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Reorientation of Tetradecylmethyl Viologen on Gold upon Coadsorption of Decanethiol and Its Mediation of Electron Transfer to Nitrate Reductase§ Vytas Reipa,*,† S.-M. Laura Yeh,† Harold G. Monbouquette,† and Vincent L. Vilker‡ Chemical Engineering Department, University of California, Los Angeles, California 90095-1592, and Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Received October 2, 1998. In Final Form: July 6, 1999 Decanethiol was coadsorbed with tetradecylmethyl viologen (C14MV; 1-Methyl-1′-tetradecyl-4,4′bipyridinium chloride) on gold electrodes to improve stability of C14MV as an electrochemical mediator for coupling to nitrate reductase enzymes. Surface-enhanced Raman spectroscopy (SERS) and in situ spectroscopic ellipsometry were used to monitor the structural properties of surface-confined C14MV during its redox conversion. The potential range investigated was limited to that of the first electron transfer to give V•+, as the subsequent reduction to the neutral species is irreversible and could not be used for electron-transfer mediation to redox enzymes. When C14MV was adsorbed by itself, in the absence of C14MV solution species, the in situ optical studies showed the loss of initial electroactivity was due to the bipyridinium rings being oriented parallel to the electrode plane. This configuration is thought to be unfavorable for the anion (Cl-) transport in and out of the film, which is essential for the redox reaction. The electroactivity in the adsorbed film was restored by coadsorbing decanethiol (C10T) with C14MV. This gave an intercalated film with the end-on, bipyridinium ring oriented vertically relative to the electrode surface. In this film, the smaller methyl group is positioned closer to the electrode surface, and the bipyridinium electroactive groups are surrounded by longer decanethiol molecules. Both SERS and spectroscopic ellipsometry measurements show the presence of radical dimers in reduced surface films. Intercalated C14MV is stable for several thousand voltammetry scans and was found to be an efficient electron-transfer mediator to soluble nitrate reductase, despite being embedded in a decanethiolate layer.
Introduction As the accumulation of nitrate in water becomes an increasingly severe problem,1 there is a growing need for both nitrate sensors2 and for systems that promote nitrate elimination from contaminated water.3 Efficient, stable electronic coupling of nitrate reductase to electrodes could lead to such devices. Viologens, 1,1′ disubstituted 4,4′bipyridinium compounds, have been utilized extensively as electron donors to oxidoreductase enzymes catalyzing reduction reactions. Both the viologen dication/radical monocation (V2+/V•+) and radical monocation/neutral species (V•+/V°) redox couples have very negative redox potentials4 that are affected by the chemistry of 1,1′ substituents,4 oligomerization,5 and solution anion.4 Generally, viologens with longer 1,1′ substituents are more likely to form adsorbed films on electrodes upon reduction. The reduced forms of viologen, V•+ and V°, are less soluble in water than V2+ and may precipitate with counterion on * Author to whom correspondence should be addressed. † University of California, Los Angeles. ‡ National Institute of Standards and Technology. § Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment is necessarily the best available for the purpose. (1) Cosnier, S.; Innocent, C.; Jouanneau, Y. Anal. Chem. 1994, 66, 3198. (2) Smil, V. Sci. Am. 1997, July, 76-81. (3) Mellor, R. B.; Ronnenberg, J.; Campbell, W. H.; Diekmann, S. Nature 1992, 355, 717. (4) Bird, C. L.; Kuhn, A. T. Chem. Soc. Rev. 1981, 10, 49. (5) Sato, H.; Tamamura, T. J. Appl. Polym. Sci. 1979, 24, 2075.
