APPENDIX II-BJ: Searching for an Inexpensive Test for WNV 1: Ionescu, et al, “Amperometric Immunosensor for the Detection of Anti-West Nile Virus IgG Using a Photoactive Copolymer,” Enzyme and Macrobial Technology, volume 40, issue 3, 5 February 2007, pages 403-408.
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Copyright © 2006 Elsevier Inc. All rights reserved.
Amperometric immunosensor for the detection of anti-West
Rodica E. Ionescua, Serge Cosniera, , , Gregoire
Gorgya, Boaz Leshemb, Sebastien
Herrmannb and Robert S. Marksb, c, d
aLaboratoire d’Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moléculaire de Grenoble, FR CNRS 2607, Université Joseph Fourier Grenoble I, BP 53, 38041 Grenoble Cedex 9, France
bDepartment of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, 84105 Beer-Sheva Israel
cIlse Katz Center for Meso- and Nano-scale Science and Technology, Ben-Gurion University, P.O. Box 653, 84105 Beer-Sheva, Israel
dThe National Institute for Biotechnology in the Negev, P.O. Box 653, 84105 Beer-Sheva, Israel
Available online 10 July 2006.
A polymeric film consisting of two distinct monomers such as pyrrole–benzophenone and tris(bipyridine pyrrole) ruthenium (II) units was electrogenerated and used for the photochemical grafting of T7 bacteriophages displaying a specific West Nile virus (WNV) epitope. The resulting electrodes were applied to the amperometric detection of WNV antibody at 0 V in aqueous electrolyte via secondary peroxidase-labeled antibody, hydrogen peroxide and hydroquinone as the enzyme substrate. A calibration curve based on different WNV–antibody dilutions ranging from 10 to 106 was created with a relatively short response time of 30 s after substrate injection.
WNV infection has typical clinical symptoms and positive results can be obtained with specific laboratory tests . Serum or cerebrospinal fluids are either tested for the presence of IgM and IgG antibodies by ELISA immunoassays or for viral RNA detection with RT-PCR in reference laboratories. Today, there are six major types of assays for the detection of WNV antibodies such as complement fixation, hemagglutination, the plaque reduction neutralization test, immuno-fluorescence assays, enzyme-linked immunosorbent assays (ELISA) and microsphere immunoassays . More progress has been recently reported by creating a sensitive ELISA–optical fiber methodology using immobilization of inactivated virions via argon-phase silanization which provided a lower detection limit of WNV antibodies such as an analyte dilution by 106 .
With the aim to develop a portable and inexpensive immunosensor for the determination of WNV antibody, our work has also focused on an amperometric transduction of the immunoreaction with the WNV phage-displayed epitope immobilized on an electrode surface. Recently, biomolecule immobilization onto electropolymerized photoactivable films was reported, combining elegantly the advantages of the photografting process to those of the electrochemical addressing . In particular, the immobilization of biomolecules by light is topically addressable and easily applicable to a wide variety of biomolecules. The electropolymerization of a pyrrole–benzophenone derivative provided thus the first example of a polymer film allowing upon irradiation the reagentless covalent grafting of proteins and enzymes ,  and . The generation of the benzophenone excited triplet state, requires a low activation energy and is chemically stable. Electropolymerized poly(pyrrole–benzophenone) films provide similar photografting properties as benzophenone solutions do, leading to the anchoring of a protein monolayer.
However, the high hydrophobic character of these polymers leads in aqueous media to non-permeable films. This constitutes a major drawback for the development of immunosensors based on an amperometric transduction. The latter requires indeed the permeation of electroactive compounds through the polymer to reach the underlying electrode surface where an oxidation or reduction process must take place.
Taking this into consideration, we report here the design of copolymeric films electrogenerated from two pyrrolic monomers: a photoactivable pyrrole–benzophenone (monomer 1) and a cationic tris (bipyridine–pyrrole) ruthenium complex (monomer 2) (Fig. 1).
