Sensitive Human Interleukin 5 Impedimetric ... - ACS Publications

As a model, a human interleukin 5 (IL-5) immunosensor, an important sensor for disease pathology study, clinic diagnosis, and pharmaceutical research,...
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Anal. Chem. 2008, 80, 8485–8492

Sensitive Human Interleukin 5 Impedimetric Sensor Based on Polypyrrole-Pyrrolepropylic Acid-Gold Nanocomposite Wei Chen, Zhisong Lu, and Chang Ming Li* School of Chemical and Biomedical Engineering and Center for Advanced Bionanosystems, Nanyang Technological University, Nanyang Avenue, Singapore 639798 A sensitive impedimetric immunosensor was constructed by using an electropolymerized nanocomposite film containing polypyrrole (PPy), polypyrrolepropylic acid (PPa), and Au nanoparticles. The nanocomposite exhibits good stability, high porosity, high hydrophilicity, and efficient probe immobilization capability. In the film, PPa enhances the hydrophilicity while providing covalent probe attachment linkers, PPy promotes the conductivity and electroactivity, and Au nanoparticles result in good conductivity, high stability, and covalent binding linkers. These combined advantages significantly improve the detection sensitivity in comparison to the conventional methods. As a model, a human interleukin 5 (IL-5) immunosensor, an important sensor for disease pathology study, clinic diagnosis, and pharmaceutical research, was fabricated with the new nanocomposite film. Various optimization works were conducted to improve the detection sensitivity. With the optimal fabrication parameters, the detection limit for IL-5 was 10 fg/mL in phosphate buffered saline (PBS) and 1 pg/mL in 1% human serum with good specificity and a dynamic range of 3 orders of magnitude. This work demonstrates a new approach to develop a sensitive and labeless impedimetric immunosensor for potential broad applications in clinical diagnosis and drug discovery. Impedimetric immunosensors have attracted increasing interest because of their potential advantages over amperometric and potentiometric ones with respect to labeless detection, reduced assay time, multiplexing sensing, and simple operation.1-3 However, they often suffer from low sensitivity and reproducibility. A 100 pM detection limit has been achieved with a new sensing method,4 but it is not good enough to meet the requirements for most clinical diagnostic applications. Thus, there is a great need to significantly improve their sensitivities. Conductive polymers such as polyaniline, polythiophene, and polypyrrole (PPy) have been extensively investigated for applica* To whom correspondence should be addressed. E-mail: [email protected]. (1) Li, C. M.; Chen, W.; Yang, X.; Sun, C. Q.; Gao, C.; Zheng, Z. X.; Sawyer, J. Front. Biosci. 2005, 10, 2518–2526. (2) Jie, M.; Ming, C. Y.; Jing, D.; Cheng, L. S.; Na, L. H.; Jun, F.; Xiang, C. Y. Electrochem. Commun. 1999, 1, 425–428. (3) Ouerghi, O.; Touhami, A.; Jaffrezic-Renault, N.; Martelet, C.; Ben Ouada, H.; Cosnier, S. Bioelectrochemistry 2002, 56, 131–133. (4) Bakker, E.; Qin, Y. Anal. Chem. 2006, 78, 3965–3983. 10.1021/ac8012225 CCC: $40.75  2008 American Chemical Society Published on Web 10/24/2008