a bare electrode surface. Unfortunately, this often results in electrochemical irreversibility6 that limits the utility of viologens as electron-transfer mediators to redox enzymes. If this electrochemical instability could be overcome while still maintaining the convenience of an electrode system, viologens could find applications as electronic mediators for several redox enzymes having negative reduction potentials, e.g., nitrate reductases3 or ferredoxins.7 Recently, the redox activity of covalently attached asymmetric viologens8-10 and their use as electron-transfer mediators to nitrate reductase were demonstrated.9 A chemisorbed sulfide derivative of viologen initially was electroinactive on gold, but it reorganized into an electroactive state upon coadsorption of a long-chain alkanethiol.9 Reorientation of the adsorbed viologen bipyridinium residues after coadsorption of the alkyl mercaptan was hypothesized to be responsible for the improved electroactivity observed.9 Similar behavior was reported for the mixed monolayers of tetradecylmethyl viologen (C14MV) and L-R-dimyristoylphosphatidic acid on In and Sn oxides.11 Also, an increase in molecular ordering has been observed upon mixing of galvinol-substituted and decanethiol (C10T) monolayers on gold.12 Although these (6) Wang, H. X.; Sagara, T.; Sato, H.; Niki, K. J. Electroanal. Chem. 1992, 331, 925. (7) Landrum, H. L.; Salmon, R. T.; Hawkridge, F. M. J. Am. Chem. Soc. 1977, 99, 3154. (8) Bunding-Lee, K. A. Langmuir 1990, 6, 709. (9) Katz, E.; Itzhak, N.; Willner, I. J. Electroanal. Chem. 1992, 336, 357. (10) Katz, E.; Itzhak, N.; Willner, I. Langmuir 1993, 9, 1392. (11) Fernandez, A. J.; Martin, M. T.; Ruiz, J. J.; Munoz, E.; Camacho, L. J. Phys. Chem. B 1998, 102, 6799.
10.1021/la981371k CCC: $18.00 © 1999 American Chemical Society Published on Web 09/16/1999
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studies demonstrated the apparent close relationship between viologen molecular conformation on the electrode surface and electroactivity, such a relationship has not been confirmed and characterized. The study presented here was undertaken to elucidate the difference in molecular orientation of C14MV when adsorbed alone on bare gold and when coadsorbed with decanethiol (C10T), which is reflected in different redox behavior in each of the two cases. The potential range investigated was limited to that of the first electron transfer to give V•+, as the subsequent reduction to the neutral species is irreversible and could not be used for electron-transfer mediation to redox enzymes. Surfaceenhanced Raman spectroscopy (SERS) and in situ spectroscopic ellipsometry were used to monitor the structural properties of surface-confined C14MV during its redox conversion. Earlier, we demonstrated the advantages of these techniques for elucidation of biomolecule-electrode interactions when used together with traditional electrochemical methods.13-15 C14MV optical absorption in the UV-visible range is determined by the electronic state of the bipyridinium chromophore and is sensitive to the interaction between those groups that occurs during oligomerization. The dication species is transparent in the visible range, but has an absorption band due to a π-π* transition at ∼265 nm, while the monocation radical has absorption bands at 360 and 600 nm,16 which enables resonant Raman excitation in the visible range. Viologen derivatives previously were characterized by Raman spectroscopy both in solution17-19 and as surface adsorbates, particularly on silver electrodes.18-20 It was observed that substituents on the two nitrogen atoms of the viologen do not affect its overall D2h symmetry. Only the Raman bands which contain major contributions from N-alkyl stretching vibrations (∼1200 cm-1) are influenced by asymmetric substituents on the bipyridinium rings.18 Spectra are sensitive