Fig. 1. Structure of monomers 1 and 2.
Thanks to the presence of three pyrrole groups, monomer 2 can confer, beside a polycationic character, a rigid cross-linked structure to the resulting copolymer. In contrast to the linear skeleton of the poly(pyrrole–benzophenone) films reported until now, this effect may be exploited to imprint an expanded structure to the polymeric film for facilitating the permeation of redox species.
The immobilization of a WNV phage-displayed epitope onto photoactivable copolymers able to generate under irradiation several covalent bindings between polymerized benzophenone groups and a phage entity was then investigated. In addition, the potentialities of the resulting phage electrode were examined for the amperometric immunosensing of WNV antibody.
Bovine serum albumin (BSA, A-3803), and polyoxyethylenesorbitan monolaurate (Tween 20, P7949) were purchased from Sigma while goat anti-human IgG H&L HRP are from OEM-Concepts (G5-G10-2).
IgG preparations from pooled Israeli donors (IVIG-IL; Omr-IgG-am 5% intravenous IgG, lot E09071) and American donors (IVIG-US, lot G09402) containing 50 mg/ml IgG were a gift from Pr. Orgad Laub of Omrix Biopharmaceuticals (Ness-Ziona, Israel). IVIG-IL already showed prophylactic and therapeutic efficacy in treating WNV infection in mice  and was successfully employed in clinical treatment of patients with immunosuppression who had WN fever  and .
2.2. Cloning of p15x2 in T7 phage
The sequence of the WNV epitope
Ep15 consists in a sequence of 15 amino acids from the domain III of the WNV envelope
protein. A double-strand DNA corresponding to the peptide GGG-p15-GGG-p15
(Ep15x2) was synthesized (
2.3. Amplification and purification of p15x2-T7 phages
A volume of 50 ml LB-kanamycin was inoculated with a single colony of OrigamiB and incubated overnight at 37 °C. Then, 5 ml was introduced in 500 ml fresh LB-kanamycin. At OD600 = 1, the bacteria were infected with 0.001 MOI of phage. The solution was incubated in a rotary shaker at 37 °C until cell lysis was observed. The lysate was then clarified by centrifugation at 8000 × g during 10 min and the supernatant containing the Ep15x2-T7 phages was mixed with 50 g PEG-8000. 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 1 M NaCl, 10 mM Tris–HCl pH8 and 1 mM EDTA. The suspension was finally centrifuged 10 min at 10,000 rpm to remove the PEG and the supernatant transferred in a sterile 1.5 ml tube for further use.
studies were undertaken in a standard three-electrode cell using an EG&G
173 potentiostat/galvanostat and an EG&G Parc Model 175 Universal Programmer (from Princeton Applied
2.5. Modification of the electrodes
The electropolymerization of monomers or monomer mixtures (2 mM) was carried out in organic solution (CH3CN + 0.1 M TBAP) by controlled potential electrolysis at +0.85 V versus Ag | Ag+ reference electrode. The polymerization process was stopped when an anodic charge density of 2.5 mC cm−2 was reached. The resulting modified electrodes were characterized in CH3CN + 0.1 M TBAP free of monomer and then transferred in CH3CN containing 0.1 M LiClO4. Cation exchange was performed by cycling the resulting modified electrode between −1.0 and +1.3 V. The upper limit of the potential scan region was set at +1.3 V to over-oxidize the polypyrrole skeleton and hence to suppress the electrochemical signal of the polypyrrole electroactivity.
2.6. Immobilization of bacteriophages
A phage aqueous solution (20 μL) was deposited on the electrode modified by copolymeric films and left to evaporate. The resulting electrodes were then irradiated under Ar atmosphere at λ = 355 nm for 5 min. The irradiation of modified electrodes covered by adsorbed phages was performed through an optical fiber using a mercury lamp (medium pressure, 500 W) with a 68910 Arc lamp power supply, both from Oriel Instruments. Filters from Oriel instruments are used to select the wavelength at 355 nm and to block IR rays, preventing overheating of the optical fiber and electrode surface.
