Surface Plasmon Resonance Spectroscopy and Electrochemistry

Mar 9, 2006 - Yang, W. S.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russ...
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Langmuir 2006, 22, 3929-3935

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Surface Plasmon Resonance Spectroscopy and Electrochemistry Study of 4-Nitro-1,2-phenylenediamine: A Switchable Redox Polymer with Nitro Functional Groups Huiru Gu,†,‡ Zhaoyue Ng,† T. C. Deivaraj,§ Xiaodi Su,*,‡ and Kian Ping Loh*,† Department of Chemistry, National UniVersity of Singapore, 3 Science DriVe 3, Singapore 117543, Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, and Singapore Institute of Manufacturing, Nanyang DriVe, Singapore ReceiVed NoVember 9, 2005. In Final Form: January 27, 2006 Electrochemistry and electrochemical surface plasmon resonance (SPR) spectroscopy have been applied to study the electrochemical deposition and the redox transition of poly(4-nitro-1,2-phenylenediamine) (P4NoPD) on gold disk. It was shown that SPR can be the signal transducer for the different redox states of P4NoPD. Using a model biomolecular system, involving streptavidin, biotinylated DNA, and its complementary target DNA, it was found that the presence of nitro groups in P4NoPD allows the biorecognition events to be modulated by voltages. There is minimal nonspecific binding of biomolecules on oxidized (+0.2 V) or as-prepared P4NoPD, and binding occurs more significantly on the reduced P4NoPD (-0.2 to -0.6 V) with the presence of amine groups. The electrochemical deposition of P4NoPD film was also conducted on boron-doped diamond (BDD) electrode. The stability of the reduced P4NoPD film on gold and BDD was comparatively evaluated by electrochemical impedance spectroscopy (EIS). The result showed that BDD allows the electrochemical reduction of the P4NoPD film at wider cathodic limits than gold.

Introduction Studies of conducting polymers such as polypyrrole, polyaniline, and polythiophene and their derivatives have been widely reported because of the high conductivities of these polymers at their oxidized states and because the conductivities of these polymers can be switched between conducting and insulating (less conductive) states by electrochemical doping and dedoping. Various applications of these polymers have been proposed in electrochromic devices, organic batteries, sensors, biosensors, and electrocatalysis1-4 Among these polymers, poly(orthophenylenediamine) (PoPD), a ladder polymer with a phenoxiazine-like chain structure,5-8 has received attentions since its extended π-conjugated system is expected to improve the thermal stability and mechanical strength.9 4-Nitro-1,2-phenylenediamine (4NoPD), with a nitro-group substituent in the benzene ring, is an analogue to ortho-phenylenediamine (oPD). Unlike oPD, the electropolymerization of this monomer was considered difficult because the strong electron-withdrawing nature of the nitrogroup could destabilize the initially formed cation-radicals and limit the coupling reaction of polymerization.10,11 However, recently it was reported that the electropolymerization of this * To whom correspondence should be addressed. E-mail: chmlohkp@ nus.edu.sg (K.P.L.); [email protected] (X.D.S.). † National University of Singapore. ‡ Institute of Materials Research and Engineering. § Singapore Institute of Manufacturing. (1) Inzelt, G., Bard, A. J., Ed.; Electroanalytical Chemistry; Marcel Dekker: New York, 1994; Vol. 18, p 90. (2) Wieck, H. J.; Yacynych, A. M. Anal. Chem. 1980, 52, 345. (3) Ohnuki, Y.; Matsuda, H.; Ohsaka, T.; Oyama, N. J. Electroanal. Chem. 1983, 158, 55. (4) Lowry, J. P.; McAteer, K.; EI Atrash, S. S.; Duff, A.; O’Neill, R. D. Anal. Chem. 1994, 66, 1754. (5) Barbero, C.; Silber, J. J.; Sereno, L. J. Electroanal. Chem. 1990, 291, 81. (6) Kinimura, S.; Ohsaka, T.; Oyama, N. Macromolecules 1988, 21, 894. (7) Jackowska, K.; Bukowska, J.; Kudelski, A. (8) Jackowska, K.; Bukowska, J.; Kudelski, A. Pol. J. Chem. 1994, 68, 141. (9) Komura, T.; Funahasi, Y.; Yamaguti, K. J. Electroanal. Chem. 1998, 446, 113. (10) Salmon, M.; Diaz, A.; Coitia, J. ACS Symp. Ser. 1982, 192, 65. (11) Kokkinidis, G.; Kelaidopoulou, A. J. Electroanal. Chem. 1996, 414, 197.

