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Electrochemical impedance characterization of antibody–antigen interaction with signal amplification based on polypyrrole–streptavidin
Biosensors and Bioelectronics 22 (2007) 3161–3166
Electrochemical impedance characterization of antibody–antigen interaction with signal amplification based on polypyrrole–streptavidin Yinghong Xiao a,b,c , Chang Ming Li a,b,∗ , Yingshuai Liu a,b a
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore b Center for Advanced Bionanosystems, Nanyang Technological University, Singapore 637457, Singapore c School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210014, China Received 5 October 2006; received in revised form 14 December 2006; accepted 9 February 2007 Available online 17 February 2007
Abstract Streptavidin, as a dopant, has been incorporated into a polypyrrole film to bind biotinylated antibody onto the electrode surface. With four biotin binding sites, the incorporation of streptavdin, as confirmed by FTIR and impedance spectroscopy, provided a new method to amplify the response signal from antibody–antigen interaction. Biotinylated anti-goat IgG, as a probe, and goat IgG, as a target, were employed to evaluate the characteristics of the biosensor. With the amplification strategy, the detection sensitivity of the electrochemical impedance spectroscopy was significantly improved. A linear relationship between the charge transfer resistance change (Rt ) and the concentration of goat IgG ranging from 10 pg/ml to100 ng/ml was obtained. © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemical impedance; Polypyrrole; Streptavidin; Immunoreaction
1. Introduction The utilization of conducting electroactive polymers in electrochemical sensors has been increasing over the past decades (Berdichevsky and Lo, 2006; Kros et al., 2005; Li et al., 2005a,b; Arora et al., 2006). The unique feature of conducting polymer applications derives from the ability of well tuning the polymers with different counter ions including biomolecules, making it possible to detect a wide range of analytes. Although the use of conducting polymers has been restricted by the limited choice of dopants (electronegativity, molecular weight, etc.), the electrochemical formation of the polymer layer with controlled thickness could effectively construct a reproducible biosensor (Cosnier, 1999). The barriers can be circumvented by attaching biomolecules to the surface of the polymer film through specific coupling (George et al., 2006) or ion exchanging (Xiao et al., 2006). Polypyrrole (PPy) has been widely used for biological applications due to its beneficial chemical properties, aque∗ Corresponding author at: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore. Tel.: +65 67904485; fax: +65 67911761. E-mail address: [email protected] (C.M. Li).
0956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2007.02.008
ous solubility, simplicity of preparation, biocompatibility, and easy immobilization of biological molecules (Shi et al., 2004; Ramanavicius et al., 2006; Bousalem et al., 2003; Geetha et al., 2006). Thus, the immobilization of biomolecules in electropolymerized PPy films is gaining importance apart from conventional methods such as covalent binding, etc. (Cosnier, 1999). Immunosensors have aroused great interest with expectations of providing fast and highly sensitive immunological response. Immunosensors have widespread applications in clinical diagnostics, food safety and quality, biological analysis, and environmental monitoring (Livache et al., 1998; Terry et al., 2005; Ronkainen-Matsuno et al., 2002; Gonzalez-Martinez et al., 1999). Electrochemical methods appear very promising due to the relatively simple and compact equipment required. In particular, electrochemical impedance spectroscopy (EIS) represents a powerful method for probing the interfacial reaction mechanisms of modified electrodes (Xiao and Mansfeld, 1994; Katz and Willner, 2003), providing a rapid approach for monitoring the dynamics of biomolecular interaction. Thus, EIS is often used for investigating chemical transformations and processes associated with the conductive supports (Li et al., 2005a,b; Lee and Shim, 2001; Brillas et al., 1997). Electrochemical impedance immunosensing has attracted extensive interest
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Scheme 1. Schematic diagram of the principle and sequence of steps for the proposed protocol.
for quantifying the interaction between antibody and antigen (Darain et al., 2004). Impedance immunosensing could provide a general method to follow the procedure of antigen–antibody reaction at modified electrode surfaces. It is known that the antigen–antibody complex acts as a blocking layer to effect a change in the measured impedance. However, the immune reaction of antigen with antibody directly incorporated in conducting polymers is usually not sufficient to generate a significant and detectable signal change for impedance measurements. In this study, we modified the electrode surface according to the protocol illustrated in Scheme 1. PPy was electrochemically deposited on a gold electrode with streptavidin as a dopant to form an electropolymerized PPy/streptavidin film. It was plausible to use streptavidin, negatively charged, as a counter anion for PPy synthesis and biotinylated antibody was immobilized on the PPy/streptavidin film via well-known streptavidin–biotin interaction. The attachment of antibody to the electrode surface through the specific avidin (or streptavidin)–biotin molecular interaction has been revealed as an effective and reliable approach (Wilchek and Bayer, 1988). Streptavidin with four biotin binding sites, adheres to the biotinylated antibodies and thus increases the antibody loading. 2. Experiments 2.1. Materials Pyrrole monomer (>98%) and sodium dodecyl sulfate (SDS) were purchased from Aldrich (Milwaukee, WI). Streptavidin was from Sigma–Aldrich, of which 55.5 g (1 unit) will bind 1.0 g biotin at pH 7.5. Biotinylated monoclonal anti-goat immunoglobulin G (IgG) antibody, rabbit anti-goat IgG antibody and goat IgG from goat serum were purchased from Sigma (St. Louis, MO) and diluted with 0.01 M phosphate buffer saline solution (PBS, pH 7.4) to different concentrations. 2.2. Instrumentation Electrochemical deposition of PPy and electrochemical measurements (impedance and cyclic voltammetry) were conducted
