SAM based immunosensor, from initial mixed SAM formation, to the complete sensor which has bound the analyte (atrazine). In the last few years, impedance spectroscopy was used as an sensitive method to probe the interface properties of surface-modified electrodes [15–17]. Several authors demonstrated the possible use of this technique for a direct sensing of the interaction between immunospecies, and since then it has been expanding continuously. The complex impedance can be presented as a combination of the real, Zre , and imaginary, Zim , components originating mainly from the resistance and capacitance of the cell, respectively. A typical shape of a non-faradic impedance spectrum (presented in the form of a Nyquist plot) includes a semicircle region lying on the Zre -axis followed by a straight line. The semicircle portion, observed at higher frequencies, corresponds to the membrane process, whereas the linear part is characteristic of the lower frequencies range and represents the diffusion process. The semicircle diameter equals the membrane resistance, Rm . To fit much better the impedance spectrum, the constant phase element impedance (CPE, Eq. (1)) was introduced into the circuit instead of a capacitance: CPE = A−1 (jω)−n
parameter n in Eq. (1). The CPE becomes equal to capacitance when n = 1. The experimental non-faradic impedance spectra were fitted with computer simulated spectra using an electronic circuit shown in Fig. 2. This equivalent circuit includes the ohmic resistance of the electrolyte solution, Rs , the Warburg impedance, Zw , from the diffusion, the constant phase element, CPE, and membrane resistance, Rm . The latter three components, Zw , CPE and Rm , represent interfacial properties of the electrode, and they are affected by the surface modification. An excellent fitting between the simulated and experimental spectra was obtained for the bare Au-electrode and the mixed monolayer-modified Au-electrode Fig. 3. It can be seen that the diameter of semicircle at high frequency increases upon the stepwise formation of modifier on the electrode surface. The membrane resistance values, Rm , were extracted from the computer simulated spectra which are 410 and 1552 cm2 for bare Au-electrode and mixed modified Au-electrode, respectively. The values of the fractional coverage area of the mixed monolayer (θ) can be calculated from the impedance diagrams using equation (2) [30]: θ =1−
Rm R∗m
(2)
where Rm and R∗m are the values of the membrane resistance derived from the impedance diagram of the bare gold elec-
(1)
The constant phase element reflects an inhomogeneity and defect area of the layer [22–29]. The extent of the deviation from the Randles and Ershler model is controlled by the
Fig. 2. Equivalent circuit used to model impedance data in PBS solution.
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Fig. 3. Nyquist diagram (Zr vs. Zi ) for the non-faradic impedance measurements corresponding to (a) bare Au-electrode and (b) mixed self-assembled monolayer functionalized Au-electrode. All measurements were performed in a PBS pH 7.0 solution. Amplitude of alternating voltage 10 mV. Symbols show the experimental data in PBS solution. Solid curves show the computer fitting of the data using the equivalent circuits shown in Fig. 2.
trode and mixed self-assembled monolayer, respectively. In our system, the fractional coverage area was equal to 0.69. The Warburg impedance values, W, were extracted from computer simulated spectra which are 218.7 and 2043 cm2 for bare Au-electrode and mixed modified Au-electrode, respectively. As can be seen, the high value of the Warburg impedance of the gold electrode with the covered SAM shows that the layer is not acting as a blocking layer but as a diffusion layer because of the low percentage of area coverage. The experimental variations of impedance versus time show a stability in the range of 8%, which proves the mixed SAMs is quite stable. Such stability of the mixed monolayer offers us a very good basis for further construction. Moreover, cyclic voltammetry experiments further confirmed that the mixed SAM layer was successfully formed on the gold surface. When the electrode surface was modified by addition of material, the electron transfer kinetics of Fe(CN)6 4− /3− were perturbed. Fig. 4 shows the cyclic voltammograms of Fe(CN)6 4− /3− at the bare gold electrode (curve a) and mixed SAM covered electrode (curve b). As
Fig. 4. Cyclic voltammetric measurement with the presence of the 5 mM redox-probe K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]: (a) bare gold electrode; (b) mixed SAM/Au-electrode. All experiments were performed in PBS pH 7.0, scan rate 50 mV/s.
Fig. 5. Nyquist diagram (Zr vs. Zi ) for the non-faradic impedance measurements corresponding to: (a) mixed SAM/Au-electrode; (b) blocked with non-specific IgG/mixed SAM/Au-electrode; (c) neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode; (d) Fab fragment K47 antibody/neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode. Solid curves show the computer fitting of the data using the equivalent circuits shown in Fig. 2. Symbols show the experimental data.