monomer on gold and glassy carbon electrodes could be realized in acidic solutions. The ability to introduce nitro groups on the electrode surface via the conducting polymer film is interesting because the nitro-groups can be reduced to amine groups, and potentially provides a basis for covalent immobilization of biomolecules for individually addressable electrochemical sensing in immunoassays and biochemical screening arrays. The fact that the P4NoPD polymer is conducting allows a lower voltage to be applied to induce the switching, as opposed to nonconducting molecular chains. Detailed investigations into the physical structure, conduction and optical properties of the different redox states in these films have not been carried out. Surface plasmon resonance (SPR) spectroscopy has been used as a unique tool to study ultrathin organic films on noble metal surfaces. The in situ real-time measurement of SPR provides researchers with quantitative and qualitative information of the solid/liquid interface.13 The combination of SPR with electrochemical techniques provides a powerful tool to study the electrochemically initiated surface modification processes for immobilization of biomolecules on electrodes. A gold film on glass slide is usually used as both the surface plasmon medium and the working electrode in electrochemistry. An electrochemical process occurring on the gold film can affect surface plasmons located on the gold surface.14 A number of works have been carried out to incorporate electrochemical processes with SPR measurement for trace metals detection,15,16 diffusion and adsorption processes monitoring,17-19 doping/dedoping process (12) Yu, B.; Khoo, S. B. Electrochim. Acta 2005, 50, 1917. (13) Knoll, W. Annu. ReV. Phys. Chem. 1998, 49, 569. (14) Kang, X. F.; Jin, Y. D.; Cheng, G. J.; Dong, S. J. Langmuir 2002, 18, 1713. (15) Chinowsky, T. M.; Saban, S. B.; Yee, S. S. Sens. Actuators, B 1996, 35, 37. (16) Jung, C. C.; Saban, S. B.; Yee, S. S.; Darling, R. B. Sens. Actuators, B 1996, 32, 143. (17) Iwasaki, Y.; Horiuchi, T.; Morita, M.; Niwa, O. Surf. Sci. 1999, 427, 195. (18) Iwasaki, Y.; Horiuchi, T.; Morita, M.; Niwa, O. Sens. Actuators, B 1998, 50, 145. (19) Iwasaki, Y.; Horiuchi, T.; Morita, M.; Niwa, O. Elecroanalysis 1997, 9, 1239.

10.1021/la053014x CCC: $33.50 © 2006 American Chemical Society Published on Web 03/09/2006

