Anal. Chem. 2006, 78, 3577-3582
Surface Modification of Polystyrene Using Polyaniline Nanostructures for Biomolecule Adhesion in Radioimmunoassays Tarveen Karir,† P. A. Hassan,‡ S. K. Kulshreshtha,‡ G. Samuel,*,† N. Sivaprasad,† and V. Meera†
Radiopharmaceuticals Program, Board of Radiation and Isotope Technology, Department of Atomic Energy, Navi Mumbai, India, and Chemistry Division, Bhabha Atomic Research Centre, Mumbai, India
The selection of an appropriate surface as a solid phase for coupling antibodies is a critical step in the development of solid-phase immunoassays. Availability of a new method of preactivating the surface of polystyrene tubes with a layer of another polymer for enhanced immobilization of antibodies seems to be promising. In this paper, we report the activation of a polystyrene surface using a layer of polyaniline and its effect on immobilizing antibodies for use as a solid phase in a T3 immunoassay. The modified surface on the polystyrene was characterized by optical absorption, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The modified tubes were coated with antibody and evaluated for their performance in the assay and validated for radioimmunoassay of T3. AFM images of the modified surface showed an enhancement in the surface roughness (Ra of 20.2 nm), as compared to an unmodified surface (Ra of 6 nm), allowing more adsorption of antibodies to the surface. XPS revealed the presence of N (binding energy ∼400 eV) on the modified surface, which could help the antibody molecules to bind to these preactivated (modified) tubes. The modified tubes, when coated with antibody, not only showed an increase in the binding with the radioiodinated tracer but also improved the precision of coating the antibody. The present method of activating polystyrene surfaces is simple, does not involve severe chemical treatment, and may have wide applicability to functionalize other supports for immobilizing biomolecules. Immobilization of one of the assay reagents on a solid support is a key requirement for some immunometric assays, such as enzyme-linked immunosorbent assays (ELISA) and solid-phase radioimmunoassays (RIA). The widely used solid-phase materials are plastics, especially thermoplastics, such as polystyrene, polypropylene, polysulfone, or polycarbonate, which can be moulded into an unlimited variety of shapes. Each has distinctive chemical and physical properties that make it useful as a support. Polystyrene is a commonly used plastic for manufacture of tubes * Corresponding author. Fax: 91-22-27887218. E-mail: grace_samuel1955@ rediffmail.com. † Department of Atomic Energy. ‡ Bhabha Atomic Research Centre. 10.1021/ac052032g CCC: $33.50 Published on Web 05/03/2006
© 2006 American Chemical Society
or beads to be used in immunoassays.1-3 There are different approaches for immobilizing biomolecules on polystyrene surfaces. Among them, passive adsorption and covalent coupling are the most common methods. Passive adsorption predominantly makes use of the hydrophobic interaction between the solid phase and the biomolecules.4,5 Different covalent immobilization procedures have been reported in the literature. An active amino group can be introduced to polystyrene by nitration of the aromatic ring, followed by reduction. The amino polystyrene is further activated by chemical reactions, such as diazotization, and the resulting surface is used for antibody immobilization.6 Covalent binding using bifunctional cross-linking agents, such as glutaraldehyde,7-9 and activation of the surface using isocyanate10 or carbodiimide11,12 are also employed. Most of the covalent immobilization approaches are tedious and involve severe chemical treatment or multistep procedures. Thus, there is a need to develop simple, yet efficient immobilization strategies, since this could offer immense potential in diagnostic applications. Recently, microcontact printing has been employed to pattern surfaces with functionalized molecules for cost-effective biosensor applications.13-16 Alternatively, functional groups for covalent or ionic binding