with an electrochemical workstation CHI 760B (CH Instruments, Austin, TX). EIS was performed in 0.01 M PBS, pH 7.4 over a wide range of frequencies from 100 to 105 Hz. Cyclic voltammetry was conducted by potential scanning between −0.9 and 0.8 V, with a scan rate of 100 mV/s. The UV–vis Hitachi spectrometer (U-2800, Tokyo, Japan) was used for the stability studies of the doped streptavidin. Incorporation of streptavidin in the PPy film was demonstrated by using FTIR spectroscopy (Spectrum GX, Perkin-Elmer, Wellesley, MA). Samples were prepared as KBr pellets. The morphology of the PPy film incorporated with streptavidin was studied with a scanning electron microscope (JEOL JSM-6700F FEG, Japan), operating at 5.0 kV. Milli-Q water of 18.2 M cm (Millipore, Inc.) was used in all experiments. 2.3. Electropolymerization of the PPy/streptavidin film on gold electrodes Electropolymerization was conducted in a three-electrode electrochemical cell with a gold electrode, platinum wire, and saturated calomel electrode (SCE) as the working, counter, and reference electrode, respectively. Before electropolymerization, the gold electrode (diameter of 2 mm) was polished with 1.0, 0.3, and 0.05 m alumina slurry, respectively, followed by rinsing with deionized water and cleaning in an ultrasonic bath for 10 min. Electropolymerization was performed at 0.8 V for 8 min in a solution containing 0.1 M pyrrole, 0.02 M SDS, and 0.2 mg/ml streptavidin. The solution was bubbled with nitrogen for 20 min before use. 2.4. Immobilization of biotinylated anti-goat IgG on the PPy/streptavidin film The PPy/streptavidin modified electrode was immersed in casein solution (0.3%, w/v) for 1 h at room temperature to eliminate nonspecific adsorption. After thoroughly rinsed with copious PBS for 5 min, the electrode was dried with a gentle nitrogen stream. Biotinylated anti-goat IgG (1.7 g/ml) was immobilized on PPy/streptavidin films at different incubation times, followed by thorough rinsing with PBS.
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2.5. Incubation of goat IgG on the immobilized biotinylated anti-goat IgG Before reacted with goat IgG, the antibody-immobilized electrode was blocked with casein for 1 h at room temperature, followed by thorough rinse with PBS. Goat IgG at different concentrations was incubated on immobilized biotinylated antigoat IgG for 1 h at room temperature, followed by rinsing with PBS. 3. Results and discussion 3.1. Electrochemical deposition of the PPy/streptavidin thin film PPy can be electrochemically synthesized on different substrates (Li et al., 2005a,b). In solutions of neutral pH proteins are usually not highly ionic and therefore, not readily incorporated into the polymer. Surfactants possess both hydrophobic and hydrophilic regions within the molecule. It is known that the hydrophobic portion of surfactants interacts with proteins (McCabe et al., 1988) which should leave the ionic or polar portion of the surfactant available to facilitate incorporation into the polymer. The use of an anionic surfactant to add ionic character to the proteins and act as carriers was, therefore, considered. In the course of the electropolymerization process, the anionic surfactant SDS was selected as a counter-ion (co-dopant) as it becomes immobile within PPy (Matencio et al., 1995) and favors the incorporation of biomolecules when in low concentration (Jang et al., 2004). Reproducibility of the process is very good because the thickness of the film, one of the important parameters for immunosensors, can be very well controlled through the passed charge during the deposition process. The film thickness can be estimated through its relation with the charge consumed in polymerization. Regarding PPy, the film thickness was calculated by assuming that 240 mC/cm2 yields a 1 m layer (West et al., 1993; Diaz and Castillo, 1980). Thus, a black film of PPy, ca. 8 m thick, was grown on a gold surface by passing 62.8 mC of charge under the potentiostatic mode. SEM imaging revealed the typical ‘cauliflower-like’ structure, a common feature of PPy (Sutton and Vaughan, 1996). The cauliflowers grew uniformly over the whole electrode, with an average size of 500 nm. Streptavidin did not have a noticeable effect, i.e., pseudo-template, on the PPy surface morphology (figure not shown). In order to confirm the incorporation of streptavidin into PPy, chemical composition of the PPy/streptavidin film was determined using FTIR. Fig. 1 shows the IR spectrum of a PPy/streptavidin film compared with PPy doped with LiClO4 . As commonly known, proteins have three characteristic absorption peaks at 1650, 1550, and 1450 cm−1 , corresponding to Amides I, II, and III bands, respectively (Byler and Susi, 1986). Fig. 1(curve a) shows that PPy/LiClO4 has an absorption band between 1560 and 1680 cm−1 , which comes from pure PPy since LiClO4 does not have absorption over 1000 cm−1 (Shen et al., 2004). The spectrum of PPy/streptavidin should be a combination of PPy and streptavidin as SDS does not have absorption
Fig. 1. FTIR spectrum of PPy/streptavidin with PPy/LiClO4 as control: (a) PPy/LiClO4 and (b) PPy/streptavidin.
in this range (1400–1700 cm−1 ). The peaks at 1432, 1548, and 1662 cm−1 observed in Fig. 1(curve b) are attributed to the three characteristic absorptions of protein while peaks at 1520 and 1580 cm−1 were transformation of the absorption band, which is caused by the conformation change of the polymer due to electrostatic attraction between polycations and doped anions. This result thus confirmed the incorporation of streptavidin in the polymer film. In order to illustrate the dopant stability, UV–vis was used to verify the incorporation of streptavidin into the deposited PPy films over a period of 1 week. After deposition, the streptavidindoped PPy was stored in PBS at 4 ◦ C for 1 and 7 days, respectively. At each specified time point, the PPy/streptavidin coated electrode was removed from the PBS solution and the bath solution was used for UV characterization. Streptavidin has a characteristic absorption peak at 280 nm. As shown in Fig. 2, after 1-day (curve a) and 1-week (curve b) soaking in PBS, respectively, no absorption peak was observed. The results indicated that streptavidin doped in PPy remained stable in the polymer and was not dissociated from the PPy film without acti-
Fig. 2. UV spectra of streptavidin released from PPy after 1-day soaking in PBS (a), 1-week soaking in PBS (b), de-doping under a negative bias (c), and pure streptavidin (d).