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study,20-23 and in situ study of conducting polymer film growth.14 SPR spectroscopy has also been used to study the optical properties of the conducting polymer such as poly(pyrrole), poly(aniline), and poly(thiophene) at different doping levels.20-23 In this work, electrochemical SPR was used to monitor the electropolymerization of poly(4-nitro-1,2-phenylenediamine) (P4NoPD) as well as the redox states transition. P4NoPD was found to exhibit a voltage-dependent, specific binding toward biomolecules. The application of a cathodic voltage converts the nitro to amine groups and allows specific binding of biomolecules (e.g., streptavidin for DNA assembly and hybridization) through covalent linkages, otherwise there is minimal nonspecific bonding of biomolecules on the as-prepared or oxidized P4NoPD. To our knowledge, this is the first SPR study documenting changes in reflectivity arising from transition between different functional groups on a conducting polymer. Boron-doped diamond (BDD) electrodes were also involved in this study for electrodeposition of P4NoPD films. The stability of the reduced P4NoPD film on gold and BDD was evaluated by electrochemical impedance spectroscopy (EIS). XPS and elliposimetry were used for characterization of chemical composition before and after redox transition and the thickness of the polymer film, respectively. Experimental Section Materials. 4NoPD was purchased from Merck (Gemany). Streptavidin (SA) was purchased from Sigma-Aldrich (St. Louis, MO). 30 mer oligonucleotides were obtained from MWG Biotech (Germany). The probe DNA was tagged with biotin at the 5′ end (5′-biotin-GCA CCT GAC TCC TGT GGA GAA GTC TGC CGT3′), and the target DNA contained a fully complementary sequence to the probe DNA (3′-CGT GGA CTG AGG ACA CCT CTT CAG AGC GCA-5′). Phosphate buffer solution (PBS, pH 7.4) was obtained by dissolving one tablet of the solid phosphate (Sigma) in 200 mL of Millipore water (18.2 mΩ‚cm). This buffer was used as the medium for streptavidin immobilization, DNA assembly, and hybridization. Glutaraldehyde (GA, Sigma-Aldrich) is used as a cross-linking reagent for covalent immobilization of SA. 50 µm-thick BDD films were grown on p-doped Si substrates in a commercial 2.45 GHz microwave plasma reactor (Astex) using methanol and boron oxide mixtures, following the established procedure.24 The BDD diamond has a resistivity of 10 Ω‚cm, and the boron doping level is approximately 1020 cm-3. The films consist of 200 nm sized sharply faceted crystals with roughness of about 60 nm (data from AFM). The resulting substrates were cut into 5 × 5 mm2 squares and used as working electrodes. Gold working electrodes with diameters of 2 mm were purchased from CH Instrument (Austin, TA). Prior to each experiment, gold working electrodes were pretreated by polishing with alumina slurry. The gold working electrodes were then rinsed thoroughly with Millipore water and sonicated in a Millipore water bath for 5 min. Finally, the working electrodes were electrochemically conditioned in 0.5 M H2SO4 by potential cycling from 0.00 to 1.40 V at a scan rate of 10 mV/s for 30 min. Electrochemical Instrument and Electropolymerization of P4NoPD. A three-electrode system was used for polymer electropolymerization and EIS measurements. Gold or BDD electrodes were used as working electrode, whereas saturated Ag/AgCl electrode and platinum were used as the reference and counter electrodes, respectively. The cyclic voltammograms were recorded with a Potentiostat/Galvanostat and a Lock-In Amplifier (PAR EG&G model (20) Georgiadis, R.; Peterlinz, K. A.; Rahn, J. R.; Peterson, A. W.; Grassi, J. H. Langmuir 2000, 16, 6759. (21) Chegel, V.; Raitman, O.; Katz, E.; Gabai, R.; Willner, I. Chem. Commun. 2001, 883. (22) Xia, C.; Advincula, R. C. Langmuir 2002, 18, 3555. (23) Baba, A.; Park, M. K.; Advincula, R. C.; Knoll, W. Langmuir 2002, 18, 4648. (24) Rao, T. N.; Yagi, I.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal. Chem 1999, 71, 2506.

Gu et al. 273A). The impedance spectra for the P4NoPD-modified gold electrode and the BDD electrode were measured at a given open circuit voltage from 100 kHz down to 0.02 Hz at a sampling rate of 10 points per decade (AC amplitude 10 mV). Electropolymerization of P4NoPD was carried out by continuous potential cycling between -0.15 and +1.10 V at 50 mV/s in 0.5 M H2SO4 solution containing 4.5 mM 4NoPD monomer. The same electropolymerization conditions were applied on both gold and diamond electrodes. SPR Equipment. SPR measurements were carried out using a double-channel Autolab ESPR instrument (Eco Chemie, The Netherlands). This instrument is based on the Kretschmann configuration, with a scanning angle setup. The principle of SPR detection has been described in detail by others.25,26 Essentially, the incident p-polarized laser light (λ ) 670 nm) is directed onto a 1 mm × 2 mm spot on the SPR sensor disk through a hemicylindrical prism of BK7 glass. The incident angle is varied by a vibrating mirror which rotates over an angle of 4° at 44 Hz. At a certain incident angle, the so-called resonance angle, energy will be transferred from the light to surface plasmons and the intensity of the reflected light will decrease to a minimum; hence, a dip will be observed in the plot of intensity versus angle. In an angular scan mode, the reflectivity is recorded as a function of incident angle (R - θ) and in a kinetic measurement mode, the SPR angle (the angle of the reflectivity minimal) is recorded as a function of time (θ - t). SPR Measurement of Streptavidin Immobilization on P4NoPD for DNA Assembly and Hybridization. The SPR gold disks were first cleaned with piranha solution (mixture of H2SO4 and H2O2 3:1). 4NoPD was then electropolymerized onto the SPR gold disk using the conditions mentioned earlier. After activation of the asprepared polymer film with glutaraldehyde (30 min exposure to 25% GA aqueous solution: PBS ) 1: 10), the surfaces were calibrated in PBS buffer, and streptavidin (0.2 mg/mL in PBS buffer) was then immobilized on the modified electrode. After saturation, the surfaces were rinsed with PBS buffer. Biotinylated DNA probe and DNA target (1 µM in PBS) were applied for assembly and hybridization consecutively. Rinsing by PBS buffer was conducted after saturation was achieved in each binding step. Ellipsometry and X-ray Photoelectron Spectroscopy Characterization of the Polymer Films. The layer thickness of P4NoPD was estimated using a Spectroscopic Ellipsometer (VB-250, J. A. Woollam) at an angle of incidence of 75°. The wavelength range used in the ellipsometry experiments was 300-900 nm. Measurements of three separate points were carried out on each sample. Prior to the measurement of the thickness, the optical constants of the clean gold substrate were measured and used as a model for the subsequent thickness analysis. The surface functional groups of P4NoPD films on BDD before and after reduction were characterized using X-ray photoelectron spectroscopy (XPS).