can (1) Gosling, J. P., Ed., Solid-phase reagents. In Immunoassays, A Practical Approach; Oxford: New York, 2000; pp 129-163. (2) Kakabakos, S. E.; Livanlou, E.; Evangelatos, G. P.; Ithakissios, D. S. Clin. Chem. 1990, 36, 492. (3) Kakabakos, S. E.; Evangelatos, G. P.; Ithakissios, D. S. Clin. Chem. 1990, 36, 497. (4) Molima, B. J. A.; Galisteo, G. F.; Hidalgo, A. R. J. Biomater. Sci., Polym. Ed. 1998, 9, 1103. (5) Molima, B. J. A.; Galisteo, G. F.; Hidalgo, A. R. J. Biomater. Sci., Polym. Ed. 1998, 9, 1089. (6) Tarcha, P. J. In Immunochemistry of Solid-Phase Immunoassays; Butler, J. E., Ed.; CRC Press: Boca Raton, FL, 1991; Chapter 2, p 39. (7) Besselink, G. A. J.; Schasfoort, R. B. M.; Bergveld, P. Biosens. Bioelectron. 2003, 18, 1109. (8) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emneus, J. Anal. Chem. 2002, 74, 2994. (9) Bataillard, P.; Gardies, F.; Jaffrezic-Renault, N.; Martelet, C.; Colin, B. Mandrand, B. Anal. Chem. 1988, 60, 2374. (10) Saito, T.; Nagai, F. Clin. Chim. Acta 1983, 133, 301. (11) Drgoev, A. B.; Gazaryan, I. G.; Lagrimini, L. M.; Ramanathan, K.; Danielsson, B. Anal. Chem. 1999, 71, 5258. (12) Su, X.; Chew, F. T.; Li, S. F. Y. Anal. Biochem. 1999, 273, 66. (13) Willner, I.; Schlittner, A.; Doron, A.; Joselevich, E. Langmuir 1999, 15, 2766. (14) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055. (15) Mrksich, M.; Whitesides, G. M. Trends Biotechnol. 1995, 13, 228. (16) Stjohn, P. M.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108.
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be introduced by adsorption of high molecular weight polypeptides.17,18 Poly-L-lysine, originally used to enhance cell adhesion to glass, has been employed to adsorb antibodies or DNA to plastic beads.19-21 Polyaniline (PANI) is a hydrophobic conducting polymer that has been immensely studied for its applications as substrates for biosensors and matrixes for immobilizing proteins. Polyanilinepolystyrene sulfonate composites synthesized within the pores of polycarbonate membranes are found to be efficient in immobilizing enzymes for glucose sensing.22 Improved biosensor sensitivity to hydrogen peroxide was observed when sulfonated polyaniline was coated on a polycationic film.23 Polyaniline is used as an efficient surface for the electrochemical reduction of horseradish peroxidase.24 An assay for ascorbic acid using polyaniline-coated microtiter plates has been reported.25 Recently, functionalized conducting polymer substrates were employed for immobilizing enzymes in electrochemical sensors.26,27 Polypropylene membranes functionalized with polyaniline demonstrated a high affinity toward different proteins, which can be immobilized onto its surface through physical adsorption or covalent immobilization.28 Polyaniline may act both as a chemically active matrix and as a matrix for immobilization of specific reagents and enzymes. Primary amines being convenient groups for cross-linking proteins, the terminal amino groups in PANI will be useful for coupling antibodies. Thus, a thin layer of PANI film coated onto the surface of polystyrene is expected to alter the immunoreactivity of immobilized antibodies. The changes in the physical nature of the surface due to PANI coating, such as surface roughness, surface charge, etc., can also affect the quantity and reactivity of the adsorbed antibodies. Nanostructured surfaces are expected to provide improved performance over their bulk counterparts for the applications as sensors due to their greater specific surface area. For application of these modified surfaces as substrates for solid-phase immunoassays (SPI) and biosensors, the topography of the surface may also play an important role. This is because biomolecular recognition of polystyrene is influenced by both its (bio)chemical nature and its three-dimensional topographic aspects.29 The aim of this work was to investigate the use of polyaniline in activating the surface of the polystyrene by introducing amino groups that are amenable for antibody immobilization. The modified polystyrene surface was characterized by absorption (17) Hobbs, R. N. J. Immunol. Methods 1989, 117, 257. (18) Papadea, C.; Check, I. J.; Reimer, C. B. Clin. Chem. 1985, 31, 1940. (19) Rasmussen, S. R.; Larson, M. R.; Rasmussan, S. E. Anal. Biochem. 