2.7. WNV-immunosensor preparation
The polymer-phage-modified glassy carbon electrodes were then used in the creation of a series of WNV-immunosensors. To fabricate highly specific immunosensors, a blocking step using phosphate-buffered saline (0.1 M PBS, pH 7.2) containing 5% (w/v) bovine serum albumin (BSA) was performed. This blocking solution was prepared daily. The WNV–antibody was diluted with 1% (w/v) BSA/PBST (PBS containing 0.05% (v/v) Tween-20), which was kept at 4 °C for 1 week.
The concrete steps for a WNV-immunosensor fabrication are: deposition of 20 μl of the blocking solution onto the surface of a polymer-phage modified glassy carbon electrode which is left to react for 1 h at room temperature. Then, the electrodes are rinsed with PBS (pH 7.2) for 5 min before adding 20 μl of several analyte (anti-WNV-IgG) dilution ranging from 10 to 106. The modified glassy carbon electrodes were then rinsed and washed once with PBST for 10 min and then thrice for 3 min each. Subsequently, the electrodes were incubated with 20 μl of a solution containing the goat anti-mouse IgG peroxidase-labeled antibodies (102) for 20 min and then rinsed and washed once with PBST for 10 min and then thrice for 3 min before performing the amperometric measurements.
3.1. Electrochemical polymerization and characterization of copolymer films poly(1–2)
After transfer in a monomer-free electrolyte solution, electrodes modified by a pure poly2 film exhibited three successive reversible couples corresponding to the one-electron reduction of each bipyridine ligand at −1.68, −1.90, and −2.20 V, respectively. It was previously demonstrated that poly1 films presented in the negative region, only one redox system at −1.85 V, attributed to the one-electron reduction of the polymerized benzophenone groups . Poly(1–2) films were electrogenerated from acetonitrile solutions containing various mixtures of monomers (2 mM), namely 1–1, 3–1 and 6–1 ratios of monomers 1 and 2 (1/2). Fig. 2 presents the electrochemical characteristics of the three poly(1–2) films, in CH3CN + 0.1 M TBAP. For an equimolar mixture of both monomers, redox responses of the benzophenone groups and bipyridine ligands overlay in the negative potential region. As expected, the current intensity of the redox system at −1.83 V, ascribed to the polymerized benzophenone, increases with the increase in monomer 1 proportion while the three successive reversible peak systems due to the ruthenium complex tend to disappear. The polymerization process was carried out in the presence of a bulky electrolyte cation (TBA+) to create an expanded polymeric structure. The modified electrodes were then transferred into CH3CN + 0.1 M LiClO4, to exchange the incorporated TBA+ by Li+ cations. In addition, the potential applied to these electrodes was repeatedly cycled between −1.0 and +1.3 V. This induces, via the Ru (II/III) transition, an electrolyte permeation through the coating to maintain its electro-neutrality. As a consequence, the bulky and hydrophobic TBA+ were replaced by the smaller and hydrophilic Li+ cations within the films. Since the Ru complex contains 3D pre-oriented pyrrole groups through its three bipyridine ligands, its presence in the copolymers led to a cross-linked structure that should retain the imprinting effect of TBA+ . The replacement of TBA+ by smaller Li+ thus may provide cavities within the cross-linked polymeric structure that would enhance the permeability of the polypyrrole films.
