Indium Tin Oxide-Coated Optical Fiber Tips Conjugated with an

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Anal. Chem. 2003, 75, 2633-2639

Development of an “Electroptode” Immunosensor: Indium Tin Oxide-Coated Optical Fiber Tips Conjugated with an Electropolymerized Thin Film with Conjugated Cholera Toxin B Subunit Tania Konry, Andres Novoa, Serge Cosnier,† and Robert S. Marks*

The Institute for Applied Biosciences and the Department of Biotechnology Engineering, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105, Israel

We demonstrate that it is possible to create surfaceconductive fiber optics, upon which may be electropolymerized a biotinylated polypyrrole thin film, which may then be used to affinity coat the fiber with molecular recognition probes. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide. Thereafter, biotin-pyrrole monomers are electropolymerized onto the conductive metal oxide surface and then exposed to avidin. Avidinbiotin interactions were used to modify the fiber optics with biotin-conjugated cholera toxin B subunit molecules, for the construction of an immunosensor to detect cholera antitoxin antibodies. The biosensor was tested for sensitivity, nonspecificity, and overall practicality. Increasing preoccupation in diagnostic testing paved the way for the elaboration of alternative, state-of-the-art analytical devices, known as biosensors. These will one day constitute a major component of clinical diagnostics, environmental monitoring, onsite food diagnostics, and biodefense. A biosensor usually couples an immobilized biospecific recognition entity to the surface of a transducer, which “transduces” a molecular recognition event into a measurable electrical signal, pinpointing the presence of the target measurand.1 The generated electrical signal is oftentimes directly proportional to the analyte concentration. One of the great challenges met by biosensors is the immobilization strategy used to conjugate the biospecific entity onto the transducer,2 be it an electrode, a piezoelectric crystal, or, as in the case of the present study, an optical fiber. The preparation of fiber-optic biosensors usually requires the immobilization of bioreceptors on or near the optical fiber surface. Reported methods of immobilizing bioreceptors, include the following: adsorption,3 entrapment in a gel matrix,4-6 cross-linking * Corresponding author. E-mail: [email protected]. Tel.: +972 (8) 6477182. Fax: +972 (8) 6472857. † Laboratoire d'Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Universite´ Joseph Fourier Grenoble 1, 301 Rue de la Chimie, BP 53, Grenoble Cedex 9, 38041, France. (1) Marks, R. S.; Bassis, E.; Bychenko, A.; Levine, M. M. Opt. Eng. 1997, 36, 3258-3264. (2) Scouten, W. H.; Luong, J. H. T.; Brown, R. S. Trends Biotechnol. 1995, 13, 178-185. 10.1021/ac026444q CCC: $25.00 Published on Web 05/03/2003

© 2003 American Chemical Society

by a multifunctional reagent,1,7-12 and covalent bonding onto a membrane13 or through an avidin-biotin linkage.13-17 This last method, avidin-biotin immobilization, has achieved wide acceptance in recent years in binding biological species to surfaces in both analytical and preparative applications.18,19 The avidinbiotin immobilization technique offers various advantages over other immobilization techniques. Such advantages are as follows: (a) it is an extremely specific and strong noncovalent binding method (association constants, Ka, of the glycoprotein avidin for biotin in either solution or immobilized onto a surface, are ∼1015 and 1010 M-1, respectively19,20); (b) avidin provides a passivation layer over the substrate surface, which subsequently helps prevent nonspecific adsorption of biomolecules to the surface; (c) the avidin-coated substrate will be homogeneously linked with biotinylated antibodies, antigen, or enzymes; (d) the avidin-biotin linkage is carried out at very mild conditions; and (e) the avidin-biotin immobilization procedure generally maintains bioreceptor binding activity more successfully that other regular methods.3,13,14 (3) Liu, X. J.; Farmerie, W.; Schuster, S.; Tan, W. H. Anal. Biochem. 2000, 283, 56-63. (4) Navas-Dı´az, A.; Ramos-Peinado, M. C. Sens. Actuators, B 1997, 39, 426431. (5) Polyak, B.; Bassis, E.; Novodvorets, A.; Belkin, S.; Marks, R. S. Water Sci. Technol. 2000, 42, 305-311. (6) Polyak, B.; Bassis, E.; Novodvorets, A.; Belkin, S.; Marks, R. S. Sens. Actuators, B 2001, 74, 18-26. (7) Henke, L.; Piuno, P. A. E.; McClure, A. C.; Krull, U. J. Anal. Chim. Acta 1997, 344, 201-213. (8) Cordek, J.; Wang, X.; Tan, W. Anal. Chem. 1999, 71, 1529-1533. (9) Walt, D. R. Acc. Chem. Res. 1998, 31, 267-278. (10) Steemers, F. J.; Walt, D. R. Mikrochim. Acta 1999, 131, 99-105. (11) Lee, M.; Walt, D. R. Anal. Biochem. 2000, 282, 142-146. (12) Szunerits, S.; Walt, D. R. Anal. Chem. 2002, 74, 1718-1723. (13) Rubtsova, M. Y.; Kovba, G. V.; Egorov, A. M. Biosens. Bioelectron. 1998, 13, 75-85. (14) Narang, U.; Anderson, G. P.; Ligler, F. S.; Burans, J. Biosens. Bioelectron. 1997, 12, 937-945. (15) Ramos, M. C.; Torijas, M. C.; Diaz, A. N. Sens. Actuators, B 2001, 73, 7175. (16) Cosnier, S.; Gondran, C.; Gorgy, K.; Wessel, R.; Montforts, F.-P.; Wedel, M. Electrochem. Commun. 2002, 4, 426-430. (17) Cosnier, S.; Stoytcheva, M.; Senillou, A.; Perrot, H.; Furriel, R. P. M.; Leone, F. A. Anal. Chem. 1999, 71, 3692-3697. (18) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (19) Savage, D.; Mattson, G.; Desai, S.; Nielander, G.; Morgensen, S.; Conklin, E. Avidin-biotin chemistry: A handbook; Pierce: Rockford, IL, 1992. (20) Zhao, S.; Reichert, W. M. Langmuir 1992, 8, 2785-2791.