to radical aggregation and were used to distinguish between face-to-face and oblique stacking of radical dimers.19 Experimental Section 1-Methyl-1′-tetradecyl-4,4′-bipyridinium dichloride was purchased from Fluka and used without further purification. Most measurements were done in 0.1 M KCl + 0.1 M KHPO4 buffer adjusted to pH 7.4. Solutions were prepared from analyticalgrade reagents in purified water (18.2 MOhm, Millipore) and were deaerated at least 30 min prior to and throughout experiments by sparging with argon. Nitrate reductase (from E. coli, EC # 1.9.6.1, 45 U/g) was obtained from Sigma and used without further purification. Ellipsometric measurements were conducted using the Woollam M-44 ellipsometer (J. A. Woollam Co., Lincoln, NE) in the vertical arrangement employing the custom-designed quartz spectroelectrochemical cell and measurement procedure de(12) Sagara, T.; Midorikawa, T.; Shultz, D. A.; Zhao, Q. Langmuir 1993, 14, 3682. (13) Reipa, V.; Gaigalas, A.; Abramowitz, S. J. Electroanal. Chem. 1993, 348, 413. (14) Reipa, V.; Gaigalas, A. K.; Edwards, J. J.; Vilker, V. L. J. Electroanal. Chem. 1995, 395, 299. (15) Reipa, V.; Gaigalas, A. K.; Vilker, V. L. Langmuir 1997, 13, 3508. (16) Mizuguchi, J.; Karfunkel, H. Ber. Bunsen-Ges. Phys. Chem. 1993, 97, 1466. (17) Ghoshal, S.; Lu, T.; Feng, Q.; Cotton, T. M. Spectrochim. Acta 1988, 44a, 651. (18) Lu, T.; Cotton, T. J. Phys. Chem. 1987, 91, 5978. (19) Lee, C.; Lee, Y. M.; Moon, M. S.; Park, S. H.; Park, J. W.; Kim, K. G.; Jeon, S. J. J. Electroanal. Chem. 1996, 416, 139. (20) Datta, M.; Vansson, R. E.; Freeman, J. J. Appl. Spec. 1986, 40, 251.
Langmuir, Vol. 15, No. 23, 1999 8127 scribed previously.15 Electrodes consisted of glass slides coated with 300 nm thermally evaporated gold (99.99%, JohnsonMatthey) on a Cr underlayer for better adhesion. These slides were stored in distilled water prior to use and were flame-cleaned according to ref 22. Following cleaning, electrodes were placed in 10-mM C14MV solution for 2 h under open circuit conditions. After thorough washing with distilled water and ethanol, the slides were immersed in 1 mM decanethiol (C10T; 1-decanethiol, Aldrich) for 1 h, rinsed in ethanol and water, and mounted in a cell. A water film was maintained on the electrode surface during mounting to prevent contamination. Raman measurements were conducted at 647-nm excitation with a Coherent Kr ion laser (Innova 300) using a system described earlier.14 Laser power, as measured at the sample, did not exceed 5 mW for SERS measurements. After polishing and sonication, gold electrodes (end face of 3-mm-diam gold rod) were subjected to five oxidation/reduction cycles in 0.1 M KCl to make them SERS active. Each cycle consisted of a 50 mV/s linear potential scan between -0.5 and 1.3 V with a 10-s delay at the positive potential limit. Electrodes were stored in buffer solution before experiments. C14MV was adsorbed by soaking a SERSactive electrode in a 10-mM C14MV aqueous solution for 2 h with or without a subsequent 1-h immersion in 1 mM C10T solution in ethanol. Every procedure was followed by thorough washing in pure water and/or ethanol. Electrochemical control was rendered by an EG&G M273A system using 0.5-mm-diam Pt wire counter and Ag/AgCl reference electrodes (Abtech model Re803). A 590 Hz low-pass filter was used in the current measurement circuit to reduce noise. All potential values are given relative to the Ag/AgCl (sat. KCl) electrode. Some voltammetry measurements were conducted using a custom designed rotating electrode cell. Nitrate reductase experiments were executed using tightly sealed 1-cm spectrophotometric cells. Due to the high sensitivity of C14MV to residual oxygen, all solution manipulations were performed in a chamber filled with argon. Control experiments with thoroughly deaerated buffers demonstrated negligible current response.