shown in Fig. 4, the stepwise assembly of bare gold and mixed SAMs is accompanied by a decrease in the peak to peak separation between the cathodic and anodic waves of redox probe. This shows the formation of the mixed monolayer. 3.2. Detection of antibody and antigen–antibody interaction by impedance spectroscopy As previously indicated, the schematic diagram for sensor fabrication and antibody binding is shown in Fig. 1. Impedance measurements were performed in the frequency range 0.5 Hz–100 kHz. Complex impedance plots of the successive building-up of the sensing layers: mixed self-assembled monolayers/gold electrode, IgG/ mixed self-assembled monolayers/gold electrode, neutravidin/IgG/mixed self-assembled monolayers/gold electrode, Fab fragment K47/neutravidin/IgG/mixed self-assembled monolayers/gold electrode are shown in Fig. 5. Using the same equivalent circuit model described in Fig. 2, an excellent fitting between the simulation and experimental spectra was obtained. The parameter n was equal to 0.905, 0.91, 0.91 and 0.918 for the mixed modified electrode, successively blocked with IgG, neutravidin and Fab fragment K47 antibody, respectively. All these values are close to 1 and it means that CPE is essentially a capacitance. The membrane resistance value, Rm , were extracted from the respective computer simulated spectra as well. The formation of a new layer after the mixed self-assembled monolayer, induces decrease of membrane resistance. The membrane resistance was respectively equal to 1552, 1193, 1061 and 768.2 cm2 for the successive steps of modification electrode: mixed monolayer,
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Fig. 6. The constant phase element, CPE, at the electrode upon the stepwise assembly: (a) bare Au-electrode; (b) mixed SAM/Au-electrode; (c) blocked with non-specific IgG/mixed SAM/Au-electrode; (d) neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode; (e) Fab fragment K47 antibody/neutravidin/blocked with non-specific IgG/mixed SAM/Au-electrode.
blocking with IgG, neutravidin and Fab fragment K47 antibody. This decrease could be attributed to change in structure of mixed modified gold electrode, and inhomogeneity of the layer. Fig. 6 shows the constant phase element extracted from the computer fitting at the functionalized electrode upon the build-up of the blocking monolayer with IgG, neutravidin and association of biotinyl antibody. It is evident that the constant phase element at the electrode surface decreases. The electrode modified with Fab fragment K47 antibody was dipped into an electrochemical cell containing 5 ml of PBS at pH 7.0. Different volumes of 0.01 mg/ml of atrazine corresponding to different concentrations of atrazine in the cell were added into electrochemical cell at room temperature. Antigen–antibody interactions were monitored by impedance spectroscopy in PBS. The impedance measurement results of antigen in range of 0–1100 ng/ml are shown in Fig. 7. The semicircle diameter in the Nyquist plot seems to decrease with the antigen concentration, implying that more amount of antigen was linked to the interface and the mixed SAMs change its structure with different concentration adding of antigen as the antigen was not immobilized on the entire surface and thus do not act as a blocking layer. When the concentration of antigen was increased over 300 ng/ml, the change of impedance spectroscopy become gradually weak; showing that immobilization of the antibody on a gold electrode trends to saturation situation. In order to obtain the calibration data set, the values of membrane resistance differences Rm versus the added atrazine concentration were plotted in Fig. 8 (curve a). The change of membrane resistance was calculated according the equation: Rm = Rm(Ab) − Rm(Ab–Ag)
(3)
Fig. 7. Complex impedance plots of antigen–antibody/neutravidin/blocking with IgG/mixed SAM/gold electrode under various concentrations of antigen. The concentrations of antigen (ng/ml): (a) 0; (b) 10; (c) 30; (d) 50; (e) 80; (f) 120; (g) 200; (h) 600; (i) 1100. Applied frequency was from 0.5 Hz to 100 kHz.
Fig. 8. Calibration plots of the variation of membrane resistance Rm with the concentration of antigen.
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where Rm(Ab) is the value of membrane resistance as antibody immobilized on the electrode, Rm(Ab–Ag) the value of the membrane resistance after antigen binding to antibody. As can be seen in the figure, the plot is linear for high concentrations of atrazine and then reaches saturation. A linear relationship between the Rm values and the concentration of atrazine was established in the range from 10 to 300 ng/ml. The plot is represented by a linear regression equation, Rm = 1.23[atrazine](ng/ml) − 3.38, the minimum detectable concentration was around 20 ng/ml. To confirm that the above-observed impedance changes generated from the result of specific antibody–antigen interaction, the sensor was exposed to a solution of haemoglobin that are expected to bind non-specifically. Fig. 8 (curve b) shows the membrane resistance differences versus the added non-specific antigen concentration. As can be seen, the sensor was not sensitive to the non-specific binding and thus applicable for a selective determination of atrazine.
4. Conclusion Well controlled methods for the formation of sensitive membranes based on mixed self-assembled monolayers can lead to effective and highly sensitive molecular devices. The application of this innovative way for designing pesticide biosensors was proven to be quite efficient as associated with impedance spectroscopy measurement it allows a selective detection of atrazine up to the nano molar range. Furthermore impedance spectroscopy and cyclic voltammetry allow to characterize and to follow the building up of the successive layers, thus a coverage rate of 0.69 was found for the mixed self-assembled formed by MHDA and biotinyl-PE. Such a strategy can be transposed to other immunosensing couples and will be applied in the future for the GMOs detection.
Acknowledgment This work was financially supported by the European community under framework VI within project IMAGEMO (Contract: QLK3-2001-02141).
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