1991, 198, 138. (20) Mazia, D.; Schatten, G.; Sale, W. J. Cell Biol. 1975, 66, 198. (21) Fritz, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalis, S. R. Proc. Natl. Acad. Sci. 2002, 99, 14142. (22) Kanungo, M.; Kumar, A.; Contractor, A. Q. Anal. Chem. 2003, 75, 5673. (23) Yu, X.; Sotzing, G. A.; Papadimitrakopoulos, F.; Rusling, J. Anal. Chem. 2003, 75, 4565. (24) Bartlett, P. N.; Birkin, P. R.; Wang, J. H.; Palmisano, F.; De Benedett, G. Anal. Chem. 1998, 70, 3685. (25) Bossi, A.; Piletsky, S. A.; Piletska, E. V.; Righetti, P. G.; Turner, A. P. F. Anal. Chem. 2000, 72, 4296. (26) Rahman, M. A.; Kwon, N.; Won, M.; Choe, E. S.; Shim, Y. Anal. Chem. 2005, 77, 4854. (27) Cosnier, S.; Stoytcheva, M.; Senillou, A.; Perrot, H.; Furriel, R. P. M.; Leone, F. A. Anal. Chem. 1999, 71, 3692. (28) Piletsky, S.; Piletska, E.; Bossi, A.; Turner; N.; Turner, A. Biotechnol. Bioeng. 2003, 82, 86. (29) Hlady, V.; Buijs, J. Curr. Opin. Biotechnol. 1996, 7, 72.
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spectroscopy, AFM, XPS, and contact angle measurement. The specific T3 antibodies were immobilized on the polyanilinemodified surface of indigenously available polystyrene tubes, both through physical adsorption and through a bifunctional spacer, glutaraldehyde. MATERIALS AND METHODS Aniline hydrochloride and ammonium peroxodisulfate were purchased from Fluka, Germany. Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium azide, sodium carbonate, sodium hydrogen carbonate, and bovine serum albumin (BSA) were procured from Sigma Chemicals, U.S.A. Glutaraldehyde and Tris salt were purchased from S. D. Fine Chemicals, Mumbai, India. Concentrated HCl was from Qualigens, Mumbai, India. Radioiodinated T3 having a specific activity of 100 MBq/µg and T3 standards in serum were prepared at ILCJ, BRIT, Mumbai, India. T3 antiserum from rabbit was obtained from Radiopharmaceuticals Division, BARC. Polystyrene star bottom tubes for RIA (12-mm diameter and 75-mm length) were purchased from Tarsons, India. Optical absorption measurements were carried out on a Chemito Spectrascan UV2600 spectrophotometer in the wavelength range of 400-1100 nm. Atomic force microscopy (AFM) measurements were carried out in noncontact mode using a scanning probe microscope (Solver P47, NT-MDT, Russia). Silicon cantilevers (Ultrasharp) having a force constant of 3 N/m and frequency of 60 kHz were used for noncontact imaging. XPS spectra were recorded using a Mg KR (1253.6 eV) source in a Riber XPS system consisting of a twin anode X-ray source (model CX700) and a MAC-2 electron analyzer. The binding energy scale was calibrated to a Au 4f7/2 line of 84 eV. Contact angle measurements were carried out using a Digidrop contact angle meter from GBX Instruments, France. The NaI (Tl) scintillation counter was from ECIL, India. Surface Modification of Polystyrene with Polyaniline. Modification of the inner surface of the polystyrene tubes with a layer of polyaniline was achieved by polymerization of aniline in polystyrene tubes using ammonium peroxodisulfate as the initiator at 25 °C. Typically, 0.5 mL each of aniline hydrochloride in HCl and ammonium peroxodisulfate in distilled water (molar ratio, 1:0.08) were added to the polystyrene tubes and allowed to react for a definite period of time until the visual greenish-blue color of the polymer was observed. The reaction was arrested to avoid further polymerization by simple aspiration or decantation of the solution, followed by immediate repeated washings of the tubes with distilled water. Parameters such as the time required (3-20 min) to stop the polymerization reaction, effect of varying the acidity of the medium (0.0-1.0 M), effect of mixing the two reagents prior to addition into the tubes were studied to optimize the protocol for the formation of polyaniline onto the surface of the polystyrene. Immobilization of T3 Antibodies and T3 Assay. T3 antibody in 0.1 M bicarbonate buffer, pH 9, at a dilution of 1:10 000 was adsorbed onto the PANI-modified surface through direct physical adsorption as well as through glutaraldehyde coupling. The physical adsorption of the antibody involved incubation of the T3 antibody solution in the tubes for 24 h with shaking. The tubes were washed and used for testing its immunological binding with the T3 tracer (%B0/T). For glutaraldehyde activation of the