tions in electrochemical sensors,5-23 of which PPy has advantages of good biocompatibility, simple synthesis, and easy biomolecule immobilization.24-28 Previously, Li et al.1,14 have demonstrated impedimetric DNA and protein sensors based on pristine PPy thin films. However, the sensors are limited by insufficient stability and sensitivity. The low sensitivity can be attributed to the poor permeability of PPy, which hinders the diffusion of the analyte.29 Pyrrolepropylic acid (Pa), a derivative of pyrrole (Py), can form a highly porous and hydrophilic thin polymer film and is used to (5) Li, C. M.; Sun, C. Q.; Chen, W.; Pan, L. Surf. Coat. Technol. 2005, 198, 474–477. (6) Chen, W.; Li, C. M.; Chen, P.; Sun, C. Q. Electrochim. Acta 2007, 52, 2845– 2849. (7) Chen, W.; Li, C. M.; Yu, L.; Lu, Z.; Zhou, Q. Electrochem. Commun. 2008, 10, 1340–1343. (8) Dong, H.; Li, C. M.; Chen, W.; Zhou, Q.; Zeng, Z. X.; Luong, J. H. T. Anal. Chem. 2006, 78, 7424–7431. (9) Dong, H.; Cao, X. D.; Li, C. M.; Hu, W. H. Biosens. Bioelectron. 2008, 23, 1055–1062. (10) Hu, W. H.; Li, C. M.; Cui, X. Q.; Dong, H.; Zhou, Q. Langmuir 2007, 23, 2761–2767. (11) Wang, J.; Jiang, M.; Fortes, A.; Mukherjee, B. Anal. Chim. Acta 1999, 402, 7–12. (12) Miao, Y. Q.; Guan, J. G. Anal. Lett. 2004, 37, 1053–1062. (13) Kanungo, M.; Srivastava, D. N.; Kumar, A.; Contractor, A. Q. Chem. Commun. 2002, 680–681. (14) Li, C. M.; Sun, C. Q.; Song, S.; Choong, V. E.; Maracas, G.; Zhang, X. J. Front. Biosci. 2005, 10, 180–186. (15) Adeloju, S. B.; Shaw, S. J.; Wallace, G. G. Anal. Chim. Acta 1993, 281, 621–627. (16) Adeloju, S. B.; Barisci, J. N.; Wallace, G. G. Anal. Chim. Acta 1996, 332, 145–153. (17) Aizawa, M.; Haruyama, T.; Khan, G. F.; Kobatake, E.; Ikariyama, Y. Biosens. Bioelectron. 1994, 9, 601–610. (18) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291–293. (19) Ramanaviciene, A.; Ramanavicius, A. Crit. Rev. Anal. Chem. 2002, 32, 245– 252. (20) Fare, T. L.; Cabelli, M. D.; Dallas, S. M.; Herzog, D. P. Biosens. Bioelectron. 1998, 13, 459–470. (21) Garnier, F.; Youssoufi, H. K.; Srivastava, P.; Yassar, A. J. Am. Chem. Soc. 1994, 116, 8813–8814. (22) Youssoufi, H. K.; Hmyene, M.; Garnier, F.; Delabouglise, D. J. Chem. Soc., Chem. Commun. 1993, 1550–1552. (23) Bauerle, P.; Scheib, S. Adv. Mater. 1993, 5, 848–853. (24) John, R.; Spencer, M.; Wallace, G. G.; Smyth, M. R. Anal. Chim. Acta 1991, 249, 381–385. (25) Sadik, O. A.; Wallace, G. G. Anal. Chim. Acta 1993, 279, 209–212. (26) Sadik, O. A.; John, M. J.; Wallace, G. G.; Barnett, D.; Clarke, C.; Laing, D. G. Analyst 1994, 119, 1997–2000. (27) Aoki, T.; Tanino, M.; Sanui, K.; Ogata, N.; Kumakura, K. Biomaterials 1996, 17, 1971–1974. (28) Niwa, O.; Morita, M.; Tabei, H. Anal. Chem. 1990, 62, 447–452. (29) Ionescu, R. E.; Gondran, C.; Gheber, L. A.; Cosnier, S.; Marks, R. S. Anal. Chem. 2004, 76, 6808–6813.

