In Situ Studies of Protein Adsorptions on Poly(pyrrole-co-pyrrole

Jan 19, 2007 - An integrated approach for the detailed characterization of an immobilized enzyme. Filipe Carvalho , Patrizia Paradiso , Benilde Sarama...
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Langmuir 2007, 23, 2761-2767

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In Situ Studies of Protein Adsorptions on Poly(pyrrole-co-pyrrole propylic acid) Film by Electrochemical Surface Plasmon Resonance Weihua Hu, Chang Ming Li,* Xiaoqiang Cui, Hua Dong, and Qin Zhou School of Chemical & Biomedical Engineering, Center for AdVanced Bionanosystems, Nanyang Technological UniVersity, 70 Nanyang DriVe, Singapore 637457 ReceiVed October 14, 2006. In Final Form: NoVember 23, 2006

Poly(pyrrole-co-pyrrole propylic acid) (PPy/PPa) composite films were prepared for the first time by electrochemical copolymerization in mixed pyrrole propylic acid (Pa) and pyrrole solutions. The electrochemical growth process was investigated by in situ electrochemical surface plasmon resonance (ESPR). Atomic force microscopy and Fourier transform infrared spectroscopy were applied to characterize the prepared films. Using bovine serum albumin as a model protein, the adsorption kinetics of the protein on PPy/PPa films were studied in situ by SPR. The composition of Pa, the isoelectric point of proteins, the pH of buffers, and surfactant treatment showed dramatic effects on the protein adsorption on the PPy/PPa film. Experimental results indicated that the electrostatic interaction between the PPy/PPa film and proteins plays a critical role in protein adsorption and provided a novel strategy to efficiently immobilize proteins and to reduce nonspecific bindings of proteins in an immunobiosensor.

Introduction Conducting polymers have attracted considerable attention for their promising applications in biosensors,1 electronics,2 and energy storage and conversion devices.3 Polypyrrole (PPy) is one of the most intensively investigated and widely used conducting polymers for biosensors,4 mainly owing to its stability, conductivity, and biocompatibility. PPy can be synthesized by either electrochemical or chemical methods. The electrochemical method is more suitable for fabricating biosensors due to its advantages of being a simple process and low cost. The effects of substrate materials,5 synthesis methods,6 and dopants on the properties of electrodeposited PPy7 have been extensively studied. A major challenge for the construction of PPy-based biosensors is to immobilize highly concentrated probe molecules such as DNA, enzymes, antibodies, or antigens into its matrix with an entire retention of their biological activity and to have good accessibility for the target molecules. Some strategies, including entrapment,8 adsorption,9 affinity interaction,10 and covalent binding,11 have been successfully conducted for the effective immobilization of probe molecules. The entrapment of probe * Corresponding author. Tel.: +65 67904485; fax: +65 67911761; e-mail address: [email protected]. (1) Chen, W.; Li, C. M.; Yang, X.; Sun, C. Q.; Gao, C.; Zeng, Z. X.; Sawyer, J. Front. Biosci. 2005, 10, 2518. (2) Potember, R. S.; Hoffman, R. C.; Hu, H. S.; Cocchiaro, J. E.; Viands, C. A.; Murphy, R. A.; Poehler, T. O. Polymer 1987, 28, 574-580. (3) Novak, P.; Muller, K.; Santhanam, K. S. V.; Haas, O. Chem. ReV. 1997, 97, 207-282. (4) Ramanavicius, A.; Ramanaviciene, A.; Malinauskas, A. Electrochim. Acta 2006, 51, 6025-6037. (5) Li, C. M.; Sun, C. Q.; Chen, W.; Pan, L. Surf. Coat. Technol. 2005, 198, 474-477. (6) Damos, F. S.; Luz, R. C. S.; Kubota, L. T. Electrochim. Acta 2006, 51, 1304-1312. (7) Wang, L. X.; Li, X. G.; Yang, Y. L. React. Funct. Polym. 2001, 47, 125139. (8) Li, C. M.; Sun, C. Q.; Choong, V. E.; Maracas, G.; Zhang, X. J. Front. Biosci. 2005, 10, 180-186. (9) Purvis, D.; Leonardova, O.; Farmakovsky, D.; Cherkasov, V. Biosens. Bioelectron. 2003, 18, 1385-1390. (10) Ionescu, R. E.; Gondran, C.; Gheber, L. A.; Cosnier, S.; Marks, R. S. Anal. Chem. 2004, 76, 6808-6813. (11) Schuhmann, W.; Lammert, R.; Uhe, B.; Schmidt, H. L. Sens. Actuators B 1990, 1, 537-541.