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vation. However, after the modified electrode was subject to a negative voltage bias (−0.8 V) in PBS for 8 min, a clear absorption peak at 280 nm was observed (Fig. 2, curve c). The negative bias rendered the counterions (streptavidin) dedoped from the PPy matrix. The result implies the incorporated biomolecules could be released from the polymer matrix upon external stimulation. 3.2. Immobilization of biotinylated antibody on the PPy/streptavidin film Avidin (streptavidin)–biotin bonding is commonly used to capture the biotinylated proteins. The avidin (streptavidin)– biotin complex bond formation is very rapid and the strongest known non-covalent binding, with a dissociation constant of 10−15 M (Lee et al., 2005). Therefore, biotinylated anti-goat IgG was strongly attached to the PPy/streptavidin film. EIS is an effective method to study the properties of surface-modified electrodes. The dynamics of charge transfer at the electrode interface is strongly influenced by the nature of the electrode surface and the structure of the double layer. In our study, PPy/streptavidin/biotinylated anti-goat IgG was constructed on gold electrodes. Fig. 3 shows the impedance spectrum of a PPy/streptavidin coated gold electrode (curve a, straight line) compared with an antibody-immobilized surface (the cluster group of curve b). The PPy/streptavidin film exhibited a straight line, which is characteristic of a capacitor. However, after antibody immobilization, the Nyquist plots exhibited a semicircle shape at the higher frequency range as well as a straight line at the lower frequency portion. The diameter of the semicircle provides an estimate of the film charge-transfer resistance. In the PPy/streptavidin film, this part of the resistance was insignificant. With antibody attached on the surface of the PPy/streptavidin film, the semicircle diameter at high frequencies increased. The results indicated that the antibody immobilized electrode was not as electroconductive as the PPy/streptavidin film. It was expected since antibody introduces an insulating protein layer on the PPy/streptavidin surface,
Fig. 3. Nyquist plots of PPy/streptavidin (a) and biotinylated anti-goat IgG layer with different immobilization time (b). Inset is the amplification of (b).
inducing a high charge transfer resistance. The relationship between impedance signals and immobilization time of the antibody was investigated. The antibody was immobilized on the PPy–streptavidin film for different time (5 min, curve b1, 10 min, curve b2, 15 min, curve b3, 20 min, curve b4, 30 min, curve b5, and 60 min, curve b6) with the results displayed as the inset of Fig. 3. The gradually increased charge transfer resistance with time indicated that more and more antibody was immobilized on the PPy–streptavidin film. When immobilization time was over 30 min, the resistance did not change noticeably with increasing time, indicating the antibody immobilization process was completed. 3.3. Incubation of goat IgG on biotinylated anti-goat IgG immobilized electrode For the control experiment, the procedure was the same as that of PPy/streptavidin except that anti-goat IgG was used as the co-dopant instead of streptavidin. After deposition of PPy/anti-goat IgG on the gold electrode, goat IgG was incu-
Fig. 4. Cyclic voltammetry (A) and impedance spectroscopy (B) of PPy/anti-goat IgG before (a) and after (b) goat IgG incubation.
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Fig. 5. Rt of the PPy/streptavidin film (a), biotinylated anti-goat IgG immobilized electrode (b), and immune complex with 0.01 ng/ml antigen (c).
bated on the PPy/anti-goat IgG film. Notice that after antigen incubation the enclosed area of the cyclic voltammogram was smaller than before (Fig. 4A) indicating that the switching property of the coat became worse. This confirmed the presence of a blocking layer, i.e., antigen on the electrode. However, impedance spectroscopy shows that the charge transfer resistance (Rt ) remained almost constant (Fig. 4B). Such behavior indicates that although the electrochemical properties could be changed upon the interaction of antibody and antigen, the binding reaction of antigen at the antibody-incorporated surface is often insufficient, corresponding to a small impedance change. After antibody–antigen interaction on the electrode, there is a small increase of Rt , reflecting the limit of the direct immune detection in sensitivity. With the strategy of streptavidin–biotin binding, the immobilized biotinylated antibody was increased. The impedance response signals were amplified due to the increase of the antibody payload as shown in Fig. 5. Since the PPy/streptavidin film exhibited the characteristic of a capacitor in the high frequency range, the value for Rt was quite small (column a). With an organic layer of biotinylated antibody introduced onto the surface of the PPy/streptavidin film, the impedance response increased due to the charge transfer limiting process (column b). Furthermore, immune reaction with 10 pg/ml of goat IgG leads to a significant increase of Rt (column c). Goat IgG of different concentrations was incubated on biotinylated anti-goat IgG immobilized electrodes and the impedance responses were detected. The change of the charge transfer resistance Rt is obtained by subtracting the resistance of the immobilized biotinylated antibody (approximately 900 ) from the resistance of the resultant immune complex. As for the control, Rt is obtained by deducting the resistance of the PPy/anti-goat IgG film from the resistance of the antibody–antigen complex. Fig. 6 shows a linear increase in the impedance response with the increment of goat IgG concentration in the range of 10 pg/ml to 100 ng/ml, and then reaches a plateau (curve a). However, without this amplification strategy, the variation of impedance signals is insignificant indicating the low sensitivity (curve b). The results revealed that the sensitivity of the presented method was significantly improved over two orders of magnitude, with an excellent detection limit of 10 pg/ml of antigen.