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build an amperometric immunosensor with good sensitivity.8 However, polypyrrolepropylic acid (PPa) has much higher resistivity than PPy and apparently is not good to be directly used for a highly sensitive impedimetric immunosensor due to the low signal-to-noise (S/N) ratio from its high intrinsic impedance. In recent years, PPy based nanocomposites have been studied because of their unique properties inherited from individual components. Particularly PPy-metal nanocomposites are studied to enhance the physical properties.30 PPy-Au nanocomposite with improved electroactivity and stability has been developed in our previous work.6 The Au nanoparticle in the composite can function as a “large counterion” to retard PPy degradation31 and enhance the hydrophilicity by its surface hydrophilic groups, thus making the composite more suitable in the biomedical applications,32 especially in electrochemical sensors with improved conductivity and stability. Efficient probe immobilization is one of the great challenges in construction of a highly sensitive impedimetric immunosensor. Entrapment can simply and conveniently attach biomolecules in the PPy film, but the entrapped randomly oriented biomolecules may lose the bioactivities. Covalent binding is a better strategy to immobilize biomolecules for better probe orientation while increasing the probe density through increasing the linker density in the film. Pa is made in our laboratory, and its carboxyl (COOH) groups can serve as the linkers for covalent biomolecule immobilization.8 Considering the individual advantages of PPy, PPa, and the Au nanoparticle, the nanostructured PPy-PPa-Au composite is designed in this work for a highly sensitive and stable impedimetric immunosensor, in which the sensing film could benefit from the good conductivity of PPy, the enhanced hydrophilicity and covalent binding ability of PPa, and the superior stability and conductivity of Au nanoparticles. IL-5 is an interleukin produced from CD4+ T cells and a 115 amino acid long TH2 cytokine in human,33,34 which stimulates B cell growth and increases immunoglobulin secretion.35,36 IL-5 has been associated with several allergic diseases including allergic rhinitis and asthma.37 Strong evidence indicates that IL-5 plays an important role in allergic asthma pathology.38 Therefore, quantitative detection of IL-5 has great significance for disease pathology study, clinic diagnosis, and pharmaceutical research. Enzyme-linked immunosorbent spot (ELISPOT) and enzymelinked immunosorbent assay (ELISA)39-42 are conventionally (30) Bose, C. S. C.; Rajeshwar, K. J. Electroanal. Chem. 1992, 333, 235–256. (31) Skotheim, T. A., Reynolds, J., Eds.; Handbook of Conducting Polymers, 2nd ed.; Marcel Dekker: New York, 1998. (32) Sethuraman, A.; Han, M.; Kane, R. S.; Belfort, G. Langmuir 2004, 20, 7779– 7788. (33) Secor, W. E.; Stewart, S. J.; Colley, D. G. J. Immunol. 1990, 144, 1484– 1489. (34) Takatsu, K.; Tominaga, A.; Harada, N.; Mita, S.; Matsumoto, M.; Takahashi, T.; Kikuchi, Y.; Yamaguchi, N. Immunol. Rev. 1988, 102, 107–135. (35) Beagley, K. W.; Eldridge, J. H.; Kiyono, H.; Xu, J. C.; McGhee, J. R. FASEB J. 1988, 2, A666–A666. (36) Harriman, G. R.; Kunimoto, D. Y.; Elliott, J. F.; Paetkau, V.; Strober, W. J. Immunol. 1988, 140, 3033–3039. (37) Shen, H. H. H.; Ochkur, S. I.; McGarry, M. P.; Crosby, J. R.; Hines, E. M.; Borchers, M. T.; Wang, H. Y.; Biechelle, T. L.; O’Neill, K. R.; Ansay, T. L.; Colbert, D. C.; Cormier, S. A.; Justice, J. P.; Lee, N. A.; Lee, J. J. J. Immunol. 2003, 170, 3296–3305. (38) Sanderson, C. J. Blood 1992, 79, 3101–3109. (39) Koch, A.; Knobloch, J.; Dammhayn, C.; Raidl, M.; Ruppert, A.; Hag, H.; Rottlaender, D.; Muller, K.; Erdmann, E. Clin. Immunol. 2007, 125, 194– 204.

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methods for immunological detection. Although some detection kits are commercially available, the assay is time-consuming, laborintensive, operation skill demanding, and also requires expensive optical devices. In comparison to ELISA and ELISPOT, an impedimetric immunosensor can apparently offer a fast, inexpensive, convenient, and portable detection technique. In this work, we investigate the PPy-PPa-Au nanocomposite based human IL-5 labeless impedimetric immunosensor, which includes synthesis of the PPy-PPa-Au nanocomposite and its optimization for high electroactivity and stability. Raman spectroscopy, cyclic voltammetry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and alternating current (ac) impedance were used to characterize and/or optimize the nanocomposite. The IL-5 sensor calibration curves were obtained in PBS and PBS + 1% human serum solutions to evaluate their performances. EXPERIMENTAL SECTION Chemicals and Materials. Glassy carbon electrodes (3 mm in diameter), an Ag/AgCl reference electrode, and a Pt wire counter electrode were purchased from CH Instruments (Austin, TX). Rabbit IgG, antirabbit IgG, antigoat IgG, PBS (pH 7.4), acetonitrile, Py, chlorauric acid trihydrate, and human serum were received from Sigma-Aldrich. Sodium citrate tribasic dehydrate and potassium carbonate anhydrous were purchased from Fluka. Tannic acid was purchased from Riedel-de Haen. 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Merck. IL-5 and anti-IL5 IgG were provided by BD Biosciences as gifts. The deionized water (18.2 MΩ cm) was obtained from a Millipore Milli-Q water purification system. All other reagents were of analytical grade. Synthesis of Au Nanoparticles and Pa. Au nanoparticles and Pa were home-synthesized according to our previous works.8,10 Nuclear magnetic resonance (NMR) was used to confirm the Pa purity. The results of 1NMR (CDCl3) are 2.83 (t, 2H, J ) 7.2 Hz), 4.20 (t, 2H, J ) 7.2 Hz), 6.14 (t, 2H, J ) 2.1 Hz), 6.67 (t, 2H, J ) 7.2 Hz), and 9.00 ppm (s, bd peak, 1H). Electrochemical Synthesis and Characterization. Electropolymerization and CV were performed with CHI660B (CH Instruments). In the experiments, the glassy carbon electrodes were polished with 0.3 µm γ-alumina powder, followed by ultrasonic clean for 10 min in deionized water, then soaked in acetone for 5 min, and followed by vigorously washing with deionized water again. Cyclic voltammograms (CVs) with the polished electrodes were measured in 5 mM Fe(CN)63-/ Fe(CN)64- with a scan rate range up to 100 mV/s to confirm that the cathodic and anodic peak potential separation was not more than 60 mV for good performance (the theoretical value is ∼59 mV). Otherwise, the electrode was reconditioned until the desired performance was achieved. All experiments were conducted using a three electrode system with glassy carbon, Pt wire, and Ag/ AgCl (saturated KCl) as the working, counter, and reference electrodes, respectively. Electrochemical impedance spectroscopy (40) Taguchi, T.; McGhee, J. R.; Coffman, R. L.; Beagley, K. W.; Eldridge, J. H.; Takatsu, K.; Kiyono, H. J. Immunol. Methods 1990, 128, 65–73. (41) Borg, L.; Kristiansen, J.; Christensen, J. M.; Jepsen, K. F.; Poulsen, L. K. Clin. Chem. Lab. Med. 2002, 40, 509–519. (42) Tary-Lehmann, M.; Hricik, D. E.; Justice, A. C.; Potter, N. S.; Heeger, P. S. Transplantation 1998, 66, 219–224.