biomolecules constitutes a simple one-step method during the electrochemical polymerization of PPy, but it suffers greatly from the poor accessibility of the target molecules due to its hydrophobicity. The simplest approach is the direct adsorption of probe biomolecules onto the film surface, but the weak stability and random orientation limit its sensing capability. A well-known method is to trap avidin into the PPy film for immobilizing biotinylated biomolecules (DNA, protein, etc.) through the affinity interaction between avidin and biotin. This method is restrained by the tedious biotinylating process. Covalent binding involves the attachment of probe molecules through chemical binding between probe proteins and the carboxyl or amino groups on the PPy surface, which are introduced either by the post-functionalization of the PPy film or the initial electropolymerization of the functionalized pyrrole. This approach allows better orientation for higher activity of probe proteins, making it a promising method for probe attachment in biosensors. Another consideration that should be taken into account for the construction of biosensors is the nonspecific adsorption of proteins on a sensing surface. In most cases, the degree of nonspecific adsorption of proteins on a solid sensing surface determines the sensitivity and specificity of a biosensor. It is important to fundamentally understand the nature of the effects of the nonspecific adsorption of proteins on the performance of biosensors. Some recognized effects were proven to be essential to protein nonspecific adsorption, such as hydrophobicity, size, stability, shape, charge of proteins, charge of adsorption surface, temperature, ionic strength, and buffer pH. The behavior of protein adsorption on some important materials used in biotechnology, such as self-assembled monolayers of different alkanethiols on gold surfaces,12-14 poly(dimethylsiloxane),15 phospholipid bi(12) Cao, C.; Kim, J. P.; Kim, B. W.; Chae, H.; Yoon, H. C.; Yang, S. S.; Sim, S. J. Biosens. Bioelectron. 2006, 21, 2106-2113. (13) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303-8304. (14) Kitano, H.; Kawasaki, A.; Kawasaki, H.; Morokoshi, S. J. Colloid Interface Sci. 2005, 282, 340-348. (15) Wu, D.; Zhao, B.; Dai, Z.; Qin, J.; Lin, B. Lab Chip 2006, 6, 942.

10.1021/la063024d CCC: $37.00 © 2007 American Chemical Society Published on Web 01/19/2007

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Figure 1. Schematic representation of the electropolymerization of a PPy/PPa composite film.