Results and Discussion Electropolymerization of P4NoPD on BDD Electrode and SPR Gold Disk. Electropolymerization of P4NoPD was carried out using 0.5 M H2SO4, which gave a good solubility of 4NoPD and a wide cathodic window. Figure 1 shows a series of cycle voltammograms (CVs) for the oxidative polymerization of 4.5 mM 4NoPD in 0.5 M H2SO4 at BDD electrode. An irreversible anodic peak (O1) at ∼0.75 V which appeared during the first forward scan revealed the oxidation of the 4NoPD monomer. The rapid decrease of this peak after the first cycle is due to the restricted access of the 4NoPD monomer to the electrode surface after the first CV cycle of polymer film formation. A reversible redox pair labeled as O2 and R2 can be seen in the CV plot in Figure 1, the intensity of the redox couple increases with the cycles of electropolymerization, suggesting that these are linked to a progressive increase in the density of redox (25) Wink, T.; Van Zuilen, S. J.; Bult, A.; van Bennekom, W. P. Anal. Chem. 1998, 70, 827. (26) Bart. M.; van Os, P. J. H. J.; Kamp, B.; Bult, A.; van Bennekom, W. P. Sens. Actuators, B 2002, 84, 129.

SPR and Electrochemical Studies of P4NoPD

Figure 1. CVs of electropolymerization of 4NoPD on BDD electrode in 0.5 M H2SO4 solution, containing 4.50 mM 4NoPD. The cycles shown are 1st, 5th, 10th, 15th, 20th, and 25th (the last cycle).

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Figure 3. CVs showing the irreversible reduction of nitro groups on P4NoPD film on BDD for 20 continuous cycles in 0.50 M H2SO4. Potential scan rate: 50 mV/s.

After the study of 4NoPD electropolymerization, the irreversible reduction of P4NoPD on the BDD electrode was investigated. According to reported studies on the reduction of aromatic nitro groups,12,27-30 the reduction scheme is given as 2e-,2H+

-H2O

RNO2 98 RN(OH)2 98 RNO 2e-,2H+

2e-,2H+, -H2O

98 RNHOH 98 RNH2

Figure 2. SPR response on gold electrode during the electropolymerization of 4NoPD (40 cycles scan from -0.15 to +1.0 V at scan rate of 50 mv/s). The inset shows the magnified region of the initial increase in SPR response. The oscillation period of the SPR signal corresponds to the period between the CV cycles.

functional groups in thicker films. This redox couple is attributed to the reversible transformation between nitroso and hydroxylamine groups in P4NoPD. During the reversed scan, two reduction peaks, R1 (Ep,R1 ) 0.07 V) and R2 (Ep,R2 ) 0.01 V) appeared and R1 became sharper at higher cycles. R1 may be due to occluded oligomers which were present in the initial stage of polymerization. The CV profile in this potential window range is similar when the electroploymerisation of P4NoPD was carried out on gold or diamond electrode. The electropolymerization process was monitored by SPR in real time on gold disk when CV was being performed. Figure 2 gives the SPR response (angular shift in milli-degree versus time) during this polymerization. A sudden increase of resonance angle at the start of the first CV cycle evidenced the formation of P4NoPD on gold. A control experiment was done by cycling the same voltages on the SPR gold disk in 0.5 M H2SO4 solution without 4NoPD monomer. The potential change itself during CV did not cause the resonance angle to shift upward, which means that the resonance angle increase observed in the electropolymerization comes indeed from the deposited polymer film. After a number of cycles, there is a change in amplitude of the oscillations. A parallel ellipsometry measurement revealed a nonlinear increase in the thickness of the films at this point, and this may be due to changes in the surface polymerization mechanism.

