Anal. Chem. 2004, 76, 6808-6813
Construction of Amperometric Immunosensors Based on the Electrogeneration of a Permeable Biotinylated Polypyrrole Film Rodica E. Ionescu,† Chantal Gondran,‡ Levi A. Gheber,† Serge Cosnier,*,‡ and Robert S. Marks†
The Institute for Applied Biosciences and Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva, 84105, Israel, and Laboratoire d'Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Mole´ culaire de Grenoble, FR CNRS 2607, Universite´ Joseph Fourier, Grenoble, France
The construction of amperometric immunosensors to cholera antitoxin immunoglobulins were shown to have improved sensitivity when the cholera toxin B subunit biorecognition entity was linked to an electrogenerated biotinylated polypyrrole film copolymerized with pyrrolelactobionamide monomer. The copolymer exhibits greater film permeability than biotinylated polypyrrolic or polyphenolic films for the permeation of electroactive species. Hence, when the presence of the HRP marker of the immunoassay was determined using hydroquinone, the production of electroactive quinone was shown to permeate faster to the electrode, thus providing a faster response time. The intimate combination of the exquisite affinity of antigenantibody interactions with the sensitivity of optical, electrochemical, or gravimetric transducers has led to the emergence of immunosensors as valuable selective tools in diagnostic laboratories and medical treatment.1-3 The growing and increasing use of biochips, as well as miniaturized immunosensors, requires that we create nonmanual methods that will provide the reproducible and localized deposition of relevant immunoagents (antigen or antibody). The electrosynthesis of organic conducting polymers is one of the few known methods that allows for the reproducible deposition of linked biological macromolecules, with controlled spatial resolution over surfaces whatever their size and geometry.4-6 However, the immunoagent immobilization by conventional entrapment within the polymer film during its electrochemical growth may prevent the formation of antigen-antibody complexes. In contrast, the combination of electrochemical addressing with the high-affinity interaction of avidin-biotin (association constant Ka ) 1015 M-1) leads to an affinity-driven immobilization * To whom correspondence should be addressed. E-mail: Serge.Cosnier@ ujf-grenoble.fr. Telephone: +33 (4) 76-51-49-98. Fax: +33 (4) 76-51-42-67. † Ben-Gurion University of the Negev. ‡ Institut de Chimie Mole´culaire de Grenoble. (1) Suzuki, H. Electroanalysis 2000, 12, 703-715. (2) Worwood, M. Ann. Clin. Biochem. 2002, 39, 221-230. (3) Shah, J.; Wilkins, E. Electroanalysis 2003, 15, 157-167. (4) Schuhmann, W. Mikrochim. Acta 1995, 121, 1-29. (5) M. Gerard, M.; Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 345-359. (6) Cosnier, S. Anal. Bioanal. Chem. 2003, 377, 507-520.