Scheme 1. Schematic of the Immobilization of the Protein Probe via the Activation of Carboxylic Functional Groups (Activated by EDC)a

a

GCE: glassy carbon electrode.

(EIS) was conducted to characterize PPy-PPa and PPy-PPa-Au nanocomposite films and antibody-antigen interaction by using CHI660B equipped with the frequency response analysis module. All EIS were measured at open circuit voltage (OCV) over a frequency range of 1 to 105 Hz in 0.01 M PBS (pH ) 7.4). Both CV and EIS characterization for the electroactivity of different nanocomposite films were conducted without using any reporter such as ferricyanide. SEM Characterization. The surface morphologies of the PPy-PPa and PPy-PPa-Au nanocomposite films were characterized by field emission (FE)-SEM (JSM-6500F Japan). Before characterization, the samples were stored in vacuum overnight. Raman Spectroscopy Characterization. Raman spectroscopy was performed with the Renishaw Micro Raman spectrometer by using 632.8 nm lines of the He-Ne laser as the excitation source over the range of 400-2000 cm-1. TEM Characterization. TEM characterization of the nanocomposite film was performed with a JEOL JEM-2010 system. The sample film was peeled off from the electrode and ground in an agate mortar with acetone. Determination of Carboxylic Group Density. The density of the carboxyl in the films was quantified by using the toluidine blue O (TBO) method.43-45 The nanocomposite coated electrodes were soaked in 5 × 10-4 M TBO with pH 10 adjusted by NaOH. The formation of ionic complexes between the COOH group and cationic dye was carried out for 5 h at room temperature. The nonspecific adsorbed dye was removed by rinsing with pH 10 NaOH solution three times. Desorption of the dye to determine the quantity of COOH group was conducted in 50 wt % acetic acid solution, which was measured by the absorbance of dye at 633 nm with a HITACHI U-2800 double beam system. Immunosensor Fabrication. A typical protocol to fabricate the PPy-PPa-Au nanocomposite immunosensor is shown in Scheme 1. The precursor solution for polymerization typically contained 0.26 M Py, 0.065 M Pa, 0.01 M PBS, and 0.15 mM Au nanoparticles, but various compositions were used for optimization experiments. The PPy-PPa-Au nanocomposite film was electrochemically deposited in one step by applying 1.5 mA cm-2 (43) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1993, 9, 1121–1124. (44) Li, B.; Ma, Y.; Moran, P. M. Biomaterials 2005, 26, 4956–4963. (45) Hu, J.; Yin, C.; Mao, H. Q.; Tamada, K.; Knoll, W. Adv. Funct. Mater. 2003, 13, 692–697.