layers,16 silica,17 and immobilized tannin18 was studied. Due to the importance of PPy in biosensors, many efforts have been focused on studies of the nonspecific adsorption of proteins on PPy surfaces. It was found that nonspecific adsorption could be suppressed effectively by doping PPy with different dopants such as dodecyl benzene sulfonate,19 chloride (Cl-), dodecyl, tosylate,20 and octadecyl sulfate. Some modification methods such as aminating PPy,21 immobilizing covalently heparin onto PPy surface,22 and bulk modifying PPy with poly(vinyl alcohol)23 can reduce the nonspecific adsorption as well. However, the mechanism of nonspecific adsorption on PPy is not completely understood. Recently, a sensitive amperometric immunosensor using poly(pyrrole propylic acid) (PPa) films for probe immobilization was reported by our group.24 The abundant carboxyl groups of PPa film provide a versatile platform for direct covalent binding of probe proteins. The hydrophilic nature of the film allows the electroactive species to diffuse to the inner film surface for greater signals. However, the relatively low conductivity of PPa makes the electrochemical growth difficult. The strong surface acid environment of PPa may denature the proteins. The poor conductivity of PPa film could reduce the signal-to-noise ratio. In order to overcome these disadvantages and take the advantage of the PPa film for biosensor applications, in this work, we used Pa as an additive in a monomer pyrrole solution to prepare PPy/ PPa composite films. The electrochemical growth process was investigated by in situ electrochemical surface plasmon resonance (ESPR). Atomic force microscopy (AFM) and Fourier transform infrared (FTIR) were applied to characterize the as-prepared films. The protein adsorption behavior on PPy/PPa composite films was studied in situ by SPR using bovine serum albumin (BSA) as a model protein. The adsorption kinetics of BSA on PPy/PPa composite films and the effects of Pa proportion, the isoelectric point (pI) of the protein, the pH of the buffer, and the surfactant on the adsorption were investigated as well. The results provide an important approach to develop more sensitive and reliable protein sensors. Experimental Section Reagents and Materials. BSA, Cytochrome c, pyrrole, Na2HPO4, citric acid, phosphate-buffered saline (PBS, pH 7.4), KCl, and Tween 20 were purchased from Sigma-Aldrich. Pyrrole was distilled before (16) Glasmastar, K.; Larsson, C.; Hook, F.; Kasemo, B. J. Colloid Interface Sci. 2002, 246, 40-47. (17) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F. J. Phys. Chem. B 1999, 103, 3727-3736. (18) Chatterjee, S.; Chowdhury, R.; Bhattacharya, P. Biochem. Eng. J. 2000, 5, 77-82. (19) Liu, M.; Zhang, Y.; Wang, M.; Deng, C.; Xie, Q.; Yao, S. Polymer 2006, 47, 3372-3381. (20) Azioune, A.; Chehimi, M. M.; Miksa, B.; Basinska, T.; Slomkowski, S. Langmuir 2002, 18, 1150-1156. (21) Zhang, X.; Bai, R.; Tong, Y. W. Sep. Purif. Technol., in press (doi: 10.1016/j. seppur.2006.04.001). (22) Li, Y.; Neoh, K. G.; Kang, E.-T. J. Colloid Interface Sci. 2004, 275, 488-495. (23) Li, Y.; Neoh, K. G.; Cen, L.; Kang, E. T. Langmuir 2005, 21, 1070210709. (24) Dong, H.; Li, C. M.; Chen, W.; Zhou, Q.; Zeng, Z. X.; Luong, J. H. T. Anal. Chem. 2006, 78, 7424-7431.

