Anal. Chem. 2007, 79, 8662-8668
Amperometric Immunosensor for the Detection of Anti-West Nile Virus IgG Rodica E. Ionescu,† Serge Cosnier,*,† Sebastien Herrmann,‡ and Robert S. Marks‡,§
De´ partement de Chimie Mole´ culaire UMR-5250, ICMG FR-2607, CNRS Universite´ Joseph Fourier, BP-53, 38041 Grenoble Cedex 9, France, Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, 84105 Beer-Sheva, Israel, and cThe National Institute for Biotechnology in the Negev, and the Ilse Katz Center for Meso- and Nano-scale Science and Technology, P.O. Box 653, 84105 Beer-Sheva, Israel
An amperometric immunosensor for the detection of West Nile virus (WNV) IgG was developed. This device was based on the immobilization of T7 phages, which were modified by an additional peptide sequence taken from the virus and used as antigen. The electropolymerization of a phage-amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provided an efficient entrapment of phages in a polypyrrole film. After incubation with a secondary peroxidase-labeled antibody, the immunosensors were applied to the quantitative amperometric determination of WNV-antibody at 0 V vs Ag/AgCl via the reduction of the enzymically generated quinone in the presence of hydroquinone and H2O2. The optimum immunosensor configuration detected low WNVantibody dilutions down to a titer of 1:107 with an excellent regeneration of the immunosensor response by glycine treatment. West Nile virus (WNV) belongs to the family Flaviridae, genus Flavivirus, being a member of the Japanese encephalitis virus serocomplex, that includes St. Louis encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, and Japanese encephalitis virus as demonstrated using hemaglutination inhibition and crossneutralization methodologies.1-3 WNV is found in Africa, the Middle East, and Western Asia and occasionally has been identified in outbreaks of disease in European countries while it has now reached the shores of North America.4-8 Human infection is commonly asymptomatic; however, meningitis, encephalitis, or * Corresponding author. Tel.: + 33 4 76 51 49 98. Fax: + 33 4 76 51 42 67. E-mail:
[email protected]. † CNRS Universite´ Joseph Fourier. ‡ Ben-Gurion University of the Negev. § The National Institute for Biotechnology in the Negev, and the Ilse Katz Center for Meso- and Nano-scale Science and Technology. (1) Ostlund, E. N.; Andresen, J. E.; Andresen, M. Vet. Clin. North Am. Equine Pract. 2000, 16, 427- 441. (2) Calisher, C. H.; Karabatsos, N.; Dalrymple, J. M.; Shope, R. E.; Porterfield, J. S.; Westaway, E. G.; Brandt, W. E. J. Gen. Virol. 1989, 70, 37-43. (3) Murphy, F. A.; Fauquet, C. M.; Bishop, D. H. L.; Ghabrial, S. A.; Jarvis, A. W.; Martelli, G. P.; Mayo, M. A.; Summers, M. D. Arch. Virol. 1995, 10, 1-586. (4) Le Guenno, B.; Bougermouh, A.; Azzam, T.; Bouakaz, R. Lancet 1996, 348, 1315-1316. (5) Hubalek, Z.; Savage, H. M.; Halouzka, J.; Juricova, Z.; Sanogo, Y. O.; Lusk, S. Czechland Viral Immunol. 2000, 13, 427-433
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meningoencephalitis may occur.9 Usually, the transmission of WNV to horses and humans occurs through the bite of infected mosquitoes, but WNV can also be transmitted through blood transfusion, organ transplantation, breast feeding, intrauterine exposure, and laboratory-acquired infection.10-14 Currently, there are no effective therapies or vaccines against WNV infection. Therefore, the prevention of WNV invasion is an important public health concern in regions that have close links with areas in which WNV is endemic. The primary tool for diagnosing a WNV infection is the detection of WNV-specific antibodies in serum, plasma, or cerebrospinal fluid. The main approaches for the detection of WNV antibodies is through a complement fixation test (CF), hemagglutination-inhibition test (HI), plaque-reduction neutralization (PRNT), immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and the microsphere immunoassay (MIA).15 Unfortunately, CF and HI assays require highly trained personnel, demand strict quality control of reagents, and are not specific while PRNT tests require long assay times (6-10 days).16-18 Although IFA assays offer some advantages over CF, HI, and PRNT, this method is faced with a problem of cross(6) Lvov, D. K.; Butenko, A. M.; Gromashevsky, V. L.; Larichev, V. P.; Gaidamovich, S. Y.; Vyshemirsky, O. I.; Zhukov, A. N.; Lazorenko, V. V; Salko, V. N.; Kovtunov, A. I.; Galimzyanov, K. M.; Platonov, A. E.; Morozova, T. N.; Knutoretskaya, N. V.; Shishkina, E. O.; Skvortsova, T. M. Emerg. Infect. Dis. 1999, 6, 373-376. (7) Weinberger, M.; Pitlik, S. D.; Gandacu, D.; Lang, R.; Nassar, F.; Ben David, D.; Rubinstein, E.; Izthaki, A.; Mishal, J.; Kitzes, R.; Siegmann-Igra, Y.; Giladi, M.; Pick, N.; Mendelson, E.; Bin, H.; Shohat, T.; Chowers, M. Y. Emerg. Infect. Dis. 2001, 7, 686-691. (8) Tsai, T. F.; Popovici, F.; Cernescu, C.; Campbell, G. I.; Nedeku, N. I. Lancet 1998, 352, 767-771. (9) Weiss, D.; Carr, D.; Kellachan, J.; Tan, C.; Phillips, M.; Bresnitz, E.; Layton, M. Emerg. Infect. Dis. 2001, 7, 654-658. (10) Murgue, B.; Murri, S.; Zientara, S.; Durand, B.; Durand, J. P.; Zeller, H. Emerg. Infect. Dis. 2001, 7, 692-696. (11) Murgue, B.; Zeller, H.; Deubel, V. Curr. Top. Microbiol. Immun. 