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The avidin-biotin immobilization method requires some process to attach the avidin onto the transducer surface as mentioned previously. These procedures, even when they have proven to be successful, have some disadvantages. The physical adsorption frequently suffers from low surface loading or stability,21 and the covalent attachment using a cross-linker regularly results in partial denaturation of the biomolecules, attachment at undesired molecular locations, or conformational changes leading to the reduction of their activity.22,23 The immobilization on porous membranes usually involves an adsorption or covalent binding step of avidin, while membranes are not usually practical. An innovative method based on avidin-biotin affinity interactions for biomolecule immobilization has been previously reported for the construction of amperometric biosensors. This alternative method consists of the electrogeneration of electronic conducting polymers functionalized by biotin moieties that allow for the subsequent attachment of avidin in a natural way. With this aim in view, biotin derivatives bearing an electropolymerizable pyrrole group have been recently synthesized and applied to the elaboration of electrically conducting poly(pyrrole) films.24-26 The latter were successfully used in the design and construction of enzymebased amperometric biosensors, highlighting the importance of a perfectly defined initial biotinylated layer.17,27,28 We have recently exploited this procedure to functionalize an optical fiber by coating optical fiber tips by chemical oxidative polymerization of pyrrole-biotin with iron(III) chloride,29 because the insulating properties of the bare optical fiber do not permit the use of the electropolymerization method. This approach, however, presented a major drawback because it was very difficult to control the reproducibility of the deposition of the biotinylated polymer. In electrochemical deposition procedures, it is possible to precisely electrogenerate a polymer coating over conductive microsurfaces of complex geometry. In addition, the polymer films exhibit a high robustness during operation in either aqueous or organic media. The advantage of the electrochemical polymerization method is that these films can be prepared easily in a rapid, well-controlled, one-step procedure. Optical fibers are made of doped silica, which are electrically inert. To secure recognition molecules through conjugation via electropolymerization, we must create an electrically conductive layer at the fiber surface. In a previous work, we reported that it is possible to create surface-conductive fiber optics, upon which may be electropolymerized a biotinylated polypyrrole thin film, which may then be used to affinity coat the fiber with recognition probes. This fiber-optic electroconductive surface modification is (21) Ulbrich, R.; Golbik, R.; Schellenberger, A. Biotechnol. Bioeng. 1991, 37, 280-287. (22) Narang, U.; Anderson, G. P.; King, K. D.; Liss, H. S.; Ligler, F. S. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 2980, 187-194. (23) Leckband, D.; Langer, R. Biotechnol. Bioeng. 1991, 37, 227-237. (24) Cosnier, S.; Galland, B.; Gondran, C.; Le Pellec, A. Electroanalysis 1998, 10, 808-813. (25) Torres-Rodriguez, L. M.; Roget, A.; Billon, M.; Bidan, G. Chem. Commun. 1998, 1993-1994. (26) Cosnier, S.; Lepellec, A. Electrochim. Acta 1999, 44, 1833-1836. (27) Mousty, C.; Bergamasco, J. L.; Wessel, R.; Perrot, H.; Cosnier, S. Anal. Chem. 2001, 73, 2890-2897. (28) Mousty, C.; Lepellec, A.; Cosnier, S.; Novoa, A.; Marks, R. S. Electrochem. Commun. 2001, 3, 727-732. (29) Marks, R. S.; Novoa, A.; Thomassey, D.; Cosnier, S. Anal. Bioanal. Chem. 2002, 374, 1056-1063.