constant current for 300 s. The coated electrode was washed three times with 0.01 M PBS and dried under a gentle nitrogen stream. The electrode was then immersed in acetonitrile containing 1.5 wt % EDC for 1.5 h at room temperature. After carefully rinsing with acetonitrile, the electrode was exposed to a solution containing 100 µg/mL IL-5 overnight, followed by washing in PBS to remove nonspecific absorbed proteins. RESULTS AND DISCUSSION Effect of Au Nanoparticle on the Nanocomposite. The effect of incorporated Au nanoparticles on the electrochemical behavior of the produced PPy-PPa-Au nanocomposite film was investigated. The E-t curve (E, deposition potential; t, deposition time) in the inset of Figure 1a shows that the deposition potential of PPy-PPa is always higher than that of PPy-PPa-Au during the whole deposition process at the constant current, indicating that the electrochemical deposition of PPy-PPa has higher polarization potential. Particularly, the PPy-PPa deposition potential sharply increases to 0.94 V, compared to 0.85 V for the PPy-PPa-Au, demonstrating an even higher polarization at the beginning. Clearly, the lower polarization potential comes from the incorporation of Au nanoparticles into the composite. In our previous work,7 the AFM in situ study reveals that the Au nanoparticle has stronger affinity with the electrode surface than Py to be a nucleus for facilitating the nucleation and growth process during the electrochemical deposition. CVs of the PPy-PPa and PPy-PPa-Au films that have the same amount of electrode material and the same thickness because of the same electricity consumed for the synthesis5 (Figure 1a) exhibit two well-defined doping/dedoping redox peaks. The different CV shapes for the PPy-PPa and PPy-PPa-Au can be ascribed to their different electroactivities, which have been also reported in our previous work.6 The PPy-PPa-Au film shows significantly higher peak current and much less peak potential separation than PPy-PPa, indicating its much higher electroactivity. This could be ascribed to the higher electrochemical reactive surface area and better catalytic properties of the PPy-PPa-Au composite. CVs obtained with different scan rates (Figure 1b) illustrate symmetric features, and the linear relationship of peak current vs the scan rate indicates the nature of surface electrode reactions with fast charge transfer rates. Analytical Chemistry, Vol. 80, No. 22, November 15, 2008

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Figure 1. The electrochemical behavior of PPy-PPa-Au nanocomposite: (a) cyclic voltammograms of PPy-PPa and PPy-PPa-Au nanoparticle film in 0.01 M PBS obtained with a scan rate of 100 mV/s. Inset: the galvanostatic deposition of the PPy-PPa film (solid line) and the PPy-PPa-Au nanoparticle film (dashed line) on the glassy carbon electrode. (b) Cyclic voltammograms of the PPy-PPa-Au film in 0.01 M PBS with different scan rates: 20 (A), 40 (B), 60 (C), 80 (D), 100 (E), 120 (F), 140 (G) mV/s. Inset: the dependence of the oxidation peak current on the scan rate.

Figure 2. SEM images of (a) the PPy-PPA film and (b) the PPy-PPA-Au nanocomposite.

Morphologies of PPy-PPa and PPy-PPa-Au Films. The morphology of PPy-PPa-Au nanocomposite film is significantly different from PPy-PPa as shown in the SEM images (Figure 2), in which the PPy-PPa-Au film (Figure 2b) shows a significantly more porous structure than the dense PPy-PPa film (Figure 2a), apparently possessing a higher specific surface area. The larger particles of PPy-PPa-Au are produced by the effect of Au nanoparticles during the polymer growth.7 The Au nanoparticles can bind with monomer Py or Pa through the carboxyl groups in their antiaggregation layer, which has been proved in our previous work,7 and further become nuclei, resulting in parallel electrodeposition processes from both Au nanoparticles and Py-Pa monomers.7 The Au nanoparticle shows faster nucleation and growth rate than Py and Pa monomers7 since it can attach a number of monomer Py or Pa molecules, resulting in larger polymerized particles than that nucleated from Py and Pa. This 8488

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is supported by in situ AFM measurements.7 This is why a less uniform, rougher, and porous morphology of the PPy-PPA-Au film is observed in Figure 2. Effect of Au Nanoparticles on Stability and Conductivity of the Nanocomposite. The electrochemical stabilities of PPy-PPa-Au and PPy-PPa films were compared by measuring their impedance changes in 0.01 M PBS at room temperature for a long period. The impedance result measured in an electrochemical system is often modeled by an electronic equivalent circuit for the solution resistance, Rs, the Warburg impedance, Zw, resulting from the diffusion of ions, the double layer capacitance, Cdl, and Rct for the electrochemical processes.1,14 The increased Rct for the doping/dedoping process of a PPy thin film for a long time measurement can be used to represent the degradation of the polymer in its electroactivity. In Li’s early work,1 a normalized resistance, ∆Rct is introduced to eliminate the variation of PPy

Figure 3. Comparison of PPy-PPa and PPy-PPa-Au nanocomposites: (a) electrochemical stability comparison of the PPy-PPa-Au and the PPy-PPa films in 0.01 M PBS at room temperature, (b) electric transport I-V curves of PPy-PPa-Au and PPy-PPa, (c) Raman spectra comparison of the PPy-PPa and the PPy-PPa-Au nanocomposites, and (d) TEM image of the PPy-PPa-Au film.

films for multiple detections with different electrodes, which is expressed as ∆Rct)(Rct2 - Rct1) ⁄ Rct1