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Fig. 6. Changes of Rt of PPy/streptavidin/biotinylated anti-goat IgG (a) and PPy/anti-goat IgG (b) immobilized electrode after incubation with goat IgG.
4. Conclusions In brief, streptavidin, as a dopant, could be incorporated into a PPy film providing a bridge to bind biotinylated antibody onto the surface of an electrode. With its four biotin binding sites, the incorporated streptavidin could bind more biotinylated antibodies, providing an amplification effect on the interaction between antibody and antigen. The attachment of insulating materials (antibody or antigen) on conductive supports is anticipated to alter the interfacial charge transfer features at the electrode surface. With the amplification strategy, the detection sensitivity of the sensor is significantly improved. Acknowledgment This work was financially supported by the Center of Advanced Bionanosystems, Nanyang Technological University. References Arora, K., Chaubey, A., Singhal, R., Singh, R.P., Pandey, M.K., Samanta, S.B., Malhotra, B.D., Chand, S., 2006. Biosens. Bioeletron. 21, 1777–1783. Berdichevsky, Y., Lo, Y.-H., 2006. Adv. Mater. 18, 122–125. Bousalem, S., Yassar, A., Basinska, T., Miksa, B., Slomkowski, S., Azioune, A., Chehimi, M.M., 2003. Polym. Adv. Technol. 14, 820–825. Brillas, E., Cabot, P.L., Garrido, A., Montilla, M., Rodriguez, R.M., Carrasco, J., 1997. J. Electroanal. Chem. 430, 133–140. Byler, D.M., Susi, H., 1986. Biopolymers 25, 469–487. Cosnier, S., 1999. Biosens. Bioelectron. 14, 443–456. Darain, F., Park, D.S., Park, J.S., Shim, Y.-B., 2004. Biosens. Bioelectron. 19, 1245–1252. Diaz, A., Castillo, J., 1980. J. Chem. Soc., Chem Commun., 397–398. Geetha, S., Rao, C.R.K., Vijayan, M., Trivedi, D.C., 2006. Anal. Chim. Acta 568, 119–125. George, P.M., LaVan, D.A., Burdick, J.A., Chen, C.-Y., Liang, E., Langer, R., 2006. Adv. Mater. 18, 577–581. Gonzalez-Martinez, M.A., Puchades, R., Maquieira, A., 1999. Trends Anal. Chem. 18, 204–218. Jang, K.S., Lee, H., Moon, B., 2004. Synth. Met. 143, 289–294. Katz, E., Willner, I., 2003. Electroanalysis 15, 913–947. Kros, A., Sommerdijk, N.A.J.M., Nolte, R.J.M., 2005. Sens. Actuators B 106, 289–295. Lee, B.-S., Gupta, S., Krishnanchettiar, S., Lateef, S.S., 2005. Anal. Biochem. 336, 312–315. Lee, T.-Y., Shim, Y.-B., 2001. Anal. Chem. 73, 5629–5632.
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Li, C.M., Sun, C.Q., Chen, W., Pan, L., 2005a. Surf. Coat. Technol. 198, 474–477. Li, C.M., Chen, W., Yang, X., Sun, C.Q., Gao, C., Zheng, Z.X., Sawyer, J., 2005b. Frontiers Biosci. 10, 2518–2526. Livache, T., Bazin, H., Mathis, G., 1998. Clin. Chim. Acta 278, 171–176. Matencio, T., Depaoli, M.A., Peres, R.C.D., Torresi, R.M., Detorresi, S.I.C., 1995. Synth. Met. 72, 59–64. McCabe, J.P., Fletcher, S.M., Jones, M.N., 1988. J. Immunol. Method 108, 129–135. Ramanavicius, A., Kausaite, A., Ramanaviciene, A., Acaite, J., Malinauskas, A., 2006. Synth. Met. 156, 409–413. Ronkainen-Matsuno, N.J., Thomas, J.H., Halsall, H.B., Heineman, W.R., 2002. Trends Anal. Chem. 21, 213–225.
Shen, Y.J., Reddy, M.J., Chu, P.P., 2004. Solid State Ionics 175, 747–750. Shi, G., Rouabhia, M., Wang, Z., Dao, L.H., Zhang, Z., 2004. Biomaterials 25, 2477–2488. Sutton, S.J., Vaughan, A.S., 1996. Polymer 37, 5367–5374. Terry, L.A., White, S.F., Tigwell, L.J., 2005. J. Agric. Food Chem. 53, 1309–1316. West, K., Jacobsen, T., Zachau-Christiansen, B., Careem, M.A., Skaarup, S., 1993. Synth. Met. 55–57, 1412–1417. Wilchek, M., Bayer, A., 1988. Anal. Biochem. 171, 1–32. Xiao, H., Mansfeld, F., 1994. J. Electrochem. Soc. 141, 2332–2337. Xiao, Y.H., Che, J.F., Li, C.M., Sun, C.Q., Chua, Y.T., Lee, V.S., Luong, J.H.T., 2006. J. Biomed. Mater. Res. A. 80A, 925–931.