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done by the deposition of a thin layer of indium tin oxide. Thereafter, biotin-pyrrole monomers are electropolymerized onto the conductive metal oxide surface. We were able to demonstrate the presence of an electropolymerized polypyrrole-biotin film by allowing horseradish peroxidase-labeled avidin to link to it and thereafter exposing it to luminol in order to obtain a chemiluminescence radiation.30 It is known that indium tin oxide, commonly referred to as ITO, is a transparent conducting oxide. We have previously demonstrated that it is possible to modify the exterior properties of an optical fiber core (doped silica) that is a transparent electrical insulator of high resistance by sputtering a thin-layer film of ITO on its thermally, sputter-robust surface.30 ITO is an indium oxide (In2O3)-based material that has been doped with tin oxide (SnO2) to improve its electrical quality. Tin acts as a cationic dopant in the In2O3 lattice and substitutes indium sites to bind with interstitial oxygen. The presence of SnO2 would result in n doping of the lattice because the dopant would add electrons to the conduction band.31 The ITO film, with an allowed wide band gap of ∼3.8 eV, is highly transmissive in the visible region32 and has low resistivity, attributed to excessive numbers of free electrons causing the Fermi level to be located in the conduction band.33 We must produce thinner films of indium tin oxide even though there is a correlation between thicker films and lower resistivity, as lower light transmission may occur due to absorption in the film.33 Also, transparency is unfortunately not perfect due to diffuse scattering from surface roughness and internal scattering from grain boundary effects.33 There are several known methods to deposit thin films of ITO, including radio frequency (rf) sputtering,34 magnetron sputtering,35 dc field sputtering,36 screen-printing technique,37 electron38 or ion39 beam deposition, chemical vapor deposition,40 activated reactive evaporation,41 and spray pyrolysis.42 We have chosen, herein, to use radio frequency sputtering. Such a method has been used on a variety of substrates, such as glass34 or polymers (acrylic, polycarbonate, poly(ethylene terephthalate)).43,44 The coating of fiber-optic waveguides with ITO for the construction of polymer electroluminescent diodes with microcylindrical geometry has been previously reported,45-47 but we report herein a pioneering (30) Marks, R. S.; Novoa, A.; Konry, T.; Krais, R.; Cosnier, S. Mater. Sci. Eng. C 2002, 21, 189-194. (31) Wu, W. F.; Chiou, B. S. Thin Solid Films 1994, 247, 201-207. (32) Naseem, S.; Coutts, T. J. Thin Solid Films 1986, 138, 65-70. (33) Knickerbocker, S. A.; Kulkarni, A. K. J. Vac. Sci. Technol., A 1995, 13, 1048-1052. (34) Chiou, B. S.; Tsai, J. H. J. Mater. Sci. Mater. El 1999, 10, 491-495. (35) Buchanan, M.; Webb, J. B.; Williams, D. F. Thin Solid Films 1981, 80, 373-382. (36) Higuchi, M.; Uekusa, S.; Nakano, R.; Yokogawa, K. Jpn. J. Appl. Phys. 1 1994, 33, 302-306. (37) Bessais, B.; Mliki, N.; Bennaceur, R. Semicond. Sci. Technol. 1993, 8, 116121. (38) Agnihotry, S. A.; Saini, K. K.; Saxena, T. K.; Nagpal, K. C.; Chandra, S. J. Phys. D 1985, 18, 2087-2096. (39) Bregman, J.; Shapira, Y.; Aharoni, H. J. Appl. Phys. 1990, 67, 3750-3753. (40) Chopra, K. L.; Major, S.; Pandya, D. K. Thin Solid Films 1983, 102, 1-46. (41) Nath, P.; Bunshah, R. F.; Basol, B. M.; Staffsud, O. M. Thin Solid Films 1980, 72, 463-468. (42) Manifacier, J. C.; Szepessy, L.; Bresse, J. F.; Perotin, M.; Stuck, R. Mater. Res. Bull. 1979, 14, 109-119. (43) Kulkarni, A. K.; Lim, T.; Khan, M.; Schulz, K. H. J. Vac. Sci. Technol., A 1998, 16, 1636-1640. (44) Shin, J. H.; Shin, S. H.; Park, J. I.; Kim, H. H. J. Appl. Phys. 2001, 89, 51995203.