Results and Discussion Voltammetry. SERS-active gold electrodes were subjected to several cyclic voltammetry scans in viologenfree buffer following exposure to 10-mM C14MV solution for 2 h in the open circuit and thorough rinsing. The initial voltammetric response showed redox peaks, but after approximately three scans, subsequent voltammograms showed no redox peaks in the potential range from -0.43 to -0.68 V (see Figure 1b), indicating only the decrease in double-layer capacitance with respect to the electrodes that had no exposure to viologen (Figure 1a). In contrast, our previous results showed well-defined current peaks in the voltammograms obtained in dilute C14MV solution.22 For electrodes with adsorbed C14MV that were exposed for 1 h to 1-mM decanethiol (C10T), reversible redox behavior was observed as shown in Figure 2. Sustained potential cycling across the potential range of the redox peaks for several days did not diminish the response, suggesting excellent stability of the adsorbed molecular layer (Figure 2). We estimate coverage of about 30%, as determined by coulometry and using a surface area of 40 Å2/molecule for C14MV adsorbed in the end-on, upright position. The midpoint potential for C14MV with coadsorbed decanethiol (-0.53 V vs Ag/AgCl, sat KCl) was shifted about 70 mV from that for C14MV in solution (-0.46 V).22 The voltammetric trace showed small anodic/cathodic peak separation, characteristic of surface-confined species, but a plot of peak current versus the square root of scan rate was linear (Figure 3), which is characteristic of a (21) Cotton, T.; Kim, J. H.; Uphaus, R. A. Microchem. J. 1990, 42, 44. (22) Reipa, V.; Monbouquette, H. G.; Vilker, V. L. Langmuir 1998, 14, 6563.
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Figure 1. Potentiodynamic curves recorded on a SERS-active gold electrode in 0.1 M KCl + 0.1 M KHPO4 buffer, pH 7.4: (a) bare gold; (b) after 2 h exposure to 10 mM tetradecylmethyl viologen (C14MV) solution with subsequent washing before the potential scan. The potential sweep rate was 0.2 V/s.
Figure 3. Cathodic peak current dependence on the square root of the potential scan rate for a gold electrode with sequentially adsorbed C14MV and decanethiol. Peak current vs rotation rate for the 0.2 V/s scan is shown in the inset.
Figure 2. Potentiodynamic curves recorded for a SERS-active gold electrode with sequentially adsorbed C14MV and decanethiol (C10T): (a) first scan, (b) 5000th scan. Experimental conditions are the same as in Figure 1.
mass transfer limitation. The voltammetric response, however, was not sensitive to electrode rotation, which suggests that bulk species diffusion is not the rate-limiting step (see inset on Figure 3). Later, we will argue that solution anion movement in the adsorbed layer25 is the rate-limiting step that is consistent with both voltammetric responses. Raman Spectroscopy. Based on the voltammetry data, we hypothesized that loss of C14MV electroactivity when adsorbed in the absence of decanethiol could be due to some surface rearrangement leading to an electroinactive species. Therefore, coupling between electroactivity and structure for C14MV with and without subsequent decanethiol adsorption was investigated using SERS. Figure 4 compares spectra for C14MV in solution with that of adsorbed C14MV with and without decanethiolate at a potential of -0.3 V. Proposed C14MV band assignments are listed in Table 1 and are based on prior analyses reported in the literature.17,19,21 The SERS spectrum recorded for C14MV-modified electrodes, without coadsorbed C10T (Figure 4b), includes (23) Brolo, G. A.; Irish, D. E.; Smith, D. B. J. Mol. Struct. 1997, 405, 29. (24) Osawa, M.; Yoshi, K. Appl. Spectrosc. 1997, 51, 512. (25) De Long, H. C.; Buttry, D. A. Langmuir 1992, 8, 2491.
Figure 4. Raman spectra for (a) 10 mM C14MV aqueous solution; (b) MRS-active gold electrode at -0.3 V with adsorbed C14MV in 0.1 KCl + 0.1 M KHPO4 buffer, pH 7.4; and (c) SERSactive gold electrode at -0.3V with adsorbed C14MV and coadsorbed decanethiol (C10T). The excitation wavelength was 647 nm at laser power of 30 mW (a) or 5 mW (b, c).