Electrochemical impedance characterization of antibody–antigen interaction with signal amplification based on polypyrrole–streptavidin Yinghong Xiao a,b,c , Chang Ming Li a,b,∗ , Yingshuai Liu a,b a
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore b Center for Advanced Bionanosystems, Nanyang Technological University, Singapore 637457, Singapore c School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210014, China Received 5 October 2006; received in revised form 14 December 2006; accepted 9 February 2007 Available online 17 February 2007
Abstract Streptavidin, as a dopant, has been incorporated into a polypyrrole film to bind biotinylated antibody onto the electrode surface. With four biotin binding sites, the incorporation of streptavdin, as confirmed by FTIR and impedance spectroscopy, provided a new method to amplify the response signal from antibody–antigen interaction. Biotinylated anti-goat IgG, as a probe, and goat IgG, as a target, were employed to evaluate the characteristics of the biosensor. With the amplification strategy, the detection sensitivity of the electrochemical impedance spectroscopy was significantly improved. A linear relationship between the charge transfer resistance change (Rt ) and the concentration of goat IgG ranging from 10 pg/ml to100 ng/ml was obtained. © 2007 Elsevier B.V. All rights reserved. Keywords: Electrochemical impedance; Polypyrrole; Streptavidin; Immunoreaction
1. Introduction The utilization of conducting electroactive polymers in electrochemical sensors has been increasing over the past decades (Berdichevsky and Lo, 2006; Kros et al., 2005; Li et al., 2005a,b; Arora et al., 2006). The unique feature of conducting polymer applications derives from the ability of well tuning the polymers with different counter ions including biomolecules, making it possible to detect a wide range of analytes. Although the use of conducting polymers has been restricted by the limited choice of dopants (electronegativity, molecular weight, etc.), the electrochemical formation of the polymer layer with controlled thickness could effectively construct a reproducible biosensor (Cosnier, 1999). The barriers can be circumvented by attaching biomolecules to the surface of the polymer film through specific coupling (George et al., 2006) or ion exchanging (Xiao et al., 2006). Polypyrrole (PPy) has been widely used for biological applications due to its beneficial chemical properties, aque∗ Corresponding author at: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore. Tel.: +65 67904485; fax: +65 67911761. E-mail address: [email protected] (C.M. Li).
0956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2007.02.008
ous solubility, simplicity of preparation, biocompatibility, and easy immobilization of biological molecules (Shi et al., 2004; Ramanavicius et al., 2006; Bousalem et al., 2003; Geetha et al., 2006). Thus, the immobilization of biomolecules in electropolymerized PPy films is gaining importance apart from conventional methods such as covalent binding, etc. (Cosnier, 1999). Immunosensors have aroused great interest with expectations of providing fast and highly sensitive immunological response. Immunosensors have widespread applications in clinical diagnostics, food safety and quality, biological analysis, and environmental monitoring (Livache et al., 1998; Terry et al., 2005; Ronkainen-Matsuno et al., 2002; Gonzalez-Martinez et al., 1999). Electrochemical methods appear very promising due to the relatively simple and compact equipment required. In particular, electrochemical impedance spectroscopy (EIS) represents a powerful method for probing the interfacial reaction mechanisms of modified electrodes (Xiao and Mansfeld, 1994; Katz and Willner, 2003), providing a rapid approach for monitoring the dynamics of biomolecular interaction. Thus, EIS is often used for investigating chemical transformations and processes associated with the conductive supports (Li et al., 2005a,b; Lee and Shim, 2001; Brillas et al., 1997). Electrochemical impedance immunosensing has attracted extensive interest
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Scheme 1. Schematic diagram of the principle and sequence of steps for the proposed protocol.
for quantifying the interaction between antibody and antigen (Darain et al., 2004). Impedance immunosensing could provide a general method to follow the procedure of antigen–antibody reaction at modified electrode surfaces. It is known that the antigen–antibody complex acts as a blocking layer to effect a change in the measured impedance. However, the immune reaction of antigen with antibody directly incorporated in conducting polymers is usually not sufficient to generate a significant and detectable signal change for impedance measurements. In this study, we modified the electrode surface according to the protocol illustrated in Scheme 1. PPy was electrochemically deposited on a gold electrode with streptavidin as a dopant to form an electropolymerized PPy/streptavidin film. It was plausible to use streptavidin, negatively charged, as a counter anion for PPy synthesis and biotinylated antibody was immobilized on the PPy/streptavidin film via well-known streptavidin–biotin interaction. The attachment of antibody to the electrode surface through the specific avidin (or streptavidin)–biotin molecular interaction has been revealed as an effective and reliable approach (Wilchek and Bayer, 1988). Streptavidin with four biotin binding sites, adheres to the biotinylated antibodies and thus increases the antibody loading. 2. Experiments 2.1. Materials Pyrrole monomer (>98%) and sodium dodecyl sulfate (SDS) were purchased from Aldrich (Milwaukee, WI). Streptavidin was from Sigma–Aldrich, of which 55.5 g (1 unit) will bind 1.0 g biotin at pH 7.5. Biotinylated monoclonal anti-goat immunoglobulin G (IgG) antibody, rabbit anti-goat IgG antibody and goat IgG from goat serum were purchased from Sigma (St. Louis, MO) and diluted with 0.01 M phosphate buffer saline solution (PBS, pH 7.4) to different concentrations. 2.2. Instrumentation Electrochemical deposition of PPy and electrochemical measurements (impedance and cyclic voltammetry) were conducted