use. The other chemicals were used without further purification. Pa was synthesized by our group recently,24 and its structure is shown in Figure 1.The buffer used in present work was 0.01 M PBS (pH 7.4), and protein solutions were prepared by dilution with PBS. In particular, Na2HPO4/citric acid buffers (pH 3.6, 4.8, and 6.0) were used to investigate the effect of buffer pH. All solutions were prepared with deionized (DI) water from a Millipore Milli-Q water purification system. Protein solutions were stored at 4 °C before use. Pyrrole and Pa solutions were stored in dark flasks at 4 °C before use. ESPR Equipment. The Autolab SPRINGLE system (Echo Chemie B.V., The Netherlands) was used in this work. Briefly, the experimental setup was based on the Kretschmann optical configuration and used a monochromatic p-polarized laser (λ ) 670 nm) as the light resource. The laser light was directed through a hemicylindrical glass prism (nd ) 1.518 at 25 °C) onto the gold film. The incidence angle (θSPR) was obtained by measuring the intensity of reflected light by a photodiode detector among a dynamic range of 4000 m° (4°) at a frequency of 10 Hz. The cuvette from Autolab SPRINGLE contained a three-electrode system. A gold disk was placed in the cuvette (3 mm diameter) as the working electrode. A platinum bar electrode and a Ag/AgCl/KCl(sat) electrode were used as the counter electrode and reference electrode, respectively. All the potentials reported in this work are relative to the Ag/AgCl/ KCl(sat) reference electrode. An Autolab potentiostat/galvanostat (PGSTAT30, Echo Chemie B.V., The Netherlands) was connected to the Autolab SPRINGLE system to enable electrochemical modulation during in situ electrochemical-optical measurements. In order to eliminate any possible contamination, prior to each experiment, the cuvette was thoroughly cleaned in ultrasonic cleaner with DI water and ethanol, each for 3 min. At the same time, the gold disk electrode was dipped into a freshly prepared H2SO4/H2O (piranha, 7:3 v/v) solution for 3 min followed by rinsing with DI water from the Milli-Q system. Electropolymerization of Polymer Films. The pure PPy film was electrochemically polymerized in a 10 mM pyrrole + 10 mM KCl solution. For preparing the PPy/PPa composite film, solutions with different Pa-to-pyrrole ratios but constant 10 mM total Pa + pyrrole concentration were used. Cyclic voltammetry was chosen to electrochemically polymerize the conducting polymer films in the cuvette from Autolab SPRINGLE, which contained 150 µL of testing solution. The potential was scanned from 0 to 0.76 V for 20 cycles at a scan rate of 100 mV/s. Then the electrolyte solution was drained out followed by intensive washing with PBS buffer. In this report, the notation R:β PPy/PPa (e.g., 9:1 PPy/PPa, 8:2 PPy/PPa, 7:3 PPy/PPa, etc.) is used to represent the corresponding composite film polymerized from the precursor solution containing R mM pyrrole, β mM Pa, and 10 mM KCl. AFM and FTIR Measurements. AFM was used to characterize the surface morphology of pure PPy and 7:3 PPy/PPa composite films for comparison. Measurements were conducted in tapping mode by a Nanoman AFM (Veeco Metrology Group, USA) at ambient temperature. Each film was prepared by the same method described in 2.3, then the gold disk was removed from the glass prism, and the optical oil was washed out carefully without damaging the above film followed by a thorough drying in a gentle N2 gas stream before AFM measurements. FTIR spectra of pure PPy and 7:3 PPy/PPa composite films were recorded with a Nicolet 5700 FTIR spectrometer (Thermo Electron Corporation) through the KBr pellet method. The conducting polymer

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Langmuir, Vol. 23, No. 5, 2007 2763 Table 1. Changes in the SPR Angle (∆θ) and Approximate Thickness of Different Polymer Films Electropolymerized by Cyclic Voltammetry from 0 to 0.76 V for 20 Cycles PPy ∆θ (m°) 1220 approx. thickness (nm) 12.2

Figure 2. SPR response (a) and simultaneous curves of current and SPR angle for the first cycle (b) in the electropolymerization of a 7:3 PPy/PPa composite film in a 10 mM KCl solution by cyclic voltammetry from 0 to 0.76 V at a scan rate of 100 mV/s. films were deposited onto a large-area gold film on wafers by cyclic voltammetry, as described in 2.3, for a long time (overnight), followed by a thorough rinsing with DI water, and then were dried in a vacuum. The dried film was peeled off carefully for FTIR measurements. The spectra were recorded from 4000 to 400 cm-1, with a 4 cm-1 resolution for 32 scans. SPR Measurements. The baseline of the SPR binding curve was obtained after injecting 150 µL of buffer, that is, 0.01 M PBS (pH 7.4), into the cuvette. In particular, in order to investigate the pH influence, Na2HPO4/citric acid solutions with pH 3.6, 4.8, and 6.0 as buffers were used. After obtaining a stable baseline, 150 µL of protein solution was added to replace the buffer solution (indicated by arrow 1 in the binding figures in the next section). After measuring the binding curve, the solution was changed back to the buffer again (indicated by arrow 2 in binding figures). All experiments were conducted at room temperature.