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protocol of enzymes involving solely a single attachment point that fully retain their biological activity.7-10 More interestingly, binding of a protein monolayer displaying excellent accessibility to each immobilized protein has been recently achieved by using avidin-biotin bridges between electropolymerized biotinylated polypyrrole films and biotinylated antibodies.11 Among conventional methods in the detection of antigenantibody binding events, the electrochemical transduction ensures attractive advantages such as its ease of use in turbid samples, portability, low cost, and compatibility with bulk manufacturing procedures. Besides the direct reagentless immunosensing based on frequency impedance measurements,12-14 the detection of an immunoreaction is commonly performed either by optical15,16 or by amperometric transduction, following the classical enzymelinked immunosorbent assay strategy.17-22 The latter approach consists of the use of antibodies labeled with enzymes catalyzing the production of electroactive species that are amperometrically monitored at the electrode surface. As a consequence, the diffusion of the electroactive probes indicates the immunoreaction through the underlying biotinylated polymer film and hence the (7) Cosnier, S.; Galland, B.; Gondran, C.; Le Pellec, A. Electroanalysis 1998, 10, 808-813. (8) Torres-Rodriguez, L. M.; Roget, A.; Billon, M.; Livache, T.; Bidan, G. J. Chem. Soc., Chem. Commun. 1998, 1993. (9) Cosnier, S.; Le Pellec, A. Electrochim. Acta 1999, 44, 1833-1836. (10) Cosnier, S.; Stoytcheva, M.; Senillou, A.; Perrot, H.; Furriel, R. P. M.; Leone, F. A. Anal. Chem. 1999, 71, 3692-3697. (11) Ouerghi, O.; Touhami, A.; Jaffrezic-Renault, N.; Martelet, C.; Ben Ouada, H.; Cosnier, S. Bioelectrochemistry 2002, 56, 131-133. (12) Sargent, A.; Loi, T.; Gal S.; Sadik O. A. J. Electroanal. Chem. 1999, 470, 144-156. (13) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913-947. (14) Kalab, T.; Skladal, P. Anal. Chim. Acta 1995, 04, 361-368. (15) Marks, R. S.; Bassis, E.; Bychenko, A.; Levine, M. M. Opt. Eng. 1997, 36, 3258-3264. (16) Konry, T.; Novoa, A.; Cosnier, S.; Marks, R. S. Anal. Chem. 2003, 75, 26332639. (17) Zayats, M.; Raitman, O. A.; Chegel, V. I.; Kharitonov, A. B.; Willner, I. Anal. Chem. 2002, 74, 4763-4773. (18) Rishpon, D. I. Biosens. Bioelectron. 1996, 11, 409-417. (19) Ghidilis, A. L.; Atanasov, P.; Wilkins, M.; Wilkins, E. Biosens. Bioelectron. 1998, 13, 113-131. (20) Santandreu, M.; Alegret, S.; Fabregas, E. Anal. Chim. Acta 1999, 396, 181-188. (21) Liu, G. D.; Wu, Z. Y.; Wang, S. P.; Shen, G. L.; Yu R. Q. Anal. Chem. 2001, 73, 3219-3226. (22) Dai, Z.; Yan, F.; Chen, J.; Ju, H. Anal. Chem. 2003, 75, 5429-5434. 10.1021/ac049413z CCC: $27.50
© 2004 American Chemical Society Published on Web 10/09/2004
Figure 1. Amperometric immunosensor-HRP setup based on a hydroquinone (HQ)/quinone (Q) electrochemical system.
sensitivity of the amperometric immunosensor are strongly related to the permeation properties of the biotinylated polymer used for the immobilization of antibody or antigen. However, the biotinylated polymers described until now exhibited poor permeabilities since their formation passivated the electrodes.23,24 Most of them were thus employed for the elaboration of affinity sensors based on gravimetric measurements.25,26 Taking the permeability issue into account, we report herein a new procedure for the fabrication of amperometric immunosensors based on the electrogeneration of a biotinylated copolymer film exhibiting a high permeability in order to enhance the transduction step. It was composed of biotin groups allowing the anchoring of antibody or antigen through avidin by affinity interactions and lactobioamide groups that improve the hydrophilic character of the organic film and hence will improve its permeability. Due to the importance of cholera as an endemic scourge or infrequently as a pandemic in a number of countries in Asia, Africa, and Latin America, the model immunosensor fabricated was based on the detection of anticholera toxin antibody.15,16,27 The transduction step involved the recognition of the captured target by a secondary cholera antitoxin antibody marker labeled by peroxidase (Figure 1). The performances of the immunosensor were investigated through the amperometric detection of the enzymatically oxidized hydroquinone in the presence of hydrogen peroxide. (23) Yang, S. T.; Witkowski, A.; Hutchins, R. S.; Scott, D. L.; Bachas, L. G. Electroanalysis 1998, 10, 58-60. (24) Kuramitz, H.; Matsuda, M.; Sugawara, K.; Tanaka, S. Electroanalysis 2003, 15, 225-229. (25) Dupont-Filliard, A.; Roget, A.; Livache, T.; Billon, M. Anal. Chim. Acta 2001, 449, 45-50. (26) Torrez Rodrigez, L. M.; Billon, M.; Roget, A.; Bidan, G. J. Electroanal. Chem. 2002, 523, 70-78.