characteristic bipyridinium Raman bands (1170, 1195, 1303, and 1532 cm-1). These bands are still present after several voltammetry scans. This persistence of viologen Raman bands in the spectrum for adsorbed C14MV, and the ability to recover a stable electrochemical response upon coadsorption of C10T, suggest that desorption of C14MV is not the cause of the electrochemical instability on gold. Although the same main bands prevail in the spectrum for C14MV in solution (Figure 4a) and in the adsorbed state at -0.3 V (Figure 4b), there are some noteworthy differences. The single band at 840 cm-1 in the solution spectra, assigned as C-C stretching, is split into two bands (825 and 845 cm-1) for the adsorbed molecule. The solution band at 1065 cm-1, the pyridinium ring breathing vibration, is also split into two bands for the adsorbed state (1027 and 1062 cm-1). The bands at 1235 and 1450 cm-1 are also split into two bands upon adsorption. Solution
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Table 1. Solution- and Surface-Enhanced Raman Vibrational Frequencies (in cm-1) of Tetradecylmethyl Viologen (C14MV) or Tetradecylmethyl Viologen and Decanethiol (C14MV + C10T) electrode SERS at -0.3 V
electrode SERS at -0.6 V
solution Raman
C14MV
C14MV + C10T
C14MV
C14MV + C10T
C14MV band assignments
840 892 1065
823; 843 892; 970 1027; 1062 1078 1116 1175 1192 1225; 1240 1303
823; 845 892 1025; 1062 1070 1115 1167 1195 1225; 1255 1303
823; 843 892 1022; 1030; 1055
ν(C-C); ν(C-N)Ag
1450
825; 845 892; 970 1027; 1062 1078 1116 1170 1195 1225; 1240 1303 1360 1420
1420; 1440
1540 1610
1532 1615
1532 1615; 1638
1420; 1440 1490 1529 1612
1175 1195 1235 1305
bands at 1540 and 1175 cm-1 are downshifted to 1532 and 1170 cm-1 in the adsorbed case, while the band at 1610 is upshifted to 1615 cm-1. The relative intensities of the 1170 cm-1 band, corresponding to the N-C14H29 stretch, and of the N-CH3 band at 1195 cm-1 are similar in both the adsorbed and solution spectra. SERS band intensity differences relative to the corresponding solution Raman bands are determined by surface selection rules, which basically favor vibrations along the surface normal.23 Also, the enhancement of the SERS signal decays rapidly with distance from the metal surface. Since both N-alkyl stretches of C14MV are along the same axis of the viologen molecule, they will have the same orientation relative to the electrode surface, yet they are the same distance from the electrode only when the bipyridinium rings are positioned parallel to the electrode surface. A comparison of SER spectra before and after decanethiol adsorption can therefore provide evidence of bipyridinium ring reorientation upon decanethiol coadsorption. We have analyzed only these bands that could be assigned to C14MV molecules in the mixed layer. In fact, the spectrum is different for C14MV with coadsorbed decanethiol (Figure 4c). The higher-frequency band at 1192 cm-1, corresponding to N-CH3 stretching, dominates the N-C14H29 stretch, indicating that C14MV is oriented with the methyl end closer to the electrode surface. Emergence of the 1116 cm-1 band, tentatively assigned to a ring C-C stretch (B2u)17 along the molecular axis, supports the conclusion that the viologen rings are oriented perpendicular to the surface. This band is absent from the solution spectrum (Figure 4a), is of moderate intensity in the adsorbed viologen spectrum (Figure 4b), and is enhanced after coadsorption of decanethiol (Figure 4c). These spectra therefore imply C14MV adsorption in an end-on position when intercalated in a thiolate layer where the C-C inter-ring stretch is oriented away from the electrode surface and the methyl group is positioned close to the surface. The SERS spectra at -0.3 V (Figure 4b-c) show the appearance of several new bands at 825, 970, 1027, 1078, 1360, 1532, and 1638 cm-1, and particularly strong new bands at 1027 and 1532 cm-1, which cannot be explained solely on the basis of surface selection rules. All of these additional bands are characteristic of the radical monocation,17 which is somewhat unexpected because the spectra were recorded at an electrode potential characteristic of fully oxidized species. We showed earlier, by spectroscopic ellipsometry,22 the transient presence of the monocation radical species on a gold electrode at this same potential (-0.3 V). The current data therefore provide additional evidence for the existence of radical
1110 1195 1240; 1255 1303; 1355 1422; 1445 1490 1525; 1537
Ring breathing ν(C-C)B2u ν(N-C14H29)Ag ν(N-CH3)Ag δ(H-C-C)Ag δ(C-C)irrAg δ(H-C-C)B1g ν(C-N)Ag ν(C-C)Ag
Figure 5. SERS spectra for (a) C14MV adsorbed alone on a bare gold SERS-active electrode and (b) C14MV with coadsorbed decanethiol on gold. Electrode potential, E ) -0.6 V. The excitation wavelength was 647 nm at a laser power of 5 mW.