2001, 267, 196. (12) Nasci, R. S.; Savage, H. M.; White, D. J.; Miller, J. R.; Cropp, B. S.; Godsey, M. S.; Kerst, A. J.; Bennett, P.; Gottfried, K.; Lanciotti, R. S. Emerg. Infect. Dis. 2001, 7, 742-744. (13) Kulasekera, V. L.; Kramer, L.; Nasci, R. S.; Mostashari, F.; Cherry, B.; Trock, S. C.; Glaser, C.; Miller, J. R. Emerg. Infect. Dis. 2001, 7, 722-725. (14) Wong, S. J.; Boyle, R. H.; Demarest, V. L.; Woodmansee, A. N.; Kramer, L. D.; Li, H.; Drebot, M.; Koski, R. A.; Fikrig, E.; Martin, D. A.; Yong, S. P. J. Clin. Microbiol. 2003, 41, 4217-4223. (15) Prince, H. E.; Hogrefe, W. R. Clin. Appl. Immun. Rev. 2005, 5, 45-63. (16) Kuno, G. Adv. Virus Res. 2003, 61, 3-65. (17) Shi, P-Y.; Wong, S. J. Expert Rev. Mol. Diagn. 2003, 3, 733-41. 10.1021/ac0707129 CCC: $37.00
© 2007 American Chemical Society Published on Web 10/23/2007
reactivity for WNV antibodies and additional serum tests are usually recommended.17 In contrast, ELISA and MIA constitute highly sensitive and specific screening tests for the detection of WNV antibodies but require two to three days to complete the assay.19 Moreover, the detection is based on fluorescence measurement that requires a complex, nonminiaturized, and costly confocal system that considerably limits the widespread use of optical readouts and the development of portable devices. In contrast, amperometric detection of antigen-antibody binding events ensures attractive advantages such as its ease of use in turbid samples, portability, low cost, and sensitivity. In particular, the detection of an immunoreaction is commonly carried out via the use of secondary antibodies labeled with enzymes.20 The latter catalyze the production of electroactive species that are amperometrically monitored at the electrode surface, the signal intensity being proportional to the amount of WNV antibody anchored on the transducer surface. With the aim to develop a portable and inexpensive amperometric immunosensor for the determination of WNV antibody, a new phage bearing a sequence of 15 amino acids of the WNV envelope protein was prepared and used as an antigen (bioreceptor) instead of the virus itself. The main advantage lies in the innocuousness of this modified phage toward humans as well as its ease of preparation. Since the transducer is an electrode, phage immobilization can be performed by electrochemical entrapment in polymeric films. The electrogeneration of polymers, indeed, leads to the simple and reproducible formation of films with precise spatial resolution over conductive surfaces and provides an easy control over the polymer thickness. In addition, the quality of the electrogenerated polymer films is characterized by the absence of manufacturing defects and a good chemical and storage stability in aqueous and organic solvents.21,22 Among the different electrochemical procedures of biomolecule entrapment, an original two-step procedure of biosensor construction, based on the electropolymerization properties of amphiphilic pyrrole monomers in their adsorbed state, requires little amounts of biomolecules and leads to a higher density of immobilized biomolecules than conventional approaches.23 This method consists first in the immobilization of monomers and biomolecules together, by adsorption, on the electrode surface. The subsequent electropolymerization step of the adsorbed monomers in an aqueous electrolyte free of biomolecules and monomers induced the physical entrapment of the adsorbed biomolecule in the “in situ” generated polypyrrole films.24 Although this approach was only applied to coenzyme and enzyme entrapments, the procedure was extended to phage immobilization. The present paper reports on the creation of an amperometric immunosensor for the fast detection of a WNV IgG-antibody, using a poly(pyrrole-alkyl ammonium) film for the retention of WNV-phage at the surface of a glassy electrode. The electro(18) Martin, D. A.; Muth, D. A.; Brown, T.; Johnson, A. J.; Karabatsos, N.; Roehrig, J. T. J Clin. Microbiol. 2000, 38, 1823-6. (19) Johnson, A. J.; Martin, D. A.; Karabatsos, N.; Roehrig. J. T. J. Clin. Microbiol. 2000, 38, 1827-1831. (20) Ionescu, R. E.; Cosnier, S.; Herzog, G.; Gorky, K.; Leshem, B.; Herrmann, S.; Marks, R. S. Enzyme Microb. Technol. 2007, 40, 403-408. (21) Cosnier, S. Electroanalysis 2005, 17, 1701-1715. (22) Grennan, K.; Killard, A. J.; Smyth, M. R. Electroanalysis 2005, 17, 13, 601369. (23) Cosnier, S. Electroanalysis 1997, 9, 894-902. (24) Cosnier, S.; Innocent, C. J. Electroanal. Chem. 1992, 328, 361-366.