modified tubes, 0.5 mL of 0.1% glutaraldehyde solution was incubated at 56°C for 2 h, followed by thorough washings of the tubes to avoid cross-linking. A 0.5-mL portion of T3 antibody in 0.1 M bicarbonate buffer, pH 9, was added to these activated tubes for covalent binding. These tubes were incubated for 24 h at 4 °C, followed by washing, and were tested for its immunological binding with T3 tracer. Eight replicate measurements of binding of both sets of the antibody-coated tubes with T3 tracer were carried out to estimate the coefficient of variation (i.e., the ratio of standard deviation to mean of the observation). These were evaluated in the entire range of standards used (0-4.8 ng/mL). The binding at zero standard gives the precision of coating the antibody to the respective tubes, whereas the results of standard curves indicate the precision in the working range. For the construction of T3 standard curves in these antibody-coated tubes (physically adsorbed or through glutaraldehyde coupling), the assay protocol used was as follows: 50 µL of T3 standards in serum and 200 µL of T3 tracer were mixed and incubated for 2 h at room temperature. At the end of the incubation, the tubes were decanted, washed, and measured for radioactivity in a NaI (Tl) scintillation counter. The percentage of the ratio of the binding of T3 standards (B) to the binding at 0-standard (B0) (%B/B0) was calculated to construct dose-response curves. Reproducibility of the coating of the polymer as well as the coating of T3 antibody was studied for eight batches. Stability of the antibody-coated tubes by direct physical adsorption as well as through glutaraldehyde was also evaluated in terms of its binding with the T3 tracer (%B0/T) at zero standard at various time spans after coating when the tubes were stored in airtight packets at 4 °C. RESULTS AND DISCUSSION Surface Modification of Polystyrene with Polyaniline. Chemical polymerization of aniline in acidic medium using ammonium peroxodisulfate in polystyrene tubes led to the deposition of the polyaniline film on the inner surface of the tubes. The onset of polymerization and subsequent deposition of polyaniline onto polystyrene could be identified from the visual observation of blue color in the solution. A fraction of the polymer particles that were formed in solution was found to retain on the surface of polystyrene. The strong adhesion of polyaniline to polystyrene was evident from the fact that the blue color of the film was retained on the tubes even after discarding the reaction mixture and successive washings. The polymerization started after a small induction period that decreased with an increase in the acidity of the reaction medium. The presence of acid increased the rate of polymerization and thereby decreased the induction period. In 0.1 M HCl, the induction period was only ∼3-4 min, and thus, in all further experiments during the preparation of polymer, aniline hydrochloride dissolved in 0.1 M HCl was used. The reaction was arrested after 5 min, thus minimizing the time required for its preparation. With an increase in polymerization time, it is likely that the thickness of the polyaniline film formed on the surface is increased. A majority of the polymerization occurred in the bulk phase, and only a small fraction of the monomer is consumed to polymerize at the interface. To stop the interfacial polymerization, the bulk reagent is discarded, and no attempts were made to estimate the conversion of monomer at the interface.
Figure 1. Absorption spectra of polyaniline-modified polystyrene surface at various pHs.