molecules in the adsorbed film, the effect being more pronounced with coadsorbed decanethiol. Although the concentration of the monocation is about 4 orders of magnitude less than that of dication, at -0.3 V (as estimated from Nernst equation), this is compensated for by the higher intensity of resonance-enhanced radical bands. According to published absorption spectra of the disubstituted viologen cation radical solutions, our excitation wavelength (647 nm) is close to the maximum of the broad visible absorption band centered at λ ) 610 nm.21 The SERS spectrum recorded at -0.6 V for C14MV adsorbed on gold without coadsorbed decanethiol (Figure 5a) contains bands characteristic of both the C14MV dication and monocation. Compared to the spectrum recorded at -0.3 V (Figure 4b), there is only minor variation in the intensity ratio of the 1065 cm-1 (radical monocation ring breathing) and 1025 cm-1 (dication ring breathing) bands. The low intensity of the radical bands suggests that only a small portion of adsorbed molecules is reduced. A notable difference between the spectra of Figure 5a and the spectra of Figure 4a is the absence of the inter-ring C-C stretch (expected at 1355 cm-1 for monocation species17 because of reduction at -0.6 V) which was present in the adsorbed dication spectrum at 1305 cm-1 (Figure 4a). Again, such an effect can be accounted for on the basis of viologen adsorbate orientation relative to the electrode surface. In the discussion above, it was noted that the parallel orientation of the bipyridinium moiety relative to the electrode plane does not favor appearance of this vibration in the SERS spectrum,
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suggesting that this is the prevailing position when C14MV is adsorbed without the decanethiol. Such an arrangement was found to be electroinactive in this work, in contrast to the case when viologen was present in solution.22 Resonant enhancement is evident in the SERS spectra for C14MV with coadsorbed decanethiol (C10T) at -0.6 V (Figure 5b), a characteristic of the monocation radical. Bands assigned to bipyridinium chromophore vibrations (1022, 1355, and 1530 cm-1) are several times more intense relative to the rest of the spectrum for C14MV intercalated in decanethiolate. Decanethiolate spectral features were not analyzed due to their significantly lower intensity. A strong band at 1022 cm-1 is a ring breathing vibration shifted from 1062 cm-1 in the dication SERS spectrum.17 This shifted band becomes a double band in the presence of decanethiolate, indicating a shift in bipyridinium ring orientation relative to the case without the adsorbed thiol. Another intense band in the adsorbed radical spectrum at 1355 cm-1 originates from the inter-ring C-C bond stretch. Reduction contributes additional electron density to this bond and increases the force constant, resulting in the shift from 1303 to 1355 cm-1. A prominent band at 1525 cm-1 is attributed mainly to the totally symmetric C-N and C-C stretches localized at the substituent ends of the bipyridinium rings. Taken together, the enhancement of these three bands suggests the end-on orientation of the double-ring system relative to the electrode plane when intercalated in decanethiolate. Also, as discussed earlier for the spectrum at -0.3 V, the presence of only one N-alkyl substituent stretch at 1195 cm-1 (N-CH3 stretch) in this spectrum is strongly indicative that the longer alkyl chain is farther from the surface. Finally, several strong bands (1022 and 1030 cm-1; 1525 and 1537 cm-1) in the spectrum shown on Figure 5b have doublet features. According to a recent study,24 these spectral features are characteristic of unaged viologen radical dimers. In a radical-radical dimer, totally symmetric (Ag) vibrational modes of two constituent molecules couple to give in-phase and out-of-phase vibrations by vibronic coupling, thereby producing doublet bands. An important feature of such surface dimers is their association with solution anion, which forms a charge-transfer complex. Spectroscopic Ellipsometry. In situ spectroscopic ellipsometry was used to detect film thickness variation during C14MV oxidation and reduction while intercalated in a