(1)

where Rct1 and Rct2 are the resistances measured at the beginning and an immersed time, respectively. Clearly, a larger ∆Rct indicates poorer stability. The change of ∆Rct of the PPy-PPa-Au film in 2.5 h is less than 10% while the PPy-PPa film increases by 23% as shown in Figure 3a. The degradation of the composite film could be ascribed to damages of the positively charged backbone of the polymer by the nucleophilic attack from the solution species.46 The results indicate that the incorporation of Au nanoparticles significantly enhances the stability of the PPy composite. It is known that a large counterion doped in a conductive polymer can effectively inhibit the nucleophilic attack6 for better stability. In PPy-PPa-Au film, it is likely that the negatively charged Au nanoparticles can function as a very large counterion doped in the polymer matrix for the improved stability. Figure 3b shows the electrical transport I-V curves of PPy-PPa and PPy-PPa-Au films under dry conditions measured (46) Beck, F.; Braun, P.; Oberst, M. Ber. Bunsen-Ges. 1987, 91, 967–974.

with conducting AFM measurements. The linear relationship of I vs V indicates the sound conducting nature of both films, and the slopes can represent their conductivities. The conductivities calculated from the I-V curves are 2.10 × 10-5 and 5.52 × 10-5 S for PPy-PPa and PPy-PPa-Au films, respectively, demonstrating that PPy-PPa-Au film has 2.6 times better conductivity than the PPy-PPa film. To study the mechanism of the conductivity improvement, the Raman spectra of these films were measured as shown in Figure 3c. The peak located in the range of 1513.5-1646.8 cm-1 represents the CdC backbone stretching of PPy,47,48 which is related to the conjugating length of the polymer chain and the conductivity of the polymer.49 The peak position of the CdC backbone shifts from 1596.0 in the PPy-PPa film to 1589.7 cm-1 in the PPy-PPa-Au film, indicating that the conjugation is shortened in the PPy-PPa-Au composite, thus resulting in higher conductivity.49 The peak in the range of 1020-1080 cm-1 can be assigned to the C-H in-plane deformation. The Raman peak intensity can characterize the size of the (47) Zhong, C. J.; Tian, Z. Q.; Tian, Z. W. J. Phys. Chem. 1990, 94, 2171–2175. (48) Schantz, S.; Torell, L. M.; Stevens, J. R. J. Appl. Phys. 1988, 64, 2038– 2043. (49) Tian, B.; Zerbi, G. J. Chem. Phys. 1990, 92, 3892–3898.

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Figure 4. CVs of PPy-PPa-Au films deposited in 0.01 M PBS with 30 mC by applying different current density: 1.77, 1.06, 0.71, 0.53, 0.35, 0.18, 0.11, and 0.035 mA cm-2 at 60, 100, 150, 200, 300, 600, 1000, and 3000 s, respectively. (Scan rate, 50 mV/s; vs Ag/AgCl, reference electrode).

anion dopant, of which the larger size can result in higher conductivity.50 The increased peak intensity at 986.7 cm-1 for the PPy-PPa-Au film shown in Figure 3c can be assigned to the larger “anion” dopant, and the negatively charged Au nanoparticles can also contribute to the improved conductivity.50 The Raman spectrum results clearly explain the mechanism of the conductivity improvement. Thus, incorporation of Au nanoparticles into the composite not only improves the stability but also the conductivity of the conductive polymer. The incorporation of Au nanoparticles was directly confirmed by the TEM, as shown in Figure 3d. The dark spot clearly demonstrates the existence of nanocrystalline Au particles in the PPy-PPa matrix. The conductive polymer composite film with significantly improved stability and conductivity is expected to have much better sensitivity and reliability in electrochemical sensor applications, particularly in an impedimetric sensor application. Effect of Deposition Current Density on Electroactivity of the Nanocomposite. PPy-PPa-Au composite deposition was carried out by using constant current to give better electroactivity.5 In order to optimize the film for the impedimetric sensor, different current densities were applied while passing the same electric charge density of 0.429 C cm-2 by controlling the deposition time. The electroactivity of the resulting film was examined by cyclic voltammetry in 0.01 M PBS as shown in Figure 4. The CV shapes in Figure 4 vary with the magnitude of the deposition current, indicating their different electroactivities as discussed above. For the two pairs of redox peaks, which have been studied in the early investigation,6 the peak current starts to decrease on the films deposited at a current density higher than 1.06 mA cm-2. The loss of the electroactivity is very likely due to overoxidation at such deposition current densities.46 The composite films produced with current densities of 0.71, 0.53, and 0.35 mA cm-2 show the best performance in both peak current and reversibility. The films deposited with low current densities of 0.18, 0.11, and 0.035 mA cm-2 show high redox peaks but have worse reversibility than those deposited with current density of 0.71, 0.53, and 0.35 mA cm-2 in terms of the peak separation. The low deposition current (50) Liu, Y. C.; Tsai, C. J. Chem. Mater. 2003, 15, 320–326.