Figure 1. Structure of pyrrole-alkyl ammonium monomer.
chemical characterizations of the polymer-phage coatings and its optimization have been investigated. In addition, the amperometric analytical performance of the optimum immunosensor configuration was determined. EXPERIMENTAL SECTION Chemicals. Bovine serum albumin (BSA, A-3803), goat antihuman IgG H&L HRP (OEM-Concepts, G5-G10-2), and polyoxyethylenesorbitan monolaurate (Tween 20, P7949) were purchased from Sigma. (11-Pyrrol-1-yldecyl)triethylammonium tetrafluoroborate (Figure 1) was synthesized as previously reported.25 LiClO4 (194711000) was provided by Acros Organics. Phage T7 (wild type) at 5.7 × 109 forming units (pfu)/mL, an SI unit for phage concentration equivalent to colony forming units (cfu)/ mL (in the case of bacteria concentration) was acquired from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). IgG preparations from pooled preselected WNV-positive Israeli donors sera (IVIG-IL; Omr-IgG-am 5% intravenous IgG, lot E09071) and WNV-negative American donors sera (IVIG-US, lot G09402) were a gift from Pr. Orgad Laub of Omrix Biopharmaceuticals (Ness-Ziona, Israel). These sera were collected respectively from Israeli and American donors that came into major blood collecting centers. The commercial IVIG product was prepared from concentrated WNV-positive human sera and is constituted by 95% IgG, for a total protein concentration of 50 mg/mL. According to the company, the WNV-positive IVIG-IL solution would contain between 0.5 and 1% specific IgG toward WNV, leading to a theoretical concentration of anti-WNV IgG in the range 240-480 µg/mL (an average value of 360 µg/mL will be taken for further calculation). IVIG-IL already showed prophylactic and therapeutic efficacy in treating WNV infection in mice26 and was successfully employed in clinical treatment of patients with immunosuppression who had WN fever.27,28 Cloning of Ep15 × 2 in T7 Phage. The sequence of the WNV epitope (Ep15) consists in a sequence of 15 amino acids from the envelope protein (E-protein) of the WNV.29 A double strand DNA corresponding to the peptide GGG-p15-GGG-p15 (p15x2) was synthesized (Danyel Biotech, Israel) with phosphorylated 5′ends and cloned into EcoRI/HindIII restriction sites of the T7Select415-1b vector (T7Select, Novagen). The ligation reaction was performed by assembling in a sterile 0.5 mL tube: 1 µL insert (0.05 pmol), 1 µL T7Select Vector Arms (0.02 pmol), 0.5 µL 10X Ligase Buffer, 0.5 µL 10 mM, 0.5 µL 100 mM (25) Coche-Guerente, L.; Deronzeir, A.; Galland, B.; Labbe, P.; Moutet J.-C.; Reverdy, G. J. Chem. Soc., Chem. Commun. 1991, 86-388. (26) Ben-Nathan, D.; Lustig, S.; Tam, G.; Robinzon, S.; Segal, S.; Rager-Zisman, B. J. Infect. Dis. 2003, 188, 5-12. (27) Hamdan, A.; Green, P.; Mendelson, E.; Kramer, M. R.; Pitlik, S.; Weinberger, M. Transplant. Infect. Dis. 2002, 4, 160-2. (28) Shimoni, Z.; Niven, M. J. S.; Pitlick, S.; Bulvik. S. Emerg. Infect. Dis. 2001, 7, 759-759. (29) Atias, D.; Lobel, L.; Virta, M.; Marks, R. S.; Cullen, D.; Lowe, C. In Handbook of Biosensors and Biochips; Weetall, H. H., Karube, I., Eds.; WileyInterscience: New York. In press.