The reversible redox couple labeled as R2 and O2 at 0.05 V corresponds to the reduction of the nitroso (RNO) group and the oxidation of the hydroxylamine (NHOH) group. Reduction at voltages greater than -0.6 V is irreversible and will produce amine as the final product. The successful irreversible reduction of P4NoPD could be further confirmed by comparing the redox properties before and after polymer film reduction. Figure 3 shows redox peaks O2 and R2 of the P4NoPD film in 0.5 M H2SO4 before and after reduction in 0.1 M H2SO4 (scan rate: 50 mV/s). It can be seen that, after the reduction, the peaks for O2 and R2 were reduced in height and the peak voltages shifted negatively. This is due to the increased film resistances following the irreversible reduction of the nitro groups.12 XPS Characterization of P4NoPD Modified BDD. XPS was used to characterize surface functional groups change before and after reduction of P4NoPD on BDD. Figure 4 shows the relevant N 1s, O 1s, and F 1s spectra. To demonstrate the successful reduction of nitro groups to amine groups in polymer chains, p-trifluoromethyl-benzaldehyde (TFMBA) was used to label each NH2 groups by three fluorine atoms, as shown in the following equation:31

If the NO2 groups in the P4NoPD chains were successfully (27) Hammerich, O., Lund, H., Eds.; Organic Chemistry; Dekker: New York, 2001. (28) Laviron, E.; Rouillier, L. J. Electroanal. Chem. 1990, 288, 165. (29) Laviron, E.; Meunuier-Prest, R.; Vallat, A.; Rouillier, L.; Lacasse, R. J. Electroanal. Chem. 1992, 341, 227. (30) Lacasse, R.; Meunuier-Prest, R.; Laviron, E.; Vallat, A. J. Electroanal. Chem. 1993, 259, 223. (31) Terlingen, J. G. A.; Brenneisen, L. M.; Super, H. T. J.; Pijpers, A. P.; Hoffman, A. S.; Feijen, J. J. Biomater. Sc., Polym. Ed. 1993, 4, 165.

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Figure 5. CVs showing scans to the cathodic limits for the irreversible reduction of nitro groups in the P4NoPD film, on gold disk and BDD electrodes. Electropolymerization was performed by 25 cycles in 4.50 mM 4NoPD in 0.50 M H2SO4. Potential scan rate: 50 mV/s. Table 1. SPR Angle Shift and Surface Coverage of Biomolecules of Each Binding Steps

Figure 4. XPS spectra showing (a) N 1s, (b) O 1s, and (c) F 1s signals on P4NoPD on BDD electrode. The black squares denote the original signals of the as-prepared P4NoPD film (25 cycles in 4.50 mM 4NoPD 0.50 M H2SO4), whereas the open circles show the signals after electrochemical reduction at -0.6 V with reference to Ag/AgCl reference electrode (reduced in 0.10 M H2SO4).

reduced to free NH2 groups, the presence of the F 1s will provide a fingerpint in the XPS spectra. In Figure 4A, the N 1s peak shifted toward lower binding energy after electrochemical reduction of P4NoPD. This could be assigned to the presence of the -NdCH-Ar-CF3 component, which gives a lower binding energy at ∼398.8 eV compared to the nitro groups. The decrease in the O 1s peak intensity, observed in Figure 4B, provides further proof of the reduction of NO2 groups. Furthermore, the presence of F 1s could be detected in

step

angle shift (mdeg)

surface coverage (× 1012 molecules/cm2)