decanethiolate layer on a gold electrode. Measurements were conducted during repeated 20 mV/s voltammetric scans between -0.2 and -0.7 V. A periodic lowamplitude variation of the ellipsometric phase and amplitude parameters, ∆ and Ψ, respectively, as a function of potential (Figure 6) indicated reversible perturbation of the electrode surface state. A certain amount of ∆ and Ψ variation can arise from recharging of the thin metal surface layer due to the so-called “electroreflectance” effect; therefore, its contribution was deduced from the total signal by subtracting the ∆, Ψ variation in the same potential range for the bare gold electrode as described in Reipa et al.15 Subsequently, the film thickness and optical constant spectrum were estimated based on a three-phase (solution, surface film, gold substrate) model15 using averaged data from five scans. We have solved for n, k, and d by fitting the linear ∆, Ψ trajectories in the δ∆-δΨ plane at each measured wavelength, thus assuming that n and k are invariant for each respective segment and only d is changing. This analysis resulted in measured average film thicknesses of 20.3 ( 0.2 Å at -0.2 V and 18.3 ( 0.1 Å at -0.7 V. These values are just slightly more
Reipa et al.
Figure 6. Ellipsometric trajectory, recorded during a potentiodynamic scan (20 mV/s) between -0.2 and -0.7 V for a gold electrode with sequentially adsorbed C14MV and decanethiol after subtraction of the gold-charging signal according to ref 33. The presented trace corresponds to the 545-nm wavelength channel.
than the approximate dimension of a fully extended C10T monolayer (18 Å). A reversible film thickness alteration (2 Å during the redox reaction could be associated with the movement of Cl- in and out of the viologen layer, as suggested the earlier in discussion of the SERS results. Dynamic ellipsometry measurements during the redox reaction of gold-surfaceconfined ferrocenyl groups showed a 3 Å layer thickness increase after oxidation.26 Such variation was related to the uptake of anions from the electrolyte, resulting in ion-pair formation between the anions and the ferricinium ions of the film. A quartz microbalance study25 of the reduction of covalently attached viologen groups on gold in chloride solution revealed large, resonant frequency variations which were ascribed to changes in associated solvent (water) due to conversion of the hydrophilic dication to the hydrophobic radical monocation. The presence of the hydrophobic decanethiolate alkyl chains among the viologen moieties on our electrodes should diminish this effect, thereby making the change in Clconcentration in the local microenvironment of the viologen monolayer a more plausible explanation for the thickness variation during reduction. Film absorption spectrum, R(λ) (Figure 7), was evaluated from the ellipsometric solution of the imaginary part of the complex index of refraction n ) n + ik, using the wellknown relation R(λ) ) 4πk(λ)/λ. The R(λ) spectrum determined from ellipsometry data at -0.7 V (Figure 7) is composed primarily of two absorption bands at ∼510 and ∼600 nm. The former has been reported19 to be characteristic of viologen dimers with oblique stacking, while the latter represents radical monomers. Therefore, the adsorbed C14MV may be in the form of dimers intercalated among decanethiolate alkyl chains. However, we have to note that gold interband transition around 500 nm could perturb the film R(λ) spectrum in this region.15 In previous work,22 we determined that C14MV forms a monolayer when exposed to gold as a dilute solution, and that it gives quasi-reversible redox behavior when equilibrated with solution species. Initially, the adsorbed viologen layer exhibits an ellipsometrically measured thickness that is characteristic of the end-on, fully extended orientation. Subsequently, this measured thick(26) Ohtsuka, T.; Sato, Y.; Uosaki, K. Langmuir 1994, 10, 3658.
Reorientation of Tetradecylmethyl Viologen
Figure 7. Spectrum of the imaginary part, k(λ), of the complex index of refraction as solved from ellipsometry data for a C14MV + C10T layer on a gold electrode at -0.7 V.
ness decreases, presumably because of reorientation of the bipyridinium moieties to a position that