with an electrochemical workstation CHI 760B (CH Instruments, Austin, TX). EIS was performed in 0.01 M PBS, pH 7.4 over a wide range of frequencies from 100 to 105 Hz. Cyclic voltammetry was conducted by potential scanning between −0.9 and 0.8 V, with a scan rate of 100 mV/s. The UV–vis Hitachi spectrometer (U-2800, Tokyo, Japan) was used for the stability studies of the doped streptavidin. Incorporation of streptavidin in the PPy film was demonstrated by using FTIR spectroscopy (Spectrum GX, Perkin-Elmer, Wellesley, MA). Samples were prepared as KBr pellets. The morphology of the PPy film incorporated with streptavidin was studied with a scanning electron microscope (JEOL JSM-6700F FEG, Japan), operating at 5.0 kV. Milli-Q water of 18.2 M cm (Millipore, Inc.) was used in all experiments. 2.3. Electropolymerization of the PPy/streptavidin film on gold electrodes Electropolymerization was conducted in a three-electrode electrochemical cell with a gold electrode, platinum wire, and saturated calomel electrode (SCE) as the working, counter, and reference electrode, respectively. Before electropolymerization, the gold electrode (diameter of 2 mm) was polished with 1.0, 0.3, and 0.05 m alumina slurry, respectively, followed by rinsing with deionized water and cleaning in an ultrasonic bath for 10 min. Electropolymerization was performed at 0.8 V for 8 min in a solution containing 0.1 M pyrrole, 0.02 M SDS, and 0.2 mg/ml streptavidin. The solution was bubbled with nitrogen for 20 min before use. 2.4. Immobilization of biotinylated anti-goat IgG on the PPy/streptavidin film The PPy/streptavidin modified electrode was immersed in casein solution (0.3%, w/v) for 1 h at room temperature to eliminate nonspecific adsorption. After thoroughly rinsed with copious PBS for 5 min, the electrode was dried with a gentle nitrogen stream. Biotinylated anti-goat IgG (1.7 g/ml) was immobilized on PPy/streptavidin films at different incubation times, followed by thorough rinsing with PBS.
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2.5. Incubation of goat IgG on the immobilized biotinylated anti-goat IgG Before reacted with goat IgG, the antibody-immobilized electrode was blocked with casein for 1 h at room temperature, followed by thorough rinse with PBS. Goat IgG at different concentrations was incubated on immobilized biotinylated antigoat IgG for 1 h at room temperature, followed by rinsing with PBS. 3. Results and discussion 3.1. Electrochemical deposition of the PPy/streptavidin thin film PPy can be electrochemically synthesized on different substrates (Li et al., 2005a,b). In solutions of neutral pH proteins are usually not highly ionic and therefore, not readily incorporated into the polymer. Surfactants possess both hydrophobic and hydrophilic regions within the molecule. It is known that the hydrophobic portion of surfactants interacts with proteins (McCabe et al., 1988) which should leave the ionic or polar portion of the surfactant available to facilitate incorporation into the polymer. The use of an anionic surfactant to add ionic character to the proteins and act as carriers was, therefore, considered. In the course of the electropolymerization process, the anionic surfactant SDS was selected as a counter-ion (co-dopant) as it becomes immobile within PPy (Matencio et al., 1995) and favors the incorporation of biomolecules when in low concentration (Jang et al., 2004). Reproducibility of the process is very good because the thickness of the film, one of the important parameters for immunosensors, can be very well controlled through the passed charge during the deposition process. The film thickness can be estimated through its relation with the charge consumed in polymerization. Regarding PPy, the film thickness was calculated by assuming that 240 mC/cm2 yields a 1 m layer (West et al., 1993; Diaz and Castillo, 1980). Thus, a black film of PPy, ca. 8 m thick, was grown on a gold surface by passing 62.8 mC of charge under the potentiostatic mode. SEM imaging revealed the typical ‘cauliflower-like’ structure, a common feature of PPy (Sutton and Vaughan, 1996). The cauliflowers grew uniformly over the whole electrode, with an average size of 500 nm. Streptavidin did not have a noticeable effect, i.e., pseudo-template, on the PPy surface morphology (figure not shown). In order to confirm the incorporation of streptavidin into PPy, chemical composition of the PPy/streptavidin film was determined using FTIR. Fig. 1 shows the IR spectrum of a PPy/streptavidin film compared with PPy doped with LiClO4 . As commonly known, proteins have three characteristic absorption peaks at 1650, 1550, and 1450 cm−1 , corresponding to Amides I, II, and III bands, respectively (Byler and Susi, 1986). Fig. 1(curve a) shows that PPy/LiClO4 has an absorption band between 1560 and 1680 cm−1 , which comes from pure PPy since LiClO4 does not have absorption over 1000 cm−1 (Shen et al., 2004). The spectrum of PPy/streptavidin should be a combination of PPy and streptavidin as SDS does not have absorption
Fig. 1. FTIR spectrum of PPy/streptavidin with PPy/LiClO4 as control: (a) PPy/LiClO4 and (b) PPy/streptavidin.
in this range (1400–1700 cm−1 ). The peaks at 1432, 1548, and 1662 cm−1 observed in Fig. 1(curve b) are attributed to the three characteristic absorptions of protein while peaks at 1520 and 1580 cm−1 were transformation of the absorption band, which is caused by the conformation change of the polymer due to electrostatic attraction between polycations and doped anions. This result thus confirmed the incorporation of streptavidin in the polymer film. In order to illustrate the dopant stability, UV–vis was used to verify the incorporation of streptavidin into the deposited PPy films over a period of 1 week. After deposition, the streptavidindoped PPy was stored in PBS at 4 ◦ C for 1 and 7 days, respectively. At each specified time point, the PPy/streptavidin coated electrode was removed from the PBS solution and the bath solution was used for UV characterization. Streptavidin has a characteristic absorption peak at 280 nm. As shown in Fig. 2, after 1-day (curve a) and 1-week (curve b) soaking in PBS, respectively, no absorption peak was observed. The results indicated that streptavidin doped in PPy remained stable in the polymer and was not dissociated from the PPy film without acti-
Fig. 2. UV spectra of streptavidin released from PPy after 1-day soaking in PBS (a), 1-week soaking in PBS (b), de-doping under a negative bias (c), and pure streptavidin (d).