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dithiothreitol (DTT), 0.5 µL sterile water and 1 µL (0.4-0.6 Weiss units) T4 DNA Ligase. The success of the ligation was checked by PCR. Briefly, 0.5 µL of the ligation mixture was diluted in 1.5 µL of distilled H2O and the following reagents were pooled together: 40 µL distilled H2O, 2 µL diluted ligation mixture, 5 µL 10X NovaTaq Buffer with MgCl2, 1 µL T7SelectUp primer (5 pmol), 1 µL T7SelectDown primer (5 pmol), 1 µL dNTP mix (10 mM each) and 1 µL NovaTaq DNA polymerase (1.25U). The PCR was performed during 35 cycles in a thermal cycler with the following temperatur: 94°C for 50 s, 50°C for 1 min, 72°C for 1 min and a final extension at 76°C for 6 min. The PCR reaction was then mixed with 5 µL loading buffer 10X and 10 µL of the final solution was allowed to migrate in a 3% (w/v) agarose gel (100 mV, 2 hours). In vitro packaging of the resulting circular phagemide was done by mixing 4 µL of the ligation mixture with 20 µL of packaging extract for 2h at room temperature. The reaction was stopped by adding 270 µL of sterile LB and the packaging efficiency was calculated by phage titration using plaque assay. The OriginalB E.Coli strain (Novagen) was inoculated in M9LB and incubated with shaking at 37°C to an OD600=1. Several dilutions of packaging mixture were prepared (10-3, 10-4, 10-5, 10-6 and 10-7), 100 µL of each solution were added separately to 250 µL of host cells followed by 3 mL of top-agarose before spreading the whole mixture on a LB-kanamycin plate. After an overnight incubation at room temperature, the plaques were counted on each plate. Amplification and Purification of T7-Ep15 × 2 Phages. A volume of 50 mL of LB-kanamycin was inoculated with a single colony of OrigamiB of a freshly streaked plate and incubated overnight at 37 °C with agitation. Then, 5 mL of this solution was introduced in 500 mL of LB-kanamycin until it reached an OD600 ) 1. The bacterial broth was then infected with 0.001 MOI of phage (1000 cells/pfu). The solution was then incubated in a rotary shaker at 37 °C until cell lysis was observed. The lysate was clarified by centrifugation at 8000g during 10 min, and the supernatant containing the p15 × 2-T7 phages was tittered by plaque assay. An amount of 20 g of PEG-8000 was added to the phages contained in solution. The mixture was gently mixed overnight at 4 °C and then centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was decanted and the PEG pellet resuspended in 1.5 mL of a solution of 1 M NaCl, 10 mM Tris-HCl pH 8, and 1 mM EDTA. Finally, the suspension was centrifuged for 10 min at 10 000 rpm to remove the PEG, and thereafter, the supernatant was transferred into a sterile 1.5 mL tube for further use. Advantages and Specificity of the T7-Ep15 × 2 Phage. PRNT serves as the gold standard for measuring WNV-specific antibodies due to its exquisite specificity, but most of the data presented this past decade are based on results from ELISA. The first ELISA developed for the detection of IgG was using native WNV antigens,30 but progress in recombinant technologies offered the opportunities to produce WNV antigens in a more convenient way and without working with potentially infectious material.31 Nowadays, the main recombinant WNV antigen is based on the coexpression of the protein pre-M and E in Cos-1 cells or (30) Feinstein, S.; Akov, Y.; Lachmi, B. E.; Lehrer, S.; Rannon, L.; Katz, D. J. Med. Virol. 1985, 17, 63-72. (31) Prince, H. E.; Lape-Nixon, M. Clin. Diagn. Lab. Immunol. 2005, 12, 231233.