Figure 1. Chemical structure of pyrrole-biotin.

work in the field of optical fiber immunosensors that demonstrates the possibilities of creating a biosensor for the detection of anticholera toxin using ITO-coated optical fibers that were subsequently electropolymerized with pyrrole-biotin to allow the immobilization of biomolecules by affinity interactions. EXPERIMENTAL SECTION Reagents. Avidin (Av, A9390, from egg white, 89% of protein by UV absorbance), bovine serum albumin (BSA, A4503, fraction V), cholera toxin B subunit, biotin-labeled (C9972, lyophilized powder containing ∼40% protein), anti-cholera toxin (anti-CTB, C3062, from rabbit, purified toxin from ,Vibrio cholerae), peroxidase-labeled IgG anti-rabbit antibody (A6154, from goat, affinityisolated antibody), and polyoxyethylene-sorbitan monolaurate (Tween 20, P7949) were purchased from Sigma. Luminescence measurements were carried out using the Western Blot Chemiluminescence Reagent Plus kit from NEN Life Science Products (NEL105, containing enhanced luminol reagent and oxidizing reagent). Acetonitrile (Rathburn) and lithium perchlorate (Fluka) were used as received. Tetrabutylammonium perchlorate (TBAP) was recrystallized from ethyl acetate/cyclohexane and vacuumdried at 80 °C. 11-(1-Pyrrolyl)undecanol was prepared as described previously.48 Pyrrole-biotin (Figure 1) was synthesized by dissolving together biotin (500 mg, 2 mmol) 11-(1-pyrrolyl)undecanol (417 mg, 1.66 mmol), 1,3-dicyclohexylcarbodiimide (413 mg, 2 mmol), and 4-(dimethylamino)pyridine (81 mg, 0.66 mmol) in dry deoxygenated CH2Cl2 (10 mL). The mixture was stirred at room temperature for 5 days under argon atmosphere. The resulting solution was filtered and evaporated. After extraction, pyrrolebiotin precipitated as a white solid. This crude product was then chromatographed on silica gel with 95:5 (v/v) CH2Cl2/Et2O mixture as eluting solvent, yielding 618 mg of pyrrole-biotin (78% yield): H(CDC13) NMR (200 MHz) (DMSO) δ 1.24-1.68 (m, 24 H), 2.30 (t, 2 H), 2.73 (m, 1 H), 2.89 (m, 1 H), 3.11 (m, 1 H), 3.83 (t, 2 H), 4.02 (t, 2 H), 4.28 (m, 1 H), 4.47 (t, 2 H), 5.50 (s, 1 H), 5.93 (s, 1 H), 6.10 (m, 2 H), 6.62 (m, 2 H). All other chemicals were obtained commercially and were of analytical grade. Optical Fiber Tip Preparation. All fibers used were SFS400/ 440B silica fibers (Fiberguide Industries) with an original numerical aperture of 0.22. Their core was 400 µm in diameter (refractive index of 1.457 at 633 nm) and was surrounded by a 40-µm silica cladding (refractive index of 1.44 at 633 nm), followed by a 150µm-thick silicon buffer, and finally a 210-µm-thick black Tefzel (45) Fujii, A.; Frolov, S. V.; Vardeny, Z. V.; Yoshino, K. Jpn. J. Appl. Phys. 2 1998, 37, L740-L742. (46) Fujii, A.; Chinn, D.; Shkunov, M. N.; Frolov, S. V.; Vardeny, Z. V.; Yoshino, K. Synth. Met. 1999, 102, 1010-1011. (47) Frolov, S. V.; Shkunov, M.; Fujii, A.; Yoshino, K.; Vardeny, Z. V. IEEE J. Quantum Electron. 2000, 36, 2-11. (48) Otten, T.; Darbre, T.; Cosnier, S.; Abrantes, L.; Correia, J.; Keese, R. Helv. Chim. Acta 1998, 81, 1117-1126.