Results and Discussion Electropolymerization of Polymer Films. PPa could be electropolymerized solely, but the anodic current of electrochemical growth was remarkably lower than that of PPy with the same monomer concentration due to its low conductivity.24 In the present work, Pa was only used as an additive into a pyrrole monomer solution for copolymerization. A schematic representation of copolymerization process is shown in Figure 1. Figure 2a shows the SPR responses during the cyclic voltammetry in a solution containing 7 mM pyrrole, 3 mM Pa,

9:1 PPy/PPa 8:2 PPy/PPa 7:3 PPy/PPa 1140 11.4

1080 10.8

950 9.5

and 10 mM KCl. During the electropolymerization, the SPR angle increased from -455 to 495 m° with a discontinuous form after 20 cycles, which indicated that the dielectric constant of 7:3 PPy/PPa film was higher than that of an aqueous solution. The SPR and electrochemical signals were recorded simultaneously during the first cycle, as shown in Figure 2b. The SPR angle increased dramatically after the potential reached 0.45 V versus Ag/AgCl during the anodic scan, suggesting that the electrooxidation of pyrrole and Pa took place at 0.45 V. On the reversal potential scan (i.e., cathodic scan), the SPR angle kept increasing until the potential was lower than 0.65 V, at which point the SPR angle decreased slightly, which could be contributed to the effects of doping and dedoping of Cl- ions on the dielectric constant of polymer films.6 For the electrochemical polymerization of PPy and other PPy/PPa films, SPR and electrochemical responses were similar to that of the 7:3 PPy/PPa film. The thickness of PPy/PPa is critical for its different applications. The SPR measurement could provide more reliable film thickness, which is calculated exactly by the change in the SPR angle (∆θ) based on the Fresnel equation for a four-layer system (prism/ gold film/polymer film/solution)25 in which the thickness of the conducting polymer films is proportional to ∆θ and inversely proportional to the difference between the two refractive indexes of the conducting polymer film phase and the aqueous solution phase. The ∆θ and thickness of each conducting film calculated by the Fresnel equation are shown in Table 1 based on the following parameters:26 n1 (glass) ) 1.518, n2 (gold film@670 nm) ) 0.1372 + 3.7852i, d2 ) 50 nm, n3 (conducting film) ) 1.423, and n4 (aqueous solution) ) 1.334. SPR measurements had a (5% standard deviation, indicating their good reproducibility. One could find from the results that the formation of conducting film causes an increase in the SPR angle at an exact ratio of 100 m°/nm. The results demonstrate that ESPR can be used to monitor in situ the PPy film growth and be further used to control its thickness for tailoring the film. In the results, the refractive index of pure PPy was used to calculate the thickness of composite films. Since the refractive index of composite films might be slightly different from that of pure PPy, the thicknesses of composite films in Table 1 could have deviations from the true values. However, due to the low ratio of PPa to PPy and the minor difference in their chemical structures, the results should have a good reference value. The reproducibility of SPR measurements in this work was good and was demonstrated to have a (5% standard deviation. Currently, we are developing a novel immunoassay sensor according to the results presented here. The details will be reported in a separate paper to be submitted. It was reported that PPy thickness could be estimated by the electric charge passed during the electrochemical polymerization.5 Since the resistance of the film increases with its growth, the thickness is not proportional to the exact passed electric charge. Thus, the thickness determined by the electric charge is not as accurate as that by SPR. Characterization of Polymer Films by AFM and FTIR. Generally, the roughness of a film surface has a notable influence (25) de Bruijn, H. E.; Altenburg, B. S. F.; Kooyman, R. P. H.; Greve, J. Opt. Commun. 1991, 82, 425-432. (26) Yu, J. C. C.; Lai, E. P. C. Synth. Met. 2004, 143, 253-258.