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vation. However, after the modified electrode was subject to a negative voltage bias (−0.8 V) in PBS for 8 min, a clear absorption peak at 280 nm was observed (Fig. 2, curve c). The negative bias rendered the counterions (streptavidin) dedoped from the PPy matrix. The result implies the incorporated biomolecules could be released from the polymer matrix upon external stimulation. 3.2. Immobilization of biotinylated antibody on the PPy/streptavidin film Avidin (streptavidin)–biotin bonding is commonly used to capture the biotinylated proteins. The avidin (streptavidin)– biotin complex bond formation is very rapid and the strongest known non-covalent binding, with a dissociation constant of 10−15 M (Lee et al., 2005). Therefore, biotinylated anti-goat IgG was strongly attached to the PPy/streptavidin film. EIS is an effective method to study the properties of surface-modified electrodes. The dynamics of charge transfer at the electrode interface is strongly influenced by the nature of the electrode surface and the structure of the double layer. In our study, PPy/streptavidin/biotinylated anti-goat IgG was constructed on gold electrodes. Fig. 3 shows the impedance spectrum of a PPy/streptavidin coated gold electrode (curve a, straight line) compared with an antibody-immobilized surface (the cluster group of curve b). The PPy/streptavidin film exhibited a straight line, which is characteristic of a capacitor. However, after antibody immobilization, the Nyquist plots exhibited a semicircle shape at the higher frequency range as well as a straight line at the lower frequency portion. The diameter of the semicircle provides an estimate of the film charge-transfer resistance. In the PPy/streptavidin film, this part of the resistance was insignificant. With antibody attached on the surface of the PPy/streptavidin film, the semicircle diameter at high frequencies increased. The results indicated that the antibody immobilized electrode was not as electroconductive as the PPy/streptavidin film. It was expected since antibody introduces an insulating protein layer on the PPy/streptavidin surface,
Fig. 3. Nyquist plots of PPy/streptavidin (a) and biotinylated anti-goat IgG layer with different immobilization time (b). Inset is the amplification of (b).
inducing a high charge transfer resistance. The relationship between impedance signals and immobilization time of the antibody was investigated. The antibody was immobilized on the PPy–streptavidin film for different time (5 min, curve b1, 10 min, curve b2, 15 min, curve b3, 20 min, curve b4, 30 min, curve b5, and 60 min, curve b6) with the results displayed as the inset of Fig. 3. The gradually increased charge transfer resistance with time indicated that more and more antibody was immobilized on the PPy–streptavidin film. When immobilization time was over 30 min, the resistance did not change noticeably with increasing time, indicating the antibody immobilization process was completed. 3.3. Incubation of goat IgG on biotinylated anti-goat IgG immobilized electrode For the control experiment, the procedure was the same as that of PPy/streptavidin except that anti-goat IgG was used as the co-dopant instead of streptavidin. After deposition of PPy/anti-goat IgG on the gold electrode, goat IgG was incu-
Fig. 4. Cyclic voltammetry (A) and impedance spectroscopy (B) of PPy/anti-goat IgG before (a) and after (b) goat IgG incubation.
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Fig. 5. Rt of the PPy/streptavidin film (a), biotinylated anti-goat IgG immobilized electrode (b), and immune complex with 0.01 ng/ml antigen (c).
bated on the PPy/anti-goat IgG film. Notice that after antigen incubation the enclosed area of the cyclic voltammogram was smaller than before (Fig. 4A) indicating that the switching property of the coat became worse. This confirmed the presence of a blocking layer, i.e., antigen on the electrode. However, impedance spectroscopy shows that the charge transfer resistance (Rt ) remained almost constant (Fig. 4B). Such behavior indicates that although the electrochemical properties could be changed upon the interaction of antibody and antigen, the binding reaction of antigen at the antibody-incorporated surface is often insufficient, corresponding to a small impedance change. After antibody–antigen interaction on the electrode, there is a small increase of Rt , reflecting the limit of the direct immune detection in sensitivity. With the strategy of streptavidin–biotin binding, the immobilized biotinylated antibody was increased. The impedance response signals were amplified due to the increase of the antibody payload as shown in Fig. 5. Since the PPy/streptavidin film exhibited the characteristic of a capacitor in the high frequency range, the value for Rt was quite small (column a). With an organic layer of biotinylated antibody introduced onto the surface of the PPy/streptavidin film, the impedance response increased due to the charge transfer limiting process (column b). Furthermore, immune reaction with 10 pg/ml of goat IgG leads to a significant increase of Rt (column c). Goat IgG of different concentrations was incubated on biotinylated anti-goat IgG immobilized electrodes and the impedance responses were detected. The change of the charge transfer resistance Rt is obtained by subtracting the resistance of the immobilized biotinylated antibody (approximately 900 ) from the resistance of the resultant immune complex. As for the control, Rt is obtained by deducting the resistance of the PPy/anti-goat IgG film from the resistance of the antibody–antigen complex. Fig. 6 shows a linear increase in the impedance response with the increment of goat IgG concentration in the range of 10 pg/ml to 100 ng/ml, and then reaches a plateau (curve a). However, without this amplification strategy, the variation of impedance signals is insignificant indicating the low sensitivity (curve b). The results revealed that the sensitivity of the presented method was significantly improved over two orders of magnitude, with an excellent detection limit of 10 pg/ml of antigen.