In order to compare the permeability of the different polymeric films, the permeation of a redox mediator (ferrocene (Fc)) and its water-soluble derivative (Fc(COOH)2) at poly(1–2) electrodes was investigated by cyclic voltammetry in both organic and aqueous media. Fig. 3 shows the cyclic voltammograms of Fc (2 mM) in CH3CN + 0.1 M LiClO4 recorded at poly(1–2) electrodes elaborated with 1–2 ratios of 1–1, 3–1 and 6–1. No appreciable change in current intensity of the peak system of ferrocene (2 mM) appeared between copolymers based on ratios 3–1 and 6–1 while a slightly more intense and reversible redox couple was observed for the equimolar ratio. This reflects a better permeability that may be attributed to a more efficient imprinted effect with increasing 2 proportion in the copolymer. Nevertheless, these similar permeabilities towards Fc were ascribed to a polymer swelling in organic solution. A similar experiment carried out with Fc(COOH)2 (2 mM in a 0.1 M LiClO4 aqueous solution) as the electroactive probe showed that the Fc(COOH)2 oxidation at the underlying glassy carbon electrode was completely suppressed by the presence of copolymers generated with an excess of 1. It appears that the high proportion of 1 in the poly(1–2) films prevented the formation of a rigid polymeric network leading thus to a polymer collapse in water that blocks the Fc(COOH)2 permeation. In contrast, the cyclic voltammogram recorded at the electrode covered by the equimolar copolymer clearly exhibits the reversible one-electron oxidation of the redox mediator at E1/2 = 0.49 V followed by a reversible peak system at E1/2 = 0.95 V due to the one-electron oxidation of the polymerized ruthenium complex (Fig. 4). This unambiguously indicates the beneficial effect on the film permeability brought by the electrolyte imprint. Consequently, only the photografting properties of poly(1–2) films made from an equimolar mixture of monomers were examined.
After the adsorption of phages onto poly(1–2) films, the latter were covalently linked through the light activation of the polymerized benzophenone groups at λ = 355nm. In order to investigate the result of the photochemical reaction, the modified electrodes were incubated with different samples of the WNV antibody, the dilution ranging from 10 to 106. Then, the resulting electrodes were incubated with a secondary antibody labeled with horseradish peroxidase acting as the marker for the WNV antibody. The recognition of the WNV antibody bound to the phage, conferred thus a peroxidase activity to the modified electrode. Subsequently, all the WNV-immunosensors were soaked into 0.1 M PBS (20 ml, pH 7.2) containing a peroxidase substrate: hydroquinone (2 mM) and potentiostated at 0 V. After the injection of hydrogen peroxide (2 mM), the amperometric signal corresponding to the reduction of the enzymatically generated quinone was recorded as a function of the WNV antibody dilution (Fig. 5). It appears that stable responses appeared with a fast response time (30 s) illustrating the copolymer permeability. The resulting maximum current values were then used for elaborating a calibration curve for the WNV antibody (Fig. 6). Each point from the calibration curve represents an average of three different immunosensor responses. A current decrease was observed for antibody titers ranging from to 1:106 that constitutes a very sensitive detection limit for WNV antibody. This reflects the efficient covalent photochemical binding of phages onto the polypyrrolic film without steric hindrance of subsequent immunoreactions.
Fig. 5. Functioning principle of the amperometric WNV-immunosensor.
Fig. 6. Calibration curve for WNV antibody obtained at poly(1–2) films (ratio 1–1) via an electro-enzymatic immunoassay. Applied potential 0 V in 0.1 M PBS (20 ml, pH 7.2).
demonstrated herein the potentialities of an electrogenerated
polypyrrole film composed of benzophenone
and tris(bipyridine pyrrole) ruthenium (II) groups, for the efficient
photochemical immobilization of large biological entities with retention of
their molecular recognition properties. Moreover, the imprinted permeability of
the copolymer due to the Ru complex as cross-linking
agent, allowed the use of amperometric transduction
for such biodevices.
thanked for funding under the 6th Framework contract: NMP-A-CT-2003-505485-1. Gregoire Herzog is grateful to the CNRS for postdoctoral
fellowship. The sequence of the putative WNV epitope
was generously provided by the Pr. Bracha Rager-Zisman (Department of Microbiology and Immunology,
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