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Figure 3. AFM images of a PPy film (a) and a 7:3 PPy/PPa composite film (b) electropolymerized in a 10 mM KCl aqueous solution by cyclic voltammetry from 0 to 0.76 V for 20 cycles at a scan rate of 100 mV/s.

protein with a molecular weight of 66 kD. Since its amino acid sequence and physical properties have been well-characterized, BSA has been widely used as a model protein. The adsorption rate constant, the most important parameter for adsorption kinetics, can be calculated from the SPR binding curves of a protein obtained under different concentrations of BSA on the film surface. We employed the integrated rate analysis method27 to determine the adsorption rate constant of BSA on the 7:3 PPy/PPa composite film. Generally, for protein adsorption on a solid surface, a simple and reversible bimolecular interaction is proposed: ka

A + B {\ } AB k d

where A is the adsorption protein in aqueous solution, B is the specific sites on the film surface, AB is the adsorption complex, ka is the association rate constant, and kd is the dissociation rate constant. Thus, the adsorption rate of complex AB is Figure 4. FTIR spectra of a PPy film (a) and a 7:3 PPy/PPa composite film (b).

on the protein adsorption. The surface morphologies of PPy and 7:3 PPy/PPa composite films were studied by AFM for comparison, as shown in Figure 3. Both surface profiles are smooth and homogeneous. There is no distinct difference between the two surface morphologies, suggesting that the copolymerization of pyrrole with Pa had no essential influence on the roughness under the preparation conditions. The FTIR spectra of bulk PPy and 7:3 PPy/PPa composite films are shown in Figure 4. The peaks at 1539 and 1411 cm-1 in the spectra could be ascribed to the pyrrole ring characteristic vibration. The peaks at 1274, 1172, 1033, and 910 cm-1 for PPy/PPa film could be signed to the C-H and C-N in-plane deformation vibrations, the N-H stretching vibration, and the out-of-plane deformation of C-H, respectively. A distinct peak around 1704 cm-1 observed only in 7:3 PPy/PPa could be assigned to CdO vibration adsorption, indicating the existence of PPa in the film. As we discussed above, the carboxyl groups introduced by copolymerization could be employed as linkers to covalently immobilize proteins on the surface of PPy/PPa composite films for immunobiosensors. Kinetics of BSA Adsorption on PPy/PPa Film. In immunosensors, the protein adsorption as the primary step in probe immobilization and the interaction between the probe and target molecules is essential. In order to study the important adsorption kinetics in PPy and PPy/PPa films, in situ SPR measurements were conducted for BSA adsorption on the films. BSA is a globular

d[AB]/dt ) ka[A][B]t - kd[AB] because

[B]t ) [B]0 - [AB] thus,

d[AB]/dt ) ka[A]([B]0 - [AB]) - kd[AB] The bulk concentration of A is assumed to be constant in time due to the negligibly small change during adsorption, and the SPR signal R represents the concentration of complex AB on the film surface; therefore,

dR/dt ) kaCRmax - (kaC + kd)Rt Thus,

Rt )

kaCRmax[1 - e-(kaC + kd)t] + R0 kaC + kd Rt ) E*(1 - e-kst) + R0

(1)

where C is the concentration of the protein, Rmax is the maximal change in response to a certain concentration, R0 is the response (27) Oshannessy, D. J.; Brighamburke, M.; Soneson, K. K.; Hensley, P.; Brooks, I. Anal. Biochem. 1993, 212, 457-468.

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Figure 5. SPR binding curves (solid lines) and fitting curves (circles) of BSA with different concentrations in 0.01 M PBS on 7:3 PPy/PPa composite films.