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density forms more dense morphology (data not shown), since it causes a slow deposition rate allowing better alignment of the polymer chains for less porosity of the film.51 The worse reversibility may possibly also come from poorer diffusion of doping ions in the denser film. To compromise electroactivity and reaction surface area from the porosity, electrodeposition at 0.35 mA cm-2 for 300 s was chosen to synthesize the PPy-PPa-Au film for other optimization works and the immunosensor as well. Effect of Py to Pa Ratio on Sensitivity of the Nanocomposite Based Impedimetric Immunosensor. Because of the nature of PPy and PPa, of which PPy is conductive but hydrophobic, while PPa is less conductive but very hydrophilic, it is critical to optimize the ratio of Py to Pa to have a composite film with good conductivity and hydrophilicity for a sensitive impedimetric immunosensor. The ratio of Py to Pa in the film was tailored by change of the ratio of Py to Pa in the deposition solution, and the electrochemical activities of the films produced with different ratios were evaluated by cyclic voltammetry as shown in Figure 5a, illustrating that the films produced with a Py percentage of Py + Pa lower than 50% have very weak redox peaks, clearly due to their poor electrochemical activities caused from high contents of the poorly conductive PPa8 in the composite. However, when the ratio is higher than 50%, the films show better electrochemical activities. The Pa to form the poorly conductive PPa can also result in high deposition potential8 to produce overoxidized PPy with poor electroactivity. However, a reasonable amount of PPa is needed to provide a high concentration of COOH groups for a large amount of covalently immobilized protein probes. The densities of COOH groups in various films were measured as shown in Figure 5b, demonstrating that the Py percentage in Py + Pa should be higher than 70% to have the highest density of COOH while producing the excellent electroactivity. Interestingly, incorporation of Au nanoparticles in the composites also significantly increases the density of COOH groups, which is caused by the COOH groups in the antiaggregation layer on the Au nanoparticles discussed above and more porous morphologies as shown in Figure 2b. The antirabbit IgG impedimetric immunosensor was fabricated from rabbit IgG-immobilized PPy-PPa-Au films to explore the optimal Py to Pa ratio in the deposition solution. Figure 5c shows the immunosensing responses to the sensor films deposited with different Py to Pa ratios. The measurements were conducted in 100 ng/mL antirabbit IgG + 0.01 M PBS. The result shows that the immunosensor response increases with the increase of the ratio until reaching its maximum value at 80% and then sharply dropping at 90%. This result indicates that when the Py ratio is smaller than 80%, the film has enough COOH groups for the efficient immobilization of the probe molecules, and the sensitivity of the impedimetric sensor is mainly dependent on the film electroactivity. However, when the ratio continues to increase to hit at a ratio of 90%, the film could not have enough COOH functional groups for the covalent bindings of the probe molecules, resulting in a significant reduction of the sensitivity. Impedimetric immunosensors made with different probe protein-immobilized PPy-PPa-Au composite films were used to investigate their optimal Py to Pa ratio, and almost the same optimal ratio value (51) Bufon, C. C. B.; Vollmer, J.; Heinzel, T.; Espindola, P.; John, H.; Heinze, J. J. Phys. Chem. B 2005, 109, 19191–19199.

Figure 5. Optimal Py ratio for PPy-PPa-Au nanocomposite films: (a) CV of the PPy-PPa-Au films in 0.01 M PBS which were electrochemically deposited in the solution containing different Py ratios from 10% to 90%, (b) COOH group density comparison of different PPy-PPa films with or without the existence of Au nanoparticles in precursor solutions, and (c) relation of the Py ratio in Py and Pa solution and the response of the immunosensor in 0.01 M PBS.

Figure 6. Optimization of the incubation time for the immunosensor: (a) immunosensor using rabbit IgG as the probe in 100 ng/mL antirabbit IgG solution and (b) immunosensor using IL-5 as the probe in 100 ng/mL anti-IL-5 IgG solution.

was obtained (data not shown). Therefore, the optimal Py to Py + Pa in the deposition solution is determined as 80%. Optimal Incubation Time for the Immunosensor. ∆Rct of the immunosensor fabricated with the optimal conditions discussed above was determined by Rct1 and Rct2 measured before and after the antibody-antigen binding for different incubation time in 100 ng/mL antirabbit IgG + 0.01 M PBS and anti-IL-5 IgG + 0.01 M PBS, respectively, to investigate the optimal incubation time, which is one of the important performance indicators since it is directly related to the assay time. Obviously, a larger ∆Rct indicates higher sensitivity. As shown in Figure 6, in the first 30 min, the ∆Rct increases rapidly, and after 30 min, this trend is slowed down and reaches plateaus to show the saturation. Therefore, we choose 60 min as the optimal incubation time for the immunosensor. IL-5 Impedimetric Immunosensor. The immunosensor performance for anti-IL-5 IgG detection was tested in both 0.01 M PBS and 1% human serum solution. The relation of response vs analyte concentration (C) for a sensor is determined by the sensing mechanism or reaction orders and could be linear or semilogarithmic as reported,8,52-54 which can be used to estimate the dynamic range. However, the determined analyte concentration for a real sample from the semilogarithmic calibration curve (log C) has to be converted back to C for the clinical analysis. The impedimetric sensor in this work shows a semilogarithmic