streptavidin biotin-DNA target DNA biotin-DNA/SA ratio

296 85 35 1.76

2.5 4.4 1.0

Figure 4C. This provides strong evidence for the existence of free NH2 groups after NO2 reduction. Using the ratio of the intensity of F/C (AF/C ) 0.0734) and correcting for atomic sensitivity factors as well as electron escape depth of C 1s, the surface density of fluorine could be estimated to be 0.127 × 1014 F atoms cm-2 or 4.25 × 1012 molecules/cm2 32 The density of the NH2 free groups should be close to 4.25 × 1012 molecules/ cm2. It should be noted that, although this value is lower than that of complete NH2 monolayer (∼1014 molecules/cm2), it is sufficient for the immobilization of a monolayer of protein biomolecule, e.g., streptavidin. This is because streptavidin is known to have dimensions of approximately 6 nm, and the upper limit for the immobilized density of a monolayer is 2.34 × 1012 molecules/cm2 (assuming each streptavidin molecule has an approximate spherical symmetry and a footprint of 42.8 nm2).33 Subsequent SPR studies showed that streptavidin immobilized on a reduced P4N PD film has a surface density at the same order of magnitude as the above value (see Table 1). Stability of P4NoPD on Gold and BDD Electrodes. The advantage of using the BDD electrode is that it provides a larger limit for cathodic polarization. Similar experiments carried out on gold electrodes show that reductive desorption of the oligomer film can occur at cathodic voltages greater than -0.4 V after multiple scans. Irreversible reduction of P4NoPD on BDD and gold electrodes is shown in Figure 5. Narrow electrochemical potential window of gold precludes the observation of the reduction peak before the hydrogen evolution but the reduction peak can be observed on BDD electrode. EIS was also employed to study the stability of the P4NoPD film formed on the gold electrode and on the BDD electrode after multiple cycles of CV reduction. Figure 6, panels A and (32) Yang, W. S.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N., Jr.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253-257. (33) Swann, M.; Freeman, N.; Carrington, S.; Ronan, G.; Barrett, P. Lett. Peptide Sci. 2003, 10, 487-494.

SPR and Electrochemical Studies of P4NoPD

Figure 6. Impedance Bode plot of P4NoPD film grown on (a) gold electrode and (b) BDD electrode, in PBS buffer pH 7.4. (9) Fresh polymer film; ([) polymer film after 2 cycles reduction; (2) polymer film after 6 cycles reduction; (1) polymer film after 20 cycles reduction.

B, depicts the impedance Bode plots of P4NoPD after multiple cycles of reduction on gold and BDD electrodes. It can be seen clearly that, after 20 cycles of CV reduction, the impedance of the polymer film on the gold electrode has changed. The modulus of the impedance and the shape of the impedance Bode plot became increasingly similar to that of the bare gold electrode, suggesting desorption of the films from the electrode. However, in the case of the polymer film on the BDD electrode, the impedance spectra remain unchanged after multiple cycles. This result indicates that the attachment of the P4NoPD film on the BDD electrode is more stable than the gold electrode, due perhaps to the stronger C-C covalent bonds. Therefore, the BDD electrode is a more stable platform than the gold electrode for the cathodic polarization of P4NoPD. SPR Measurement of Redox States of P4NoPD. The P4NoPD film was grown on a SPR gold disk by repeated cyclic potential scans. SPR angular scan curves (R-θ) were recorded at the anodic (+0.2 V) and cathodic limits (-0.2 V) of each cyclic potential scan so that the P4NoPD can be generated in the reduced or oxidized states, respectively. After the scans, the DC voltage was set to zero and the electrode was allowed to equilibrate for 5 s before recording the SPR signals. Figure 7 shows that, with higher cycles of polymerization, the SPR curves of both the anodic and cathodic scans shift toward higher angles. The anodic curves in Figure 7a also show distinct changes in the shape of the curve in terms of resonance depth and width compared to the cathodic scans in Figure 7b.

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Figure 7. (a) SPR (R-θ) curve at the open circuit potential after potential scanning to anodic limit +0.2 V (potential scan range set between -0.2 and +0.9 V, and each scan terminates at +0.2 V). (b) scanning to cathodic limit -0.2 V (potential scan range between -0.2 and +0.9 V, each scan terminates at -0.2 V). CVs were carried out on gold electrode in 0.5 M H2SO4 solution containing 4.5 mM 4NoPD monomer.

In principle, the shift in the angle of the resonance minimum can be associated with several factors such as changes in refractive index, thickness of the P4NoPD, and conductivities. The change in shape of the SPR R-θ curve is mainly determined by the variation in imaginary part of the dielectric constant of the film, i.e., the extinction coefficient K, which is a measurement of the light adsorption in a medium.34 At optical frequencies, a conducting material can be characterized by a complex dielectric constant, . For a light with wavelength λ that is incident on material with conductivity σ, the dielectric constant can be expressed as20

 ) real + (2σλ/c)i where c is the speed of light and the imaginary part of the dielectric constant is imag ) 2σλ/c, which is related to the optical absorption of the film. From the equation, it can be seen that the conductivity variation in the P4NoPD film can cause a change in the imaginary part of the dielectric constant, imag. Therefore, the redox transition between the more conductive nitro state (anodic scans) and the reduced hydroxylamine or amine states of P4NoPD (cathodic scans) can lead to a change in shape of the SPR (R-θ) curve, as we shown in Figure 7a. A previous SPR study of the electropolymerization of aniline14 also showed that the polyaniline film gave higher reflectivity at the oxidized state than at the reduced state. (34) Salamon, Z.; Macleod, H. A.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 117.