EXPERIMENTAL SECTION Reagents. Cholera toxin B subunit, biotin labeled (Catalog No. C-9972), anti-rabbit IgG-peroxidase (Catalog No. A-6154), anticholera toxin (Catalog No. C-3062), polyoxyethylenesorbitan monolaurate (Tween 20, P7949), bovine serum albumin (BSA, A4503), and avidin were purchased from Sigma. Pyrrole-lactobionamide was prepared as described previously.28 8-Pyrrol-1aminooctane (0.5 g, 2.57 mmol) was added to a solution of lactobionic acid (0.949 g, 2.57 mmol) in methanol (100 mL). The mixture was stirred under reflux for 24 h. The organic solvent was then removed under vaccum, and after freeze-drying, the product was obtained in quantitative yield (1.31 g, 95%). Until use, the pyrrole-lactobionamide was kept refrigerated. The pyrrolebiotin was synthesized according to the litterature as follows.10 Biotin (338 mg), 11-(1-pyrrolyl)dodecanol (327 mg), 1,3-dicyclohexylcarbodiimide (326 mg), and 4-(dimethylamino)pyridine (20 mg) were dissolved in dry CH2Cl2. The reaction mixture was stirred at room temperature for 5 days. After filtration and evaporation steps, a white precipitate (pyrrole-biotin) was obtained and purified by chromatography. Until use, the pyrrolebiotin monomer was kept under argon. All other reagents were of analytical grade. Electrochemical Instrumentation. Electropolymerization and cyclic voltammetry experiments were performed with a EG&G PAR model 173 potentiostat equipped with a model 175 universal programmer and a model 179 digital coulometer in conjunction with a Kipp and Zonen BD 91 XY/t recorder. All experiments were carried out using a thermostated three-electrode cell. The amperometric measurements were performed with a Tacussel PRGDL potentiostat at 23 °C. The working electrodes were glassy carbon disks (5-mm diameter) systematically polished with 1-µm diamond paste (Mecaprex Press PM) and then extensively rinsed with distilled water, ethanol, and acetone. A 10 mM Ag+/Ag electrode was used as a reference electrode in acetonitrile electrolyte for the electrogeneration of polypyrrolic films. A saturated Ag-AgCl-KCl electrode was used as a reference electrode in aqueous 0.1 M phosphate buffer electrolyte (pH 7). Potentials are reported versus the aqueous saturated calomel electrode. Gravimetric Measurements. AT-cut 9-MHz quartz crystal (CQE, Troyes, France) coated with two identical Au layers (200 nm thick) and modified onto one Au electrode by the biotinylated copolymer was mounted in a plexiglass cell. The flow-through cell (volume, 50 µL) was associated with a micropump (P1, Pharmacia) allowing a constant flow rate of 60 µL min-1. The experimental setup was built by coupling a homemade 27-MHz quartz crystal microbalance (QCM) by using the 9-MHz resonator on the third overtone and a frequency counter (HP 53132 A). Avidin solutions were injected in the continuous-flow mode after the resonance frequency had reached a steady-state value for a phosphate buffer solution (0.01 M), and the shift in the resonance frequency was monitored as a function of time. Immunosensor Preparation. The biotinylated polypyrrole films were made at room temperature under an argon atmosphere, by controlled potential oxidation at 0.8 V of pyrrole-biotin (4 mM) (27) Hale, Z. M.; Payne, F. P.; Marks, R. S.; Lowe, C. R.; Levine, M. M. Biosens. Bioelectron. 1996, 11, 137-148. (28) Cosnier, S.; Le Pellec, A.; Guidetti B.; Rico-Lattes I. J. Electroanal. Chem 1998, 449, 165-171.