jacket. The typical length of any single fiber used in our experiments was 25 cm. The black Tefzel jacket was stripped away mechanically using a fiber stripping tool (Micro-Strip, from MicroElectronics Inc.), leaving a 1-cm declad optical fiber tip. To remove the silicon cladding, the fiber-optic tips were dipped a few minutes in sulfuric acid (93.1%), rinsed with distilled water, and afterward rinsed with acetone and left to dry. The silica cladding was removed by dipping the fiber-optic tips in 5% (v/v) HF during 10 h with stirring. Next, the optical fibers were rinsed with distilled water and methanol and finally blow-dried. Indium Tin Oxide Sputtering. The depositions were carried out in an rf-sputtering Varian system. The sputtering target was a 4-in.-diameter circular disk of hot pressed powder 99.999% purity ITO (90 wt % In2O3 + 10 wt % SnO2), made by Testbourne Ltd. (Hampshire, U.K.). The sputtering chamber was evacuated to a pressure lower than 10-6 Torr. Argon and oxygen gases were introduced into the sputtering chamber with partial pressures of 2 × 10-3 and 2.5 × 10-4 Torr, respectively. The rf supply was then switched on and stabilized to a power of 400 W. The fiber tips were exposed to the sputter beam for 1-1.5 h. Therefore, the fiber core end face (important area where electropolymerization must occur) and at least one side of the cylindrical surface (for contacting and voltage supply) were targeted by the ITO. Characterization of the ITO Coating. X-ray analyses were made with a scanning electron microscope (Philips Quanta 2000). General morphology of the specimens was recorded by photographing the secondary image from the recording cathode ray tube. Micrographs were taken at 1024 × 880 resolution with an exposure time of 100 s. The specimens were analyzed using energy-dispersive X-ray spectrometry microanalysis analytical equipment installed in the scanning electron microscope. The data were collected and processed by a standardless quantitative analytical program (Quanta 200, FEI Co.) The corresponding thickness of the ITO film (carried out on glass slides coated during the same run) was measured by a Varian Å-Scope interferometer, model Nos. 980-4000/4006. A masking process, by vacuum deposition of an aluminum layer, was carried out before deposition of the ITO film in order to get the proper beveled edge shape. After the ITO deposition, parts of the deposited ITO layer, as well as the aluminum mask, are removed mechanically. Then, the edge shape is overcoated by an aluminum reflective film in order to achieve maximum quality fringe lines. The conductivity of the ITO film was tested by a standard fourpoint method. Direct current source was applied by a dc source (Yakogava 7651), and the voltage was measured by a digital voltmeter (Keythly 2000). Deposition of Biotin-Pyrrole onto the Optical Fiber Surface by Electropolymerization. All electrochemical experiments were performed with a Princeton Applied Research model 173 equipped with a model 179 digital coulometer and a model 175 universal programmer in conjunction with a Sefram TGM 164 X-Y/t recorder. The electropolymerization of pyrrole-biotin and the characterization of the resulting modified electrode were run at room temperature in a conventional three-electrode cell. A 10 mM Ag/Ag+ in CH3CN electrode was used as a reference electrode in acetonitrile electrolyte. The working electrodes were Analytical Chemistry, Vol. 75, No. 11, June 1, 2003

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Figure 2. Biosensor scheme describing the various steps involved in the immunoassay using ITO-poly(pyrrole-biotin)-coated optical fibers for the detection of anti-cholera toxin B subunit.

the ITO-modified optical fibers, an electrical contact being established with a platinum wire (diameter 50 µm) above the acetonitrile solution. The biotinylated polypyrrole films were prepared by controlled potential oxidation of the monomer (2 mM) in CH3CN + 0.1 M TBAP. FT-IR Characterization of the Poly(pyrrole-biotin)-Coated Optical Fiber Tip. Infrared reflection-absorption spectra for surfaces of the bare, ITO-coated and the poly(pyrrole-biotin)coated optical fibers were obtained using a Bruker Equinox 55 FT-IR spectrometer with a Bruker IRRAS device PME 37. The spectra were averaged over 128 scans at a resolution of 4 cm-1. IR spectra are shown by absorbance units [absorbance is defined as -log(R/Ro), where R and Ro are the reflectance of the sample and of the background, respectively]. Immunoassay Rationale and Design. The steps carried out for the construction of the biosensor are visualized in Figure 2. As in preceding experiments, a blocking treatment was previously carried out with BSA in order to help prevent nonspecific binding of avidin onto the glass fiber tips. The fibers were rinsed and washed twice with PBS-Tween for 5 min, before being incubated in 120 µL of 20 µg/mL avidin solution for 45 min. The fibers were rinsed and washed once with PBST for 15 min, to remove any nonspecifically attached avidin. Then, the fibers were dipped in 120 µL of 2 µg/mL biotinylated cholera toxin B subunit (b-CTB) solution during 20 min. The fibers were rinsed and washed once with PBST for 15 min and, after that, three times for 5 min. The analyte was anti-cholera toxin B subunit (anti-CTB) antibody elicited in a rabbit. The previously avidin-conjugated fibers were incubated for 20 min in 120 µL of several analyte solutions at concentrations ranging from 0.16 up to 640 µg/mL. Sets of two replicas were prepared for each concentration measured, and a series of fibers was set aside with the purpose of using them as blanks. Thereafter, the fibers were rinsed and washed once with PBST for 15 min and then three times for 5 min. Subsequently, the optical fiber tips were dipped into 120 µL of a solution containing the secondary antibody, horseradish peroxidase-labeled 2636