at t ) 0, and

E* ) kaCRmax /(kaC + kd) k s ) k aC + k d On the basis of eq 1, we calculated the values of ks, E*, and R0 by fitting the binding curves of a set of concentrations of the protein. Figure 5 presents the binding curves of 10, 20, and 50 µg/mL BSA in 0.01 M PBS (pH 7.4) on a 7:3 PPy/PPa composite film (solid line) and the fitting curves based on eq 1 (circles). The value of ka and kd calculated from the fitting equation are 4.477 × 102 M-1s-1 and 1.2 × 10-4 s-1, respectively. Figure 5 shows that the fitting curves are reasonably good, in agreement with the experimental binding curves. One could note that the fitting curves and the binding curves have deviations at the beginning, which might indicate that it took time for the bulk BSA concentration to become uniform after its injection into the cell. Although the nonspecific adsorption of proteins and ions will be greatly suppressed after immobilization of the probe proteins due to the occupation of binding sites and the reduction of surface charge, BSA is still used as an effective blocking agent in most immunoassays to cover the probe-attached surface to further eliminate the nonspecific adsorption. Thus, the study of BSA adsorption in this work has its applicational significance. Influence of Pa on Protein Adsorption. The amounts of adsorbed BSA were calculated by the increases in the SPR angle between two buffer phases at a ratio of 1 ng/mm2 per 120 m°.28 Figure 6a presents the binding curves of 20 µg/mL BSA in 0.01 M PBS onto PPy and three different PPy/PPa composite films. The amounts of adsorbed BSA, as shown in Figure 6a, decreased as the ratio of Pa to pyrrole in the films increased. This could be explained by electrostatic interaction between the film surface and the BSA molecules. Pa residues in the conducting polymer films possess net negative charges due to ionization of their carboxyl groups. Thus, the more Pa residues in the film, the more the negative charges present on the film surface. BSA is a protein with a pI of 4.7, which means that it has a net negative charge in neutral 0.01 M PBS. Therefore, the repulsion between the negatively charged PPa component in the PPy/PPa film and (28) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513-526.

Figure 6. SPR binding curves of 20 µg/mL BSA (a) and 20 µg/mL cytochrome c (b) in 0.01 M PBS on different conducting films.

the negatively charged BSA molecules could be used to explain the results obtained in Figure 6a. To ascertain this suggestion, cytochrome c, a protein with a pI of 10.7 and a molecular weight of 12.4 kD, was employed to conduct the same experiment. The result is shown in Figure 6b. It was observed that the adsorption behavior of cytochrome c on four different films showed a completely opposite trend in comparison to the results obtained with BSA. The adsorption amount increased as the ratio of Pa to pyrrole in the films increased. Obviously, since cytochrome c has positive charges in neutral 0.01 M PBS, there should be an electrostatic attractive force between the PPa component in the composite films and the protein molecules, leading to the results in Figure 6b. It could be concluded that the addition of Pa introduced negative charges into the PPy/PPa composite films and that electrostatic interaction played a dominant role in the process of protein adsorption. Thus, the adsorption of proteins with different pI values can be controlled. Influence of Buffer pH on Protein Adsorption. For further understanding of the adsorption behavior of proteins on composite films, 7:3 PPy/PPa composite films were used to investigate the effect of buffer pH on BSA adsorption. As shown in Figure 7, the amount of adsorbed BSA decreases as the pH value increases from 3.6 to 6.0. The results can also be attributed to the electrostatic interaction between the BSA molecules and the PPy/PPa composite film. As discussed above, a BSA molecule is negatively

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Figure 7. SPR binding curves of 20 µg/mL BSA in citric acid/ sodium dihydrogen phosphate buffer with different pH values on a 7:3 PPy/PPa composite film.