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Drosophila S-2 cells32 and is employed in the current commercial kit from Focus technologies33 and Abbot Laboratories.34 However, this recombinant antigen exhibits natural cross-reactivity with antibodies induced by other Flavivirus infections (up to 95% with dengue secondary infection) and thus necessitates additional assays based on more specific antigen in order to determine the origin of the infection.35 Recently, a T7 phage-displayed peptide was used in an anti-WNV IgG ELISA rationale and showed an improvement in the assay specificity. Moreover, this antigen is easy to produce, in a short period of time and without any safety issues, since it is not derived from the pathogen itself. In particular, a screen of a panel of 66 human samples composed of denguepositive, WNV-positive and negative sera by the T7-Ep15 × 2 phage, and comparison with a commercial kit revealed a 100% specificity whereas the dengue-positive sera were identified as WNV-positive in the commercial kit.36 Apparatus. Cyclic voltametric experiments and electropolymerization were performed with an EG&G PARC, 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 with a conventional three-electrode cell (Metrohm). The amperometric measurements were performed using a Tacussel PRG-DL potentiostat in conjuction with a thermostated electrochemical cell at 20 °C, the rotating rate of the biosensor being 300 rpm. The working electrodes were glassy carbon disks (i.d. ) 5 mm) systematically polished with 2 µm diamond paste (Mecaprex Press PM). A saturated Ag-AgCl-KCl electrode (Ag/AgCl) was used as a reference electrode, and a Pt wire was used as a counter electrode. Immunosensor Preparation. The synthesized monomer (pyrrole-alkyl ammonium) (6 mM, oily suspension) was dispersed in pure distilled water by sonication for 3 h. The working electrodes were modified at room temperature by spreading over their surface a mixture composed of 20 µL of monomer solution and various amounts of T7-Ep15 × 2 phage solution (10, 20, or 30 µL). Water was evaporated to dryness under vacuum (15 min), yielding adsorbed coatings. The resulting electrodes were transferred into a cell containing 0.1 M LiClO4 aqueous solution, and the polymerization of the adsorbed monomers was carried out at room temperature using two procedures: controlled potential electrolysis (CPE) at 0.9 V until the current decreased to zero or repetitive potential scanning (RPS) at 0.1 V‚s-1, over the range from 0.0 to 0.9 V versus Ag/AgCl for 10 min. After phage immobilization, a saturation of the potential sites of protein adsorption with BSA was performed to reduce overall background and increase the test sensitivity. The blocking solution consisted of in phosphate-buffered saline (0.1 M PBS, pH 7.2) containing (32) Davis, B. S.; Chang, G. J.; Cropp, B.; Roehrig, J. T.; Martin, D. A.; Mitchell, C. J.; Bowen, R.; Bunning, M. L. J. Virol. 2001, 75, 4040-4047. (33) Hogrefe, W. R.; Moore, R.; Lape-Nixon, M.; Wagner, M.; Prince, H. E. Clin. Microbiol. 2004, 42, 4641-4648. (34) Muerhoff, A. S.; Dawson, G. J.; Dille, B.; Gutierrez, R.; Leary, T. P.; Gupta, M. C.; Kyrk, C. R.; Kapoor, H.; Clark, P.; Schochetman, G.; Desai, S. M. Clin. Diagn. Lab. Immunol. 2004, 11, 651-657. (35) Wong, S. J.; Boyle, R. H.; Demarest, V. L.; Woodmansee, A. N.; Kramer, L. D.; Li, H.; Drebot, M.; Koski, R. A.; Fikrig, E.; Martin, D. A.; Shi, P. Y., J. Clin. Microbiol. 2003, 41, 4217-4223. (36) Herrmann, S.; Leshem, B.; Lobel, L.; Bin, H.; Mendelson, E.; Ben-Nathan, D.; Dussart, P.; Porgador, A.; Rager-Zisman, B.; Marks, R. S. J. Med. Virol. 2007, 141, 133-140.
5% (w/v) BSA and was prepared daily. The blocking solution (20 µL) was deposited on the poly(pyrrole-alkyl ammonium) polymer containing phage particles for 1 h at room temperature. Then, the immunosensors were rinsed with PBS (pH 7.2) for 5 min before being incubated with 20 µL of several anti-WNV-IgG solutions at dilutions ranging from 10 to 107. The WNV-antibody was diluted with 1% (w/v) BSA/PBST (PBS containing 0.05% (v/ v) Tween-20, PBST) and kept at 4 °C. The resulting modified electrodes were then rinsed and washed once with PBST for 10 min and then three times for 3 min each. Subsequently, the electrodes were incubated with 20 µL of a solution containing the goat anti-human IgG peroxidase-labeled antibodies (102) for 20 min, then rinsed, and washed once with PBST for 10 min and then three times for 3 min before performing the amperometric measurements. RESULTS AND DISCUSSION Elaboration and Evaluation of Polymer-Phage Electrodes. The electrochemical behavior of adsorbed coatings of pyrrole-alkyl ammonium alone or mixed with phage was investigated by cyclic voltammetry at a glassy carbon electrode surface in aqueous solution containing 0.1 M LiClO4 as a supporting electrolyte. The repetitive scanning of the electrode potential between 0 and 0.9 V induces the appearance and the growth of a quasi-reversible peak system, located ∼0.5 V (Figure 2). This evolution indicates the formation and growth of a polypyrrole film on the glassy carbon surface for all the coatings. The effect of phage on the electropolymerization capabilities of the adsorbed pyrrole derivative was examined by varying the amount of phage adsorbed on the electrode surface, the monomer amount being constant. It clearly appears that the increase in deposited amount of T7-Ep15 × 2 phages (10, 20, or 30 µL) leads to a decrease in polymer formation. The resulting electrodes were transferred into a phosphate buffer solution (0.1 M PBS, pH 7.2), and the polypyrrole electroactivity was characterized by cyclic voltammetry. The apparent surface coverage (Γ) of poly(pyrrole-alkyl ammonium) films was determined from the charge recorded under the polypyrrole oxidation and reduction waves, assuming that the electro-oxidation of the polypyrrole chain corresponds to one electron for three polymerized pyrrole groups.37 Table 1 summarizes the different film coverages showing a monotone decrease in the electrogenerated amount of poly(pyrrole-alkyl ammonium) from 1.1 × 10-7 to 0.8 × 10-8 mol cm-2 with the increase in deposited phage amount. As previously reported for the entrapment of enzymes, this phenomenon may indicate a diffusion of a part of the adsorbed monomer and phage to the bulk solution during the electropolymerization process, this effect increasing with the increase in the amount of adsorbed phage.38 Owing to the hydrophilic character of the phage, the increase in phage loading should counterbalance the adsorption properties of the monomer due to its hydrophobic tail. The electrochemical polymerization of the adsorbed pyrrole monomer can also be performed by CPE at 0.9 V. As a consequence, the effect of the polymerization procedure, CPE or RPS, and the deposited amount of T7-Ep15 × 2 phage on the recognition properties of the resulting electrodes were examined by amperometric measure(37) Cosnier, S.; Innocent, C. Bioelectrochem. Bioenerg. 1983, 31, 147-160. (38) Cosnier, S.; Fombon, J. J.; Labbe´, P.; Limosin, D. Sens. Actuators, B 1999, 59, 134-139.