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in CH3CN + 0.1 M LiClO4, and the charge passed during the electropolymerization step, being fixed at 1 mC. The biotinylated copolymer films (poly(pyrrole-biotin) and poly(pyrrole-lactobionamide)) were prepared in the same conditions, the concentrations of pyrrole-biotin and pyrrole-lactobionamide monomers being set at 4 mM each. The steps carried out for the construction of the immunosensor on the modified glassy carbon electrodes are the following. A blocking solution was used to prevent the nonspecific binding of avidin onto the electropolymerized films. This solution contained 5% (v/v) BSA in 0.1 M phosphate-buffered saline containing 0.5% (v/v) Tween 20 (pH 7) (PBST) and was prepared daily. The antibodies and avidin were diluted with 1% (w/v) BSA/0.1 M PBST, which was kept at 4 °C until used. The first step in creating immunosensors consisted in the deposition of a drop (20 µL) of the blocking solution on the biotinylated polymer and copolymer films. The electrodes were carefully washed several times with 0.1 M phosphate-buffered saline pH 7 (PBS) solution, and a drop (20 µL) of avidin (1 mg/mL) dissolved in 1% (w/v) BSA/PBST was then incubated for 20 min with the biotinylated films. After rinsing with PBS, the resulting modified electrodes were incubated for 20 min with 15 µL of biotinylated cholera toxin B subunit at 0.3 mg/mL dissolved in 1% (w/v) BSA/ PBST. The electrodes were rinsed with PBS and then incubated with 20 µL of anti-cholera toxin B subunit antibody elicited in a rabbit at concentrations ranging from 0.05 to 500 µg/mL. Incubation time was chosen to be 20 min. A rinsing step with PBS followed. Thereafter, the electrodes were incubated with 20 µL of a solution containing the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-cholera IgG immunoglobulin at a concentration of 0.5 mg/mL dissolved in 1% (w/v) BSA/PBST for 20 min. Finally, all the modified electrodes were washed with PBS before amperometric measurements. Amperometric Immunoassay Measurements. Batch amperometric measurements were carried out by immersing the immunosensor-HRP into 0.1 M phosphate buffer (20 mL, pH 7) containing hydroquinone (2 mM). For the response measurements, the modified electrodes were first conditioned at 0 V for 4 min to reach a stable background current. After stabilization of the electrode response, 80 µL of hydrogen peroxide (0.5 mM) was injected into the electrolyte kept under stirring conditions at 23 °C. The changes in the current were monitored as a function of anti-cholera toxin concentrations.
RESULTS AND DISCUSSION The detection of rabbit anti-cholera toxin antibody was achieved using a sandwich immunoassay based on the immobilization of the immunogenic (and safe) part of the enterotoxin, the pentameric cholera toxin (CT) B subunit protein, as the molecular recognition layer (Figure 1). The first step required the electrochemical generation of a biotinylated film on the electrode surface. This polymeric film then allowed the conjugation of avidin and the subsequent binding of cholera toxin B subunit biotin-labeled via avidin-biotin bridges. The analyte, rabbit anti-cholera toxin antibody (CT antibody), thereafter bound the corresponding immobilized CTB subunit epitopes. Following the subsequent binding of a marker goat HRP-labeled IgG anti-cholera antibody, allowed to catalyze in the presence of hydrogen peroxide, the oxidaton of various redox mediators were amperometrically 6810 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
Figure 2. Schematic representation of pyrrole-lactobioamide and pyrrole-biotin monomers.