charged in buffers with a pH value above its pI, 4.7, and positively charged in buffers with pH < 4.7. The negatively charged PPy/ PPa composite film excludes BSA molecules in buffers with pH > 4.7 but attracts those in buffers with pH < 4.7, which means the lower the buffer pH value, the more adsorbed BSA molecules are on the composite films. At the same time, it has been reported that BSA molecules undergo an irreversible structural transition at pH values below 4,29 which may also affect their adsorption amount. It is known that nonspecific adsorption is caused not only by electrostatic attraction but also by other forces such as hydrogen bonding and hydrophobic and π-π interactions. Therefore, nonspecific adsorption cannot be completely eliminated, even though there is electrostatic expulsion between the film surface and the proteins. Influence of Surfactant Treatment. Surfactant molecules have been widely used as hydrophobic probes to measure the surface hydrophobicity of films as well as biomolecules.30 In the present work, we used Tween 20, a nonionic surfactant with an average molecular weight of 1000, to evaluate the surface hydrophobicity of PPy and PPy/PPa films and to study the effect of surface treatment on the BSA adsorption. Figure 8 shows the binding curves of Tween 20 and BSA adsorptions sequentially carried out on PPy and 7:3 PPy/PPa composite films, respectively. From the Tween 20 solution part of the curves, it was observed that the adsorption of surfactant molecules on the films caused a decrease in the SPR angle, which indicated that the dielectric constant of the surfactant molecule layer on the film surface was lower than that of an aqueous solution. The decrease in the SPR angle for PPy film was larger than that for 7:3 PPy/PPa; that is, PPy film adsorbed more surfactant molecules, which indicated that PPy was more hydrophobic than the PPy/PPa composite film. This is in agreement with the reported results.24 To investigate the adsorption stability of surfactant molecules, the films with surfactant molecules were exposed to 20 µg/mL of BSA solution. As shown in the BSA part of the curves in Figure 8, the binding curve of the PPy film is flat, and there is nearly no increase in the SPR angle after contact is made with (29) Bloomfield, V. Biochemistry 1966, 5, 684. (30) Yamaguchi, S.; Mannen, T.; Nagamune, T. Biotechnol. Lett. 2004, 26, 1081.

Hu et al.

Figure 8. SPR binding curves and an illustration of the corresponding adsorption schemes for sequential adsorptions in 0.05% Tween 20 and 20 µg/mL BSA on different films.

the BSA solution, implying no BSA molecule adsorption on its surface. However, the curve of the 7:3 PPy/PPa film keeps increasing upon the injection of BSA solution. Apparently, the BSA molecules could be adsorbed on the film accompanied by a possible desorption of surfactant molecules. The increase in the SPR angle can be contributed to the adsorption of BSA and perhaps to the desorption of surfactant molecules as well. It should be noted that the sharp increase and decrease in SPR angle upon changing the solution are only due to the different refraction indexes of different solutions. The two binding curves illuminate that the PPy film is more hydrophobic and can adsorb more surfactant molecules with more stability than the PPy/PPa composite film.

Conclusions In this work we demonstrated that PPy/PPa composite films could be prepared by electrochemical copolymerization of pyrrole and Pa. The FTIR spectra indicated the presence of a carboxyl group in the PPy/PPa film and ascertained that Pa was copolymerized. Similar surface morphologies of the PPy/PPa composite film and the pure PPy film were shown by AFM images. SPR was demonstrated to be a useful method for in situ tracking of the film growth and quality control. The kinetics of BSA adsorption on the PPy/PPa film was well studied by an in situ SPR method. Experimental results indicated that the prepared PPy/PPa composite film had a more hydrophilic and negatively charged surface in comparison to that of the pure PPy film. Moreover, the hydrophilicity and charge density of the composite film could be adjusted by changing the proportion of Pa in the electrolyte solution. The adsorption behavior of proteins on the prepared films was dramatically influenced by the Pa composition. The adsorption of positively charged proteins was enhanced, but the adsorption of negatively charged proteins was suppressed. The effects of pH, pI, and Pa composition on the binding behavior of proteins studied in this work provide valuable information for protein immobilizations and target interaction detections in immunobiosensors. The sensitivity and specificity of PPy immunosensors could be significantly improved by modification of the PPy composition by copolymerization and by optimization of the adsorption conditions during probe immobilization and detection, which were demonstrated in this work. The work reported here demonstrates that in situ ESPR could have important applications in studies of electrochemical

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deposition, thickness determination, and surface adsorption kinetics. It can also be used to further develop a simple reporterless immunoassay for diagnostic applications. As follow-up research for this report, a novel ESPR immunosensor is being developed in the authors’ lab.

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Acknowledgment. This work was financially supported by the Center of Advanced Bionanosystems, Nanyang Technological University. LA063024D