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Figure 8. SPR reflectivity of P4NoPD on SPR gold disk (left plot) and bare SPR gold disk (right plot). Curves a, c, and e were collected with DC voltage of 0.2 V on gold disk and b, d, and f were collected with DC voltages of -0.2 V on gold disk.

Figure 10. Schematic illustration of the various binding reactions in SPR measurement, including glutaraldehyde activation, streptavidin immobilization, biotin-DNA assemble, and target DNA hybridization.

Figure 9. Thickness of P4NoPD films as measured by ellipsometry. Thicker films were grown using higher CV scan cycles. The films with 25 CV cycles were polarized at different potentials to see the potential-induced change in thickness. Error bars were calculated using 95% confidence limits.

The SPR response was also recorded when a DC voltage of +0.2 or -0.2 V was applied on the film. Figure 8 shows the SPR angular scan curve (R-θ) of the P4NoPD film formed on the SPR gold disk. The curve of the bare SPR gold disk subjected to the same DC voltage was used as a control (curves a, c, and e measured at +0.2 V; curves b, d, and f measured at -0.2 V). It can be seen that the reflectivity of the polymer film at +0.2 V is higher than that at -0.2 V, and that the gold control shows no changes between +0.2 and -0.2 V. This result confirms that potential-induced redox transformation of the P4NoPD film leads to changes in reflectivity, with the oxidized state having a higher reflectivity than the reduced state. Ellipsometry Characterization. The change in thickness of the P4NoPD films during electropolymerization and redox transformations was analyzed. As shown in Figure 9, the polymer film became thicker with CV cycles, and the thickness increased abruptly when the scan cycle increased above 25 cycles. This result parallels the observation in the SPR polymerization curves in Figure 2 which exhibits nonlinear shift of resonance angle at the 25th -30th potential cycles. One explanation is that the source of the polymerization changes from the 4NoPD monomer to the oligomers after these cycles, resulting in the acceleration of the rate of film growth. Variation in film thickness was also observed if the films were polarized at anodic or cathodic voltages.

P4NoPD films oxidized at +0.2 V were found to be thicker than films reduced at -0.2 or -0.6 V. Changes in the thickness of the film may result from a potential-induced piezoelectric effect, or from the swelling and de-swelling as the hydrophobicities of the films changes with changes in the functional groups. The potential induced reflectivity change (∆R) in Figure 10 should be proportional to the surface capacitance that varies as the thickness change of P4NoPD film, according to ∆q ) CDL/∆V. 14,35

SPR Measurement of Streptavidin Immobilization on P4NoPD for DNA Assembly and Hybridization. To prove that free amine groups were introduced onto P4NoPD following the reduction of nitro groups, streptavidin immobilization on the reduced polymer film was carried out using glutaraldehyde (GA) as a cross-linking reagent. GA consists of bifunctional groups and can link the lysine residues in streptavidin with the free amine groups in P4NoPD. Figure 10 shows a schematic illustration of the surface binding procedure involved in this experiment. After streptavidin immobilized on P4NoPD through GA, biotinylated DNA probe is assembled for target DNA hybridization. Figure 11 shows the SPR kinetic measurement (∆θ - t) of the sequential immobilization of streptavidin (0.2 mg/mL), assembly of biotinylated DNA probe (1 µM) and hybridization of target DNA (1 µM). The activation process of reduced P4NoPD film by GA was not presented as the angle shift observed in this step comes mainly from changes in the refractive index of liquid media (from blank PBS to GA containing PBA, then to blank PBS again). This angle shift was much larger than that of protein immobilization, which could affect the observation of the subsequent binding steps. As shown in Figure 11, on the GAactivated, reduced P4NoPD film (formed on SPR gold disk by 30 CV cycles in 0.5 M H2SO4 containing 4.5 mM 4NoPD, reduced (35) Wang, S.; Boussaad, S.; Wong, S.; Tao, N. J. Anal. Chem. 2000, 72, 4003.