ments. For this purpose, the modified electrodes were successively incubated with WNV-antibody at the saturation state (1/10 dilution) and anti-human IgG HRP-labeled IgG peroxidase-labeled antibodies. After several washing steps, the resulting electrodes were transferred into a phosphate buffer solution containing hydroquinone (2 mM) and then H2O2 (0.1 mM) potentiostated at 0 V was added to the solution. The HRP molecules immobilized by immunoreaction catalyze, in the presence of H2O2, the oxidation of hydroquinone into quinone. The latter was then electrochemically reduced at the underlying electrode surface. The presence of immobilized phages, their density, and their accessibility for the immunoreaction with the WNV-antibody were shown to be directly reflected by the current intensity of the quinone reduction. The maximum current responses (Imax) corresponding to the three amounts of phage particles (10, 20, and 30 µL) immobilized by CPE or RPS are summarized in Table 2. The comparison of six phage-modified electrodes clearly indicates that CPE provides a more efficient WNV immunosensor than RPS. This may be ascribed to a more rapid polymer growth by CPE that reduces the phenomenon of phage release into the bulk solution. In addition, it appears that the highest current response (240 nA) was recorded for the lower deposited amount of phage, namely, 10 µL. A similar evolution of the current response with the phage amount was in fact recorded for both electropolymerization procedures. This may be ascribed to a phenomenon of phage release that corroborates with the preceding monomer release observed during the polymerization process. Consequently, the polymer coating obtained by CPE and containing 10 µL of T7Ep15 × 2 phage was used for further experiments as the optimum immunosensor configuration. Since the functioning principle of this WNV immunosensor is based on an amperometric transduction at the underlying electrode surface, the influence of phage incorporation on the permeability of the polypyrrole film was investigated by rotating disk electrode (RDE) experiments. The latter were carried out at different rotation rates in the presence of hydroquinone (2 mM) in 0.1 M PBS, (pH 7.2) for electrodes modified by a polypyrrole film or a polypyrrole-phage coating. Figure 4 A shows, for instance, the rotating disk voltammograms obtained with entrapped phages. The permeability Pm of the coatings was estimated through the eqs 1-4 reported by Gough and Leypoldt39 that described the variation of steady-state limiting current ilim with the mass transport for a rotating disk electrode coated with an electroinactive membrane.
1/ilim ) 1/is + 1/im
(1)
is (Levich current) represents the current flow under the same conditions, in the absence of a membrane, and is therefore characteristic of the diffusion on the substrate in the bulk solution (eq 2). im represents the diffusion of the substrate in the membrane and depends on the product of the partition equilibrium (39) Gough, A.; Leypold, J. K. Anal. Chem. 1979, 51, 439-443.
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Table 1. Apparent Surface Coverage of the Polypyrrole Film Electropolymerized on the Glassy Carbon Electrodes in the Presence of Different Amounts of Phages phage amount (µL)
Qintegrated × 10-4 (C)
Γ (mol cm-2)
0 10 20 30
7.03 6.13 5.36 4.49
1.1 × 10-7 0.9 × 10-7 0.8 × 10-7 0.8 × 10-8
Table 2. Influence of Different Amounts of T7-Ep15 × 2 Phages Entrapped in Poly(pyrrole-alkyl ammonium) Polymer Generated by CPE or RPS on the Amperometric Immunosensor Response to WNV Antibody at the Saturation State (1/10 Dilution)a amount of T7-Ep15 × 2 phages (µL)
Imax (CPE) (nA) Imax (RPS) (nA)
10
20
30
240 40
140 15
70 10
a After labeling with HRP secondary antibody in the presence of H2O2 (0.1 mM) and hydroquinone (2 mM), the modified electrode being potentiostated at 0 V.
constant, the diffusion constant of the substrate in the membrane, and the film thickness (eq 3).