detected at the electrode surface as a function of the concentration of the captured CT anti-toxin antibody. The peroxidase indicating reaction used here the most common mediator hydroquinone. It was chosen as a redox-active substrate, the cathodic current of quinone reduction being monitored at 0 V.17,20,29-31 As a consequence, quinone permeation through the initial anchoring layer must be optimized. Electrochemical Characterization of a Copolymer Film Composed of Poly(pyrrole-biotin) and Poly(pyrrole-lactobionamide). Although the electropolymerization of pyrrolebiotin provided a compact and fully active layer of biotin, its organic character prevents the polymer from swelling in water and hence it may hinder the diffusion of quinone.32 To improve the amperometric detection of quinone, hydrophilic groups (pyrrole-lactobionamide) were introduced by copolymerization into the polymeric skeleton to counterbalance its hydrophobic character (Figure 2). Figure 3 shows the cyclic voltammograms of the electroactive permeant hydroquinone at the electrode covered by either a biotinylated polypyrrole or a biotinylated copolymer, both made with the same electropolymerization charge, namely, 1 mC. The comparison with the hydroquinone oxidation peak recorded at a bare electrode shows a slight decrease in peak current (-14%) associated with an increase of the anodic and cathodic peak separation for the copolymer while a quasidisappearance of this anodic peak is observed for the biotinylated polypyrrole. This clearly indicates that the homopolymer drastically hinders the permeation of hydroquinone while the copolymer appears much more permeable. The mass-transfer process in polymer films electrodeposited on electrode surfaces may be (29) Theegala, C. S.; Suleiman, A. A. Microchem. J. 2000, 65, 105-111. (30) Skladal, P.; Morozova, N. O.; Reshetilov, A. N. Biosens. Bioelectron. 2002, 00, 1-7. (31) Wendzinski, F.; Grundig, B.; Renneberg, R.; Spener, F. Biosens. Bioelectron. 1997, 12, 4-52. (32) Mousty, C.; Bergamasco, J. L.; Wessel, R.; Perrot, H.; Cosnier, S. Anal. Chem. 2001, 73, 2890-2897.
Figure 3. Cyclic voltammograms in the presence of hydroquinone (1 mM) and 0.1 M aqueous LiClO4 (pH 7) at (a) an uncoated glassy carbon electrode (i.d. ) 5 mm), (b) a copolymer (poly(pyrrole-biotin), poly(pyrrole-lactobionamide)) coated electrode, and (c) a biotinylated polypyrrole electrode. Scan rate at 100 mV s-1.
investigated by rotating-disk electrode (RDE) voltammetry.33-35 As a consequence, the permeability of the copolymer was determined by using RDE experiments carried out at different rotation rates with a bare electrode and a copolymer electrode. Figure 4A shows the different rotating-disk voltammograms obtained on the copolymer electrode in an aqueous solution of hydroquinone (1 mM). If the crossing of the film-solution interface is fast in both directions, its influence on the current response is simply governed by an equilibrium partition coefficient K. As described by Gough and Leypoldt,36,37 the steady-state limiting current (Ilim) is then obtained in the absence of a catalytic phenomenon, assuming that the diffusion is linear. Thus, at the plateau, the limiting current is expressed as
1 1 1 ) + Ilim Is Im
(1)
where Is and Im are two terms characterizing the diffusion of the substrate in the solution and in the film, respectively. The Levich current Is is proportional to the square root of the rotation rate: 2/3 -1/6 0
Is ) 0.62nFADs
ν
C ω1/2
(2)
and the second term depends on the product of the partition (33) Leddy, J.; Bard, A. J. Electroanal. Chem. 1983, 153, 223-242. (34) Save´ant, J. M. J. Electroanal. Chem. 1991, 302, 91-101. (35) Hofe¨r, E.; Steckhan, E.; Ramos, B.; Heineman, W. R. J. Electroanal. Chem. 1996, 402, 115-122. (36) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1979, 51, 439-443. (37) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1980, 52, 1126-1130.
Figure 4. (A) Rotating-disk electrode voltammograms of a copolymercoated glassy carbon electrode (i.d. ) 5 mm) in the presence of 1 mM solution of hydroquinone and 0.1 M aqueous LiClO4 (pH 7): (a) 3000, (b) 2500, (c) 2000, (d)1500, (e)1000, and (f) 500 rpm/min. Scan rate at 20 mV s-1. (B) Koutecky-Levich plot for an uncoated electrode (a) and a copolymer-coated electrode (b). The test solution was 1 mM hydroquinone in 0.1 M aqueous LiClO4 (pH 7).
equilibrium constant K and diffusion constant of the substrate in the film Dm.