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goat anti-rabbit IgG immunoglobulin (at a concentration of 0.08 µg/mL) for 20 min, and finally rinsed and washed once with PBST for 15 min and then three times for 5 min. Chemiluminescence Measurements. The instrument setup for the chemiluminescence measurements has been previously described.5,6 A Hamamatsu HC135-01 Photomultiplier Tube sensor module was used for chemiluminescence measurements, combining the sensitivity of a photomultiplier tube with the intelligence of a microcontroller. The detector was optimized to the blue light region and included a 21-mm-diameter active area convenient to gather light radiation without any optical focusing elements for luminescence measurements. The instrument setup was placed in a light-tight box. The far end of the fiber was held by a fiber holder (FPH-DJ, Newport) and placed into an adjustable singlefiber mount (77837, Oriel). To prevent damage to the photoncounting unit by environmental light, a manual shutter (71430, Oriel) was placed in front of the detector. To move the slide shutter, a workshop-made lever was placed outside the box. To receive and treat data, a specific driver was developed using LabView (version 3.1, National Instruments Corp.), which allowed monitoring of the chemiluminescence signal and data handling in real time. The immunosensor optical fibers were placed in a 400-µL sample tube containing the combined oxidizing reagent and enhanced luminol reagent solutions (NEN chemiluminescence reagent kit) in a 1:1 (v/v) ratio. The chemiluminescence readings were integrated for 1 s, and each measurement was obtained by taking a mean value of photon counts during 10 s. The responses of blank controls were also checked by the same procedure. RESULTS AND DISCUSSION Characterization of the ITO Coating. Sputtering from oxide targets to form transparent conducting oxide films was selected as the deposition technique, because it renders a far better control over the stoichiometry and has been shown to obviate, in most cases, the postdeposition heat treatment step. Highly transparent

Figure 3. Scanning electron micrograph of an ITO-coated optical fiber tip: (A) 300× magnification, (B) 5000× magnification, and (C) 6000× magnification in a coating border zone Table 1. X-ray Microanalysis of the ITO Coating of a Declad Optical Fiber Tip End Face element

wt %

mol %

SiO2 In2O3 SnO2

25.96 73.63 0.41

61.72 37.89 0.39

and conducting films of ITO have been previously deposited by this technique.40,49 The micrographs seen in Figure 3, depicting an ITO-coated fiber optic were taken by scanning electron microscopy. The ITO coating is clearly seen lying on the surface of the optical fiber end tip as a seemingly homogeneous layer in Figure 3A and B. Figure 3C shows a micrograph of a zone where both the bare optical fiber and the ITO-coated region can be seen side by side clearly showing the depth of the ITO thin coating film. In addition, the ITO film deposited onto the optical fiber was observed to be amorphous as others have reported for this particular deposition technique.50,51 The results of the X-ray microanalysis of the ITOcoated optical-fiber tips are shown in Table 1 and confirm that we have indeed obtained a thin layer, including indium tin oxide over a silica matrix on the fiber declad tip. As a result of the difference in the volatility and sputter rates of the indium and tin oxides, an enrichment of the film in indium is observed compared to the original composition of the ITO target. Therefore, the film composition deviates from the original target.49 The measured thickness of the ITO coating-film around the optical fiber was estimated at 260 nm, which is similar to that reported in previously published materials, which was anywhere between 250 and 600 nm.35,50 The specific electrical resistance of the ITO coating film measured by the four-point probe method was at 4.7 × 10-2 Ω‚cm. Even though this value is in the range of those reported for ITO deposition by the sputtering technique,52 it is not among the lowest demonstrated with resistance values found to be as low as 1.5 × 10-4 Ω‚cm.53 At any rate, the resistance is low enough to permit sufficient electrical conductivity to carry out the required electropolymerization of the pyrrole-biotin. On the other hand, transparency in the visible region is also strongly (49) Tahar, R. B. H.; Ban, T.; Ohya, Y.; Takahashi, Y. J. Appl. Phys. 1998, 83, 2631-2645. (50) Buchanan, M.; Webb, J. B.; Williams, D. F. Appl. Phys. Lett. 1980, 37, 213215. (51) Kawada, A. Thin Solid Films 1990, 191, 297-303. (52) Weijtens, C. H. L. J. Electrochem. Soc. 1991, 138, 3432-3434. (53) Shigesato, Y.; Takaki, S.; Haranou, T. Appl. Surf. Sci. 1991, 48-9, 269275.

Figure 4. Cyclic voltammograms recorded at a regular ITO-modified optical fiber (dashed line) and at a ITO-modified optical fiber functionalized by an electrogenerated biotinylated polypyrrole film (straight line) in CH3CN + 0.1 M TBAP; potential scans between 0 and 0.82 V. Potential measured vs Ag/Ag+ 10 mM in CH3CN + 0.1 M TBAP. Scan rate, 20 mV s-1.