Figure 2. Cyclic voltammograms of the adsorbed pyrrole-alkyl ammonium monomer (120 nmol) during the polymerization process (I) and of the resulting polymer after 33 scans (II). (A) Monomer alone, (B) monomer mixed with 10 µL of phage, (C) monomer mixed with 20 µL of phage, and (D) monomer mixed with 30 µL of phage The electropolymerization process was carried out by cycling the potential between 0 and 0.9 V vs Ag/AgCl in 0.1M LiClO4 aqueous solution (sweep rate 100 mV‚s-1) for 10 min while the polymer characterization was performed in the potential range 0-0.9 V. 8666
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is ) 0.62nFSC°Ds2/3ν-1/6ω1/2
(2)
im ) nFSC°KDm/δ ) nFSC°Pm
(3)
The terms Ds and Dm are the diffusion coefficients for the substrate in the bulk solution and in the membrane, respectively, ν is the kinematic viscosity of the solution, ω the rotation rate of the RDE, δ the thickness of the membrane, and K the partition equilibrium constant of the substrate between solution and membrane, S the electrode surface, n the number of exchanged electrons, and C° the substrate concentration. Only the first term of eq 1 is dependent upon the rotation rate of the RDE while the second reflects the permeability Pm defined as Pm ) KDm/δ cm s-1. As a consequence, experimental data from RDE voltammograms were displayed in the form of Koutecky-Levich plots. A plot of 1/ilim versus 1/ω1/2 thus exhibits a linear behavior with the same slope that obtained for a bare electrode. The value of the positive intercept represents the permeability Pm of the membranes (Figure 4). The permeability values for the poly(pyrrolealkyl ammonium) film and the polymer containing T7-Ep15 × 2 phages (10 µL) are similar, namely, 4.2 × 10-2 and 2.5 × 10-2 cm s-1, respectively. In addition, these permeability values are similar to those previously reported for the same polymer with and without entrapped glucose oxidase (3 × 10-2 and 5 × 10-2 cm s-1, respectively).40 These data seem to indicate that the phenomenon of phage entrapment, for the smallest phage amount (10 µL), did not affect the film permeability and hence its structure. (40) Cosnier, S.; Mousty, C.; Gondran, C.; Lepellec, A. Mater. Sci. Eng. C 2006, 26, 387-393.
Figure 3. Schematic representation of the functioning principle of the amperometric immunosensor for the detection of WNV antibody through HRP-conjugated secondary antibody that catalyzed the formation of electroactive quinone species (Q) in presence of hydroquinone (H2Q) and H2O2 as substrates.
Immunosensor Performance for the Detection of WNV Antibody. In order to investigate the analytical capabilities of the polypyrrole-phage coating for the amperometric detection of the WNV antibody, the phage electrodes were incubated with WNV antibody solutions diluted by a factor ranging from 10 to 107. As previously described, after incubation with anti-human IgG HRPlabeled, the current responses of the phage electrodes toward enzymatic generated quinone were recorded providing a calibration curve for WNV antibody. A maximum current response was observed for undiluted WNV antibody solution, small current differences being noticed between this solution and that diluted by a factor 10. The responses, then, were proportional to the antibody concentration (Figure 5). An amperometric immunosensor response of 40 and 22 nA was recorded for WNV-antibody titer of 1:107 and 1:108, respectively, the noise being 13 nA. The detection limit, based on a signal-to-noise ratio of 3, corresponds thus to a dilution titer of 1:107 (36 pg/mL). To validate the immunosensor response, the phage electrode was incubated with a concentrated WNV-negative IgG solution (dilution titer 1:10). The resulting weak intensity of the amperometric signal (8 nA) demonstrates the specific immune origin of the response and corroborates the value of the detection limit. This amperometric approach is more sensitive than other techniques such as colorimetric ELISA, chemiluminescent ELISA, and an optical fiber immunoassay that led to detection limits of WNV-antibody titer of 1:105 (3.6 ng/mL), 1:5 × 105 (720 pg/mL), and 1:106 (360 pg/ mL), respectively20,41. Moreover, it should be noted that the current response of the various immunosensors reached a steady-state value in a very short time, 5-20 s, illustrating the good permeability of the polymer-phage coating. It should be noted that the total time assay, 50 min, including successive incubation and (41) Herrmann, S.; Leshem, B.; Landes, S.; Rager-Zisman, B.; Marks, R. S. Talanta 2005, 66, 6-14.