The absorption spectra of polyaniline films deposited on polystyrene cuvettes monitored at three different pH conditions are shown in Figure 1. Polystyrene did not show any absorption in the visible region, and thus, the unmodified polystyrene cuvette filled with deionized water was used for background subtraction. The absorption spectrum at pH 4 showed a characteristic absorption maximum at ∼810 nm due to the formation of polaron.30 The position of the polaron band is sensitive to the doping level of polyaniline as well as the pH of the solution. Thus, with an increase in pH, the polaron band shifted to lower wavelengths. This shift of the band with an increase in pH of the solution is due to changes in the protonation of the polymer and is consistent with the reports on pH dependence of polyaniline nanoparticles.31 The topographical AFM images of the unmodified and polyaniline-modified surfaces are shown in Figure 2 (a and b, respectively). The adsorption of polyaniline onto polystyrene led to the formation of several crests and troughs on the surface with heights on the order of a few tens of nanometers. Such nanostructures on the surface increased the effective surface area of the solid phase available for antibody binding. This increased surface roughness was quantified in terms of a roughness parameter (Ra), which is defined as N
∑|Z - Z i
Ra )
av|
i)1
N
where, zi is the height of the point i in the image along the z axis from the basal plane, zav is the mean of all the zi values, and N is the total number of points in the image. The roughness parameter Ra is 20.2 nm and the zav is 85.6 nm for a polyaniline modified surface, as opposed to an Ra of 6 nm and zav of 39.7 nm for an unmodified surface. In addition to an increase in the surface area of the solid phase due to an increase in roughness, other parameters, such as electrostatic binding and coupling through (30) Han, M. G.; Cho, S. K.; Oh, S. J.; Im, S. S. Synth. Metals 2002, 126, 53-60. (31) Hassan, P. A.; Sawant, S. N.; Bagkar, N. C.; Yakhmi, J. V. Langmuir 2004, 20, 4874.
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Figure 2. Noncontact AFM image of (a) unmodified polystyrene surface and (b) polyaniline-modified polystyrene surface.
terminal amino groups of the polyaniline can also influence the binding characteristics. The electrostatic binding arises from the fact that there is a possibility of Coulombic attraction between positively charged N atoms in protonated polyaniline and negatively charged carboxylate groups in the antibody at alkaline pH.32 The existence of protonated N in polyaniline was also evident from the optical absorption spectra, as discussed earlier. An increase in hydrophilicity of the surface due to protonated PANI on polystyrene was assessed from contact angle measurements, and it was observed that the static contact angle of the surface with deionized water (pH ) 7) decreased from 67° to 57° upon surface modification. It is reported that an improved recognition of peptides can be observed when antibody immobilization is carried out in a hydrophilic environment.33 Further evidence for the presence of polyaniline on the surface and the oxidation states of N in PANI could be obtained from XPS measurements. Figure 3 shows the XPS spectra of polystyrene before and after polyaniline (PANI) modification. The binding energy of the peaks in the XPS spectra suggested the presence of N (1s) (binding energy ∼400 eV), indicating the formation and adsorption of PANI on the polystyrene surface. The asymmetry of the peak with a shoulder at binding energy >400 eV suggests that N is present in both the N and N+ states.34 This finding is consistent with the observed absorption spectra of polyaniline. A schematic representation of the effect of a polyaniline coating on polystyrene for biomolecule adhesion is shown in Figure 4. Immobilization of T3 Antibody and T3 Assay. The capability of a PANI-modified surface for enhanced biomolecule adhesion can be ascertained from the binding of specific antibodies and the subsequent assay. The percentage binding of T3 antibodies onto the polyaniline-modified polystyrene tubes through physical adsorption was 36 ( 2, as compared to 28 ( 4 that was observed for unmodified tubes (the standard deviation is obtained from six assays, with each assay comprising eight tubes). The nonspecific binding of the T3 tracer on modified as well as unmodified tubes (32) Ohki, S.; Ohshima, H. In Bioelectrochemistry: General Introduction; Caplan, S. R., Ed.; Birkhouser Verlag: Switzerland, 1995; Chapter 4. (33) Gregorius, K.; Mouritsen, S.; Elsner, H. I. J. Immunol. Methods 1995, 181, 65. (34) Kim, B. J.; Oh, S. G.; Han, M. G.; Im, S. S. Synth. Met. 2001, 122, 297304.
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Figure 3. XPS of unmodified and polyaniline-modified polystyrene surface.
Figure 4. Schematic representation of polyaniline-induced enhancement in antibody binding on polystyrene surface.
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