affected by the electrical properties of the film. The mechanism of electrical conduction and optical transmission are very much interdependent. Good electrical properties can be achieved but often at the expense of transmission.49,54 Deposition and Characterization of the Poly(pyrrolebiotin) Layer. To create a bioaffinity coating for avidin on the ITO-modified optical fibers, electropolymerization of pyrrolebiotin (2 mM) was carried out by a controlled-potential oxidation at 0.85 V in CH3CN + 0.1 M TBAP. Figure 4 (solid line) shows the cyclic voltammogram recorded for an optical fiber, functionalized with poly(pyrrole-biotin), upon transfer into a CH3CN + 0.1M TBAP solution free of monomer. The comparison with the cyclic voltammogram obtained for an ITO-coated optical fiber, not functionalized with poly(pyrrole-biotin) (Figure 4, dashed line), clearly shows the appearance of the quasi-reversible oxidation wave of the polypyrrolic film. The apparent surface coverage of electropolymerized biotin (ΓB ) 1.1 × 10-9 mol‚cm-2) was estimated by the integrated current obtained by the electroactivity of the conducting polypyrrole backbone, taking into account that 0.33 electron‚molecule-1 is involved in the electrooxidation of the polypyrrolic chain.55 An additional confirmation of the occurrence of a thin layer of poly(pyrrole-biotin) on the ITO-coated optical fiber tips was (54) Grigorovici, R. Thin Solid Films 1972, 9, 1-23. (55) Deronzier, A.; Moutet, J. C. Acc. Chem. Res. 1989, 22, 249-255.

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Table 2. Absorption Peaks Recorded by Reflection FT-IR Spectrometry of the Poly(pyrrole-biotin) Functionalized ITO-Coated Optical Fibers peak position/cm-1

assignment

2920 2850 1358 848 740-770

νas(CH2) νs(CH2) ν(C-N) in the imidazol ring of biotin ω(N-H) in the imidazol ring of biotin γ-CH or β-ring modes of pyrrole ring

Figure 5. Chemiluminescence luminol reaction catalyzed by peroxidase.

obtained by recording the infrared reflection spectra on the functionalized fiber tip surface. Because of the small quantities of poly(pyrrole-biotin) deposited by electropolymerization, the absorption intensities were low but sufficient enough to identify some absorption bands that are characteristic of the biotin and pyrrole moieties that compose the polymer. The absorption peaks and probable assignations are shown in Table 2. Immunosensor Behavior. The importance of monitoring cholera, for diagnostics or epidemiological reasons, stems from the fact that even though it is an easy disease to cure in a developed society, it is still found as an endemic scourge or infrequently as a pandemic in a number of countries in Asia, Africa, and Latin America. The etiological organism is V. cholerae and its enteropathogenic factor is cholera toxin, which induces massive intestinal fluid and electrolyte loss. Together with seral fluids, the induced loose stools are part of any monitoring or epidemiologic study. Herein, we merely utilize the immunogenic (and safe) part of the enterotoxin, the pentameric cholera toxin B subunit protein, as the antigen receptor bound to the fiber optic. The detection of rabbit anti-cholera toxin antibody was achieved using a sandwich immunoassay (Figure 2). This first step required the electropolymerized deposition of a poly(pyrrole-biotin) thin film onto an ITO-coated optical fiber tip. This thin affinity film then allowed the conjugation of avidin and the subsequent binding of cholera toxin B subunit biotin-labeled. The analyte, rabbit anticholera toxin antibody, thereafter bound the corresponding immobilized cholera toxin B subunit epitopes. The subsequent binding of a marker peroxidase-labeled IgG anti-rabbit antibody allowed us to then carry out a chemiluminescence reaction by adding an enhanced luminol solution (Figure 5).56 The light emitted, as a side reaction, is transduced by the optical fiber to the measurement instrument setup. Figure 6A shows the standard curve for the rabbit anti-cholera toxin antibody sandwich immunoassay obtained by collecting data in triplicate at antibody titers ranging from 1:30 to 1:1,200,000. (56) Freeman, T. M.; Seltz, W. R. Anal. Chem. 1978, 50, 1242-1246.

2638 Analytical Chemistry, Vol. 75, No. 11, June 1, 2003

Figure 6. (A) Calibration curve obtained by the immunoassay procedure in the detection of anti-CTB using the ITO-PPB-coated optical fibers. Titers of anti-CTB from 1:30 to 1:1 200 000 were tested in triplicate on individual fibers. (B) The curve was fitted according to the equation y ) A + B ln(x), where x is the anti-CTB dilution value and y is the chemiluminescence response. The correlation coefficient obtained was R2 ) 0.95.