washing steps, immunosensor potentiostating, and signal recording, remains markedly more rapid than those required by other WNV antibody assays. The reproducibility of the analytical performance of the immunosensor and hence the reproducibility of the recognition event between T7-Ep15 × 2 phages entrapped into a poly(pyrrole-alkyl ammonium) matrix and WNV-antibody as well as the reproducible elaboration of the sensor were examined. For this purpose, four phage electrodes were incubated with WNVantibody at the saturation state (1:10) and amperometrically checked. It appears that the four immunosensors exhibited the same current response value (240 nA, RSD ) 2.3%), demonstrating the validity of this approach. The storage stability of the immunosensors stored dry at 4 °C in the range of 10-107 was also examined by checking periodically their amperometric response to the mixture of hydroquinone and hydrogen peroxide. The immunosensor sensitivity remained identical after 6 days and then dropped by 85% after 8 days. This indicates a relatively good stability of the entrapped phage, the loss of activity being ascribable to the instability of the enzyme, the phage, or the immunological binding. The possibility to reuse the immunosensor for multiple WNV antibody detection was examined through a chemical treatment of the sensor surface. After successive incubations with WNV antibody and anti-human IgG, HRP-labeled at saturating conditions, the resulting electrode was soaked in 0.4 M glycine hydrochloride (pH 2.4) for 30 min (rotating rate of the immunosensor being 600 rpm), rinsed extensively with 0.1 M PBS (pH 7.2), and then, its amperometric response was checked. It appears that the glycine treatment totally eliminates the immunosensor response, indicating the efficient decomposition of the immunosandwich. Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
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Figure 4. (A) Rotating-disk electrode voltammograms of a glassy carbon electrode (diameter 5 mm) modified by a poly(pyrrole-alkyl ammonium) containing with T7-Ep15 × 2 phages (10 µL) in presence of a 2 mM solution of hydroquinone and 0.1 M PBS (pH 7.2): (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 (a) an uncoated electrode, (b) a polymer-coated electrode, and (c) a polymer containing T7-Ep15 × 2 phage (10 µL). The test solution was 2 mM hydroquinone in 0.1 M PBS solution (pH 7.2).
Figure 5. Amperometric current response of the immunosensor to the hydroquinone/H2O2 system as a function of WNV-antibody dilution in the range 1:10-1:107. Applied potential 0 V vs SCE.
The incubation of the same phage electrode with WNV antibody and IgG HRP-labeled, allows us to restore the initial electroenzymatic activity. Six successive cycles of WNV antibody detection at the saturation state (1:10 dilution) and electrode regeneration were carried out leading to a small decrease (20%) 8668 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
in the initial current response of the modified electrode. These results demonstrate the reversibility of the antibody immobilization onto the phage electrode. In addition, it seems that the acid treatment for the electrode surface regeneration does not affect substantially the T7-Ep15 × 2 phages entrapped into a poly(pyrrole-alkyl ammonium) film at the electrode surface. Specificity of the Amperometric Immunosensor. To ensure that the current response recorded with the immunosensor reflects the specific immunoreaction between the WNV antibody and the phage entrapped in the polypyrrole skeleton, control experiments were performed. For this purpose, four different modified electrodes were designed with the following configurations: (1) only a poly(pyrrole-alkyl ammonium) film, (2) a poly(pyrrole-alkyl ammonium) film containing T7-Ep15 × 2 phage (10 µL), (3) same as (2) but incubated with HRP-labeled antibody, and (4) a poly(pyrrole-alkyl ammonium) film containing wild-type T7 phage (10 µL) incubated successively with WNV-antibody at saturation level (1:10) and HRP-labeled antibody. It should be noted that the configurations 3 and 4 were exposed to a BSA blocking treatment and PBST washing steps identical to those applied to the phage electrodes used for WNV antibody detection. These modified electrodes were then transferred to 0.1 M PBS and potenstiostated at 0 V to record their amperometric response to a mixture of H2O2 and hydroquinone. For controls 1, 2, and 3, no cathodic current was observed. This indicates the absence of a catalytic phenomenon of H2O2 reduction by the functionalized polypyrrole film (1), that the phage exhibited no peroxidase activity (2), and the absence of an immunoreaction between T7Ep15 × 2 phage and anti-human IgG HRP-labeled (3). Configuration 4 leads to a very weak amperometric signal, namely, 4 nA, reflecting the absence of molecular recognition between WNV antibody and regular T7 phage. These control experiments demonstrate unambiguously that the amperometric responses are only triggered by the specific immunoreaction between the modified T7-Ep15 × 2 phage and the WNV antibody. CONCLUSIONS In this report, we have demonstrated that polypyrrole films can be efficiently functionalized by WNV phage with the retention of its molecular recognition properties. The designed immunosensor allows the amperometric detection of the WNV antibody with an extremely sensitive detection titer (1:107). Furthermore, the regeneration of the sensing layer was successfully reached using acidic conditions. ACKNOWLEDGMENT The sequence of the putative WNV epitope (p15) was generously given by the Pr. Bracha Rager-Zisman and Angel Porgador (Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel). EU is thanked for funding under the 6th Framework contract: NMP-A-CT-2003-505485-1 as well as the North Atlantic Treaty Organization (NATO) for the Collaborative Linkage Grant Award 981086.
Received for review April 11, 2007. Accepted September 1, 2007. AC0707129