SPR and Electrochemical Studies of P4NoPD

Figure 11. SPR responses to the sequential reaction steps of streptavidin immobilization (0.2 mg/mL), biotin-DNA assembly (1 µM), target hybridization (1 µM), rinsing of surface by PBS buffer, on (A) as-prepared P4NoPD film and (B) reduced P4NoPD film (on SPR gold disk).

in 0.1 M H2SO4 by 2 CV cycles, scan rate: 50 mV/s), immobilization of streptavidin induced a angle shift of ∆θ ) 297 mdeg. For the sequential biotin-DNA probe assembly and target DNA hybridization, the resonance angle increased by 85 mdeg and 35 mdeg, respectively. Using a factory-calibrated SPR sensitivity of 120 mdeg ) 100 ng/cm2 for protein and DNA, we converted the saturated SPR angle shift at each step to molecular binding capacities in units of molecules per square centimeter, as shown in Table 1. It can be seen that the one SA molecule binds ∼2 biotin-DNA. The four biotin binding sites on one SA molecule did not result in the binding of four biotin-DNA. This shows that the accessibility of the biotin binding sites determines the DNA binding amount. For the target hybridization, the hybridization efficiency is 43%, calculated from the quotient of the surface coverage of the target DNA/biotin-DNA. This is a reasonable hybridization efficiency value influenced by the orientation of the biotin-DNA probe mediated by the structure of SA layer.36 To make a comparison between the streptavidin binding capacity of the as-grown P4NoPD film with that of the reduced film, experiments with the same sequential binding steps were conducted on freshly formed P4NoPD subjected to the same electrochemical treatment processes. In this experiment, streptavidin adsorption on polymer covered SPR gold disk induced a resonance angle shift of 48 mdeg. This angle shift was much smaller than that on the reduced polymer film. The sequential angle shifts induced by biotin-DNA probe assembly and target DNA hybridization were also much smaller at 30 mdeg and 13 mdeg, respectively. These results indicate that the reduction of the nitro groups in as-grown films is essential to introduce a higher density of amine groups for coupling of biomolecules at a higher density. One reason for the small angle shift observed here for the as-grown films may be the due to the presence of some amine groups in the occluded oligomer. (36) Su, XD.; Wu, Y. J.; Robelek, R.; Knoll, W. Langmuir 2005, 21, 348.

Langmuir, Vol. 22, No. 8, 2006 3935

Figure 12. Surface coverage of streptavidin immobilized on reduced P4NoPD versus number of CV cycles of electropolymerization.

Control experiments were carried out to confirm that the angle shift observed in these experiments comes from the coupling between the free amines groups on the polymer film and the lysine residues. These control experiments included (1) using reduced polymer film without GA activation, this would study the effect of nonspecific binding of the streptavidin; (2) using bare SPR gold disk to study background adsorption of streptavidin on gold disk; (3) studying the nonspecific adsorption of the biotinDNA on the P4NoPD film. Angle shifts of streptavidin immobilization observed in control experiments 1 and 2 were 22 and 49 mdeg, respectively. In control experiment 3, no angle shift was detected. These results indicate that, although there could be a small amount of nonspecific streptavidin adsorption on reduced P4NoPD, biotin-DNA probes could not bind on reduced P4NoPD in the absence of immobilized streptavidin. Figure 12 shows the surface coverage of streptavidin immobilized on a reduced P4NoPD film at different CV cycles of P4NoPD polymerization. The purpose of the plot is to exhibit the relationship between the polymer film thickness and the amount of immobilized streptavidin. As shown in Figure 12, the coverage of streptavidin increased with the cycle number employed in electropolymerization.

Conclusion In summary, we have demonstrated that redox transitions between different redox states in P4NoPD films can be detected by different reflectivities in the SPR reflectivity-resonance angle curves. The oxidized state (nitro or nitroso) shows a higher reflectivity than the reduced state (hydroxylamine or amine). The binding to biomolecules on P4NoPD can be voltagemodulated: reduction of the nitro groups into amine groups switched the P4NoPD system into the state of specific binding with biomolecules. BDD provides a more stable platform for than gold electrodes for the electrochemical reduction of P4NoPD. The P4NoPD can provide a polymer matrix for individual electrochemical addressing due to its redox switching properties. LA053014X