Figure 6A shows a typical behavior for the standard calibration curve with an exponential growth as seen from the curve fit results shown in Figure 6B. The curve fit was carried out using an equation of the form y ) A + B ln(x), where x is the anti-CTB dilution and y is the corresponding response signal obtained. The standard curve was most useful for quantization of titers from 1:3000 and lower, showing in this range an acceptable square correlation coefficient, R2 ) 0.95 and a satisfactory sensitivity of 191 photocounts/s (determined within the linear concentration range of the biosensor as the slope, B of the calibration curve). At higher concentrations, the curve levels off with a response saturation observed from titers 1:150 and up. As small inaccuracies in the measurement occur at such low dilutions, measurements will lead to large errors in prediction, so such samples would have to be diluted for accurate quantization. The background signal recorded for the blank (in absence of the analyte, see Table 3, experiment 5) and its calculated standard deviation allowed us to reach a limit of quantization for a dilution as high as 1:1 200 000. The linear range obtained in the present work is wider than that achieved in our previous work, in which the poly(pyrrolebiotin) coating onto the optical fiber tip was carried out by chemical oxidative polymerization.29 In addition, the lower detection limit was improved from 1:300 00029 to 1:1 200 000 in the present work. We believe such improvements could possibly be attributed to the electrochemical procedure carried out in the present work through the poly(pyrrole-biotin) deposition, which is known to produce uniform film coatings with well-controlled thicknesses.57,58 Another advantage of the procedure reported herein resides in the fact that these fibers may be reusable. Another group has demonstrated that similar biotinylated conducting polypyrroles used for the fabrication of DNA chips showed the capability to be regenerated after “denaturation” of the biotin/ avidin links by a surfactant.59 The regenerated biotinylated (57) Deronzier, A.; Moutet, J. C. Coord. Chem. Rev. 1996, 96, 339-371. (58) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 25372574.

Table 3. Reference Experiments Carried out in Order To Determine the Possible Influence of Nonspecific Effects on the Obtained Responsea step exp

ITO sputtering

PPyB electropolymerization

Av attachment

CTB-B attachment

AntiCTB

anti-rabbit HRP

normalized responseb

1 2 3 4 5

+ + + + +

+ + + +

+ + + +

+ + + +

+ + + + -

+ + + + +

1.000 0.645 0.009 0.017 0.037

a the (+) and (-) signs indicate the steps accomplished or avoided respectively for each experiment. The response was recorded in all the experiments for an anti-CTB titer at 1:3000. b Calculated as a ratio between the response obtained from experiments 2-5 and the one obtained from experiment 1.

polypyrrole presented then the same ability to subsequently elaborate biological architectures based on avidin-biotin bridges opening the way to a reusable sensor. To check the influence of possible nonspecific interactions on the overall chemiluminescent responses, a set of reference experiments were carried out. Table 3 shows the conditions used for the set of different experiments. The steps shown in the table correspond to those illustrated in Figure 2. The results in Table 3 show that the responses from experiments 3-5 (obtained in absence of avidin, cholera toxin B subunit biotin-labeled, and rabbit anti-cholera toxin, respectively) are relatively negligible. But in the case of experiment 2 (where the electrochemical deposition of the poly(pyrrole-biotin) was avoided), a significant response was registered that reached 64.5% of that of the reference experiment 1. This is not surprising since the avidin and the cholera toxin B subunit have isoelectric points (Piso) estimated at 10 and 8.89, respectively. Given that the experiments were carried out at a pH ) 7.4, both proteins should be preferentially in the protonated form, favoring the electrostatic interactions with the negatively charged SiO2/ITO surface and, in consequence, the nonspecific adsorption of both proteins on the fiber-optic tip. It seems that the poly(pyrrole-biotin) coating provides a more hydrophobic surface (and partially positively charged) that helps reduce the nonspecific adsorption of both, the avidin and the cholera toxin B subunit biotin-labeled, as confirmed by the results of experiments 3-5 (Table 3). CONCLUSIONS We have demonstrated herein that one can modify the surface of a fiber optic, which is a transparent electrical insulator, into (59) Dupont-Filliard, A.; Roget, A.; Livache, T.; Billon, M. Anal. Chim. Acta 2001, 449, 45-50.

one that has a conductive surface by simple deposition of an indium tin oxide thin film onto its surface. This newly acquired property allows the fiber-optic tip end face to be submitted to a voltage, in the presence of affinity modified monomers, while electrogenerating an affinity polymer over its tip and end face. Once chemically modified here, with the affinity element biotin, avidin is allowed to bind, allowing the fiber to further bind to any biotinylated receptor of interest. The model analyte anti-cholera toxin immunoglobulin was detected at a low titer of 1:1 200 000. ACKNOWLEDGMENT The authors thank the Commission of the European Communities Research Directorate for their support under the PEBCAT project contract EVK1-CT-2000-00069 and the North Atlantic Treaty Organization (NATO), Scientific and Environment Affairs Division for the Collaborative Linkage Grant Award PST.CLG.976228. R.S.M. acknowledges the partial funding by the Israel Ministry of Defense, under infrastructure grant 1508852901. A.N. thanks the Israel Ministry of Absorption for partial support. We thank Svetlana Shtutina and Yakov Yuzhelevski from the Department of Physics at the Ben Gurion University of the Negev for their technical assistance for the running ITO rf sputtering instrumentation, Arielle Lepellec from the LEOPR, at Universite´ Joseph Fourier, that carried out the pyrrole-biotin electropolymerization, and Vitaly Eruhimovitch from the Multidisciplinary Center at BGU for his help with FT-IR techniques. This work is a partial completion of the M.Sc. Thesis of T.K.

Received for review December 19, 2002. Accepted March 31, 2003. AC026444Q

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