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Fig. 6. Changes of Rt of PPy/streptavidin/biotinylated anti-goat IgG (a) and PPy/anti-goat IgG (b) immobilized electrode after incubation with goat IgG.
4. Conclusions In brief, streptavidin, as a dopant, could be incorporated into a PPy film providing a bridge to bind biotinylated antibody onto the surface of an electrode. With its four biotin binding sites, the incorporated streptavidin could bind more biotinylated antibodies, providing an amplification effect on the interaction between antibody and antigen. The attachment of insulating materials (antibody or antigen) on conductive supports is anticipated to alter the interfacial charge transfer features at the electrode surface. With the amplification strategy, the detection sensitivity of the sensor is significantly improved. Acknowledgment This work was financially supported by the Center of Advanced Bionanosystems, Nanyang Technological University. References Arora, K., Chaubey, A., Singhal, R., Singh, R.P., Pandey, M.K., Samanta, S.B., Malhotra, B.D., Chand, S., 2006. Biosens. Bioeletron. 21, 1777–1783. Berdichevsky, Y., Lo, Y.-H., 2006. Adv. Mater. 18, 122–125. Bousalem, S., Yassar, A., Basinska, T., Miksa, B., Slomkowski, S., Azioune, A., Chehimi, M.M., 2003. Polym. Adv. Technol. 14, 820–825. Brillas, E., Cabot, P.L., Garrido, A., Montilla, M., Rodriguez, R.M., Carrasco, J., 1997. J. Electroanal. Chem. 430, 133–140. Byler, D.M., Susi, H., 1986. Biopolymers 25, 469–487. Cosnier, S., 1999. Biosens. Bioelectron. 14, 443–456. Darain, F., Park, D.S., Park, J.S., Shim, Y.-B., 2004. Biosens. Bioelectron. 19, 1245–1252. Diaz, A., Castillo, J., 1980. J. Chem. Soc., Chem Commun., 397–398. Geetha, S., Rao, C.R.K., Vijayan, M., Trivedi, D.C., 2006. Anal. Chim. Acta 568, 119–125. George, P.M., LaVan, D.A., Burdick, J.A., Chen, C.-Y., Liang, E., Langer, R., 2006. Adv. Mater. 18, 577–581. Gonzalez-Martinez, M.A., Puchades, R., Maquieira, A., 1999. Trends Anal. Chem. 18, 204–218. Jang, K.S., Lee, H., Moon, B., 2004. Synth. Met. 143, 289–294. Katz, E., Willner, I., 2003. Electroanalysis 15, 913–947. Kros, A., Sommerdijk, N.A.J.M., Nolte, R.J.M., 2005. Sens. Actuators B 106, 289–295. Lee, B.-S., Gupta, S., Krishnanchettiar, S., Lateef, S.S., 2005. Anal. Biochem. 336, 312–315. Lee, T.-Y., Shim, Y.-B., 2001. Anal. Chem. 73, 5629–5632.
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Y. Xiao et al. / Biosensors and Bioelectronics 22 (2007) 3161–3166
Li, C.M., Sun, C.Q., Chen, W., Pan, L., 2005a. Surf. Coat. Technol. 198, 474–477. Li, C.M., Chen, W., Yang, X., Sun, C.Q., Gao, C., Zheng, Z.X., Sawyer, J., 2005b. Frontiers Biosci. 10, 2518–2526. Livache, T., Bazin, H., Mathis, G., 1998. Clin. Chim. Acta 278, 171–176. Matencio, T., Depaoli, M.A., Peres, R.C.D., Torresi, R.M., Detorresi, S.I.C., 1995. Synth. Met. 72, 59–64. McCabe, J.P., Fletcher, S.M., Jones, M.N., 1988. J. Immunol. Method 108, 129–135. Ramanavicius, A., Kausaite, A., Ramanaviciene, A., Acaite, J., Malinauskas, A., 2006. Synth. Met. 156, 409–413. Ronkainen-Matsuno, N.J., Thomas, J.H., Halsall, H.B., Heineman, W.R., 2002. Trends Anal. Chem. 21, 213–225.
Shen, Y.J., Reddy, M.J., Chu, P.P., 2004. Solid State Ionics 175, 747–750. Shi, G., Rouabhia, M., Wang, Z., Dao, L.H., Zhang, Z., 2004. Biomaterials 25, 2477–2488. Sutton, S.J., Vaughan, A.S., 1996. Polymer 37, 5367–5374. Terry, L.A., White, S.F., Tigwell, L.J., 2005. J. Agric. Food Chem. 53, 1309–1316. West, K., Jacobsen, T., Zachau-Christiansen, B., Careem, M.A., Skaarup, S., 1993. Synth. Met. 55–57, 1412–1417. Wilchek, M., Bayer, A., 1988. Anal. Biochem. 171, 1–32. Xiao, H., Mansfeld, F., 1994. J. Electrochem. Soc. 141, 2332–2337. Xiao, Y.H., Che, J.F., Li, C.M., Sun, C.Q., Chua, Y.T., Lee, V.S., Luong, J.H.T., 2006. J. Biomed. Mater. Res. A. 80A, 925–931.