calibration curve between Rct and C. Figure 7a displays the calibration curves of ∆Rct vs different concentrations of anti-IL-5 IgG in PBS, demonstrating a linear relationship over a dynamic range of 4 orders of magnitude and a 10 fg/mL detection limit. This is a very sensitive electrochemical impedimetric sensor, which can satisfy most of the critical application requirements. The significant improvement in sensitivity can be attributed to the improved stability, good conductivity, high hydrophilicity, high porosity, and efficient covalent probe-immobilization ability of the PPy-PPa-Au composite sensing layer, which are clearly inherited from the individual components of the composites, i.e., PPy, PPa, and Au nanoparticles. The measurements were also conducted in 1% human serum to further evaluate the specificity of the immunosensor. Immunoglobulin (Ig) has five different Ig classes (IgG, IgM, IgD, IgA, and IgE) based on differences in the constant region of the heavy chain. Antigen produced by infectious diseases usually lead to a strong IgG response in human serum. Thus, the immunosensor was validated in 1% human serum for its specificity and sensitivity, and the result is shown in Figure 7b. The detection limit of the sensor is 1 pg/mL in a dynamic range between 1 pg/ (52) Toda, K.; Tsuboi, M.; Sekiya, N.; Ikeda, M.; Yoshioka, K. I. Anal. Chim. Acta 2002, 463, 219–227. (53) Bae, Y. M.; Oh, B. K.; Lee, W.; Lee, W. H.; Choi, J. W. Anal. Chem. 2004, 76, 1799–1803. (54) Chou, S. F.; Hsu, W. L.; Hwang, J. M.; Chen, C. Y. Biosens. Bioelectron. 2004, 19, 999–1005.

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Figure 7. Performance of the IL-5 sensor, anti-IL-5 antibody concentrations: 1000, 100, 10, 1, and 0 pg/mL (a) in 0.01 M PBS and (b) in 0.01 M PBS with 1% human serum. Inset: Selectivity test of the sensor in 1% human serum solution containing 1 ng/mL antirabbit antibody, antigoat antibody, and anti-IL-5 antibody, respectively.

mL and 1 ng/mL, demonstrating a good sensitivity even in the 1% human serum sample. The specificity of the immunosensor was investigated in 1% human serum solutions containing 1 ng/ mL antigoat IgG, antirabbit IgG, and anti-IL-5 IgG, respectively. The inset of Figure 7b shows that the immunosensor gives the significantly higher response to the specific analyte in comparison with nonspecific ones, indicating its good selectivity. Because of its great advantages of simplicity, low cost, fast assay time, and ability to be used in an electronic and even portable device, the impedimetric immunosensor reported in this work should have great broad applications in clinical diagnosis and environmental monitoring. However, although the device is sensitive, the detection limit in serum is significantly reduced from 10 fg/mL to 1 pg/mL. This is because in detection in serum, a much larger background is observed to significantly reduce the S/N ratio of the sensor.

contributions from the individual components in the nanocomposite, in which PPa enhances the hydrophilicity while providing covalent probe immobilization linkers, PPy promotes the conductivity and electroactivity, and Au nanoparticles offer good conductivity, high stability, and covalent binding linkers as well. Most importantly, because Au nanoparticle plays an important role in the nucleation and growth of the composite film, a highly porous nanostructured composite is formed to have high reaction surface for a high sensitivity. This work demonstrates a universal method to fabricate highly sensitive sensors through the nanocomposite approach for not only inheriting individual advantages from the composed components but also benefiting from its synergy effect such as the porosity structure of PPy-PPa-Au. The nanocomposite based immunosensor has great potential to construct sensing devices for clinical, pharmaceutical, environmental, and food safety applications.

CONCLUSION In brief, our study shows that the PPy-PPa-Au nanocomposite has much better stability, porosity, hydrophilicity, and efficient probe immobilization capability in comparison with pristine PPy material that we used previously in the construction of an immunosensor. Excellent sensitivity and selectivity of the composite based sensor are demonstrated via anti-IL5 IgG detection. The significantly improved sensitivity is ascribed to the joint

ACKNOWLEDGMENT This work was financially supported by Center of Advanced Bionanosystems, Nanyang Technological University, Singapore.

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Received for review June 16, 2008. Accepted September 14, 2008. AC8012225