Im ) nFADmKC0/δ
(3)
A is the electrode area, Ds is the diffusion coefficient for the substrate in the polymer, C0 the concentration of the substrate in the bulk of the solution, ν the kinematics viscosity of the solution, ω the rotation rate of the RDE, and δ the thickness of the polymer film. Only the first term of eq 1 is dependent upon the rotation rate of the RDE and applies to the current passed under the same conditions in the absence of film. Thus, a plot of 1/Ilim versus 1/ω1/2 should be a straight line with a positive intercept (δ/ (nFADmKC0)) whose value depends on the permeability of the polymer film: Pm ) DmK. Experimental data from RDE voltammograms were conveniently displayed in the form of KouteckyAnalytical Chemistry, Vol. 76, No. 22, November 15, 2004
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Table 1. Permeability Values of Electropolymerized Organic Films polymer poly(vinyl diquat) poly(trisvinylbipyridineruthenium) poly(cobalt porphyrin) poly(trisaminophenanthroline iron)
redox permeant
KDm (cm2 s-1)
ref
ferrocene [Ru(bpy)2Cl2] [Ru(bpy)2]2+ ferrocene
1 × 10-8 2.9 × 10-9 1.3 × 10-9 1.2 × 10-8
39
benzoquinone [Ru(bpy)2Cl2] ferrocene FcMeOH
7.8 × 10-8 7.3 × 10-10 8.2 × 10-8 6.1 × 10-8
[Ru(NH3)6]3+ [Fe(CN)6]4-
8.6 × 10-9 2.6 × 10-9
39
40 41
Levich plots of the reciprocal of the limiting current density versus the reciprocal of the square root of the rotation rate (Figure 4B). The data relative to the copolymer present a linear behavior with the same slope as for a bare electrode with a positive intercept from which the ratio (DmK)/δ (2.38 × 10-2 cm s-1) can be obtained. As previously determined for N-substituted polypyrrole films, the thickness of the polymer was estimated from the polymerization charge density to 38 nm.38 It should be noted that the presence of hydrophilic moieties in the polymeric skeleton should provide a phenomenom of polymer swelling in water that induces an underestimation of the copolymer thickness and hence an underestimation of its permeability. Nevertheless, the resulting permeability value (9.04 × 10-8 cm2 s-1) was similar or slightly higher than those reported for electropolymerized redox films in either organic or aqueous solvents illustrating thus the beneficial effect brought by the lactobionamide groups (Table 1).39-41 Interestingly, the biotinylated copolymer exhibits a better permeability than the previous biotinylated polypyrrolic and polyphenolic films that acted as insulating layers.23,24 However, the presence of the lactobionamide at a 1:1 ratio in the biotinylated copolymer may alter the affinity properties of the film and hence the quality of the subsequent avidin layer formed by avidin-biotin bridges. To examine the anchoring abilities of the biotinylated copolymer toward the attachment of avidin, gravimetric measurements were carried out using a QCM. The frequency response of QCM to a mass change was described by the Sauerbrey equation for thin, uniform, and purely elastic added layers.42,43 In particular, the binding of avidin was already investigated by gravimetric measurements with an electrode of a quartz crystal modified by the biotinylated polypyrrole. The decrease in resonance frequency corresponded to an avidin mass increase of 684 ng cm-2.32 The same experiment was performed with quartz electrodes modified by the copolymer. The coupling interaction between free avidin (38) De Gregori, I.; Carrier, M.; Deronzier, A.; Moutet, J.-C.; Bedioui, F.; Devynck, J. J. Chem. Soc., Faraday Trans 1992, 88, 1567-1572. (39) Ikeda, T.; Schmehl, P.; Denisevich, P.; Willman, K.; Murray, R. W. J. Am. Chem. Soc. 1982, 104, 2683-2691. (40) Pressprich, K. A.; Maybury, S. G.; Thomas, R. E., Linton, R. W.; Irene, E. A.; Murray, R. W. J. Phys. Chem. 1989, 93, 5568-5574. (41) Massari, A. M.; Gurney, R. W.; Schwartz, C. P.; Nguyen, S. T.; Hupp, J. T. Langmuir 2004, 20, 4422-4429. (42) Bizet, K.; Gabrielli, C.; Perrot, H.; Therasse, J. Biosens. Bioelectron. 1998, 13, 259-269. (43) O′Sullivan, C. K.; Guilbault, G. G. Biosens. Bioelectron. 1999, 14, 663670.
6812 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
Figure 5. Calibration curve for the immunosensors-HRP with antiCT ranging from 0.05 to 500 µg/mL using the hydroquinone/H2O2 system.
and biotin groups of the electrogenerated copolymer leads to a mass increase of 630 ng cm-2, thus illustrating the absence of a marked difference in the affinity properties between the two polymeric films. Calibration Curve of the Immunosensor for CT Antibody. To determine the immunosensor sensitivity to CT antibody, the immunosensors were tested with different concentrations of the target analyte (0.01-500 µg/mL). Upon immersion of the immunosensors-HRP in the buffer solution containing hydroquinone (2 mM), the baseline became stable after 4 min and the signal was recorded after the injection of H2O2. The calibration curve for the amperometric detection of CT antibody was carried out by recording, for each concentration, the response of a modified electrode (Figure 5). It should be noted that each measurement was repeated with three different immunosensors and averaged. A linear current variation was observed over the range 1-200 µg/ mL while a very sensitive detection limit of CT antibody was obtained, namely, at 50 ng/mL. This constitutes an attractive result since the detection limit obtained by a conventional spectrometric detection using the ELISA test is only 100 ng/mL anti-CT.44 It should be noted that no current response was recorded at a bare glassy carbon electrode soaked in the same solution 1 h after H2O2 injection. Moreover, each amperometric immunosensor exhibits for the whole concentration range of anti-CT (0.05-500 µg/mL) a fast response time at between 5 and 30 s. The immunosensor response and hence its construction was also quite reproducible; five immunosensors were prepared by following identical electropolymerization steps and their responses toward anti-CT (200 µg/mL) led to a relative standard deviation of only 5%. The immunosensors were also examined for their operational stability. Only a 5% decay of the initial amperometric response of the immunosensor was observed after 4 h, highlighting the stability of the supramolecular assembly as well as that of the enzymatic reaction. CONCLUSIONS We have presented herein a new strategy for the immobilization of the immunological material onto an electrode modified by an electropolymerized film. The novelty consisted in the coelectropolymerization of pyrrole-biotin and pyrrole-lactobionamide monomers as an anchoring polymeric layer. Thanks to its high hydrophilic character, the pyrrole-lactobionamide monomer (44) Leshem, B.; Sarfati, G.; Novoa, A.; Breslav, I.; Marks, R. S. Luminescence 2004, 19, 69-77.
markedly increased the permeability of the resulting biotinylated copolymer for the permeation of electroactive species. In this report, we have illustrated, in connection with a peroxidase indicator for the electrochemical reaction, the possibility for a fast and sensitive amperometric detection of cholera antitoxin antibody. It is expected that this simple procedure of immunosensor construction will be useful in the development of amperometric immunoassays.
sion of the European Communities Research Directorate for their support under the PEBCAT Project Contract EVKI-CT-2000-00069. R.S.M. thanks the Universite´ Joseph Fourier for the invited Professorship (Summer 2003) as well as the Arc-en-ciel/Keshet joint program of the France Ministry of Foreign Affairs and the Israel Ministry of Science. R.E.I. thanks the French Ministry of Foreign Affairs for funding a scientific mission at the laboratory of S.C.
ACKNOWLEDGMENT The authors thank H. Perrot (Laboratoire de Physique des Liquides et Electrochimie, UPR CNRS 15, 75252 Paris, France) for the QCM measurements. S.C. and R.S.M. thank the Commis-
Received for review April 19, 2004. Accepted August 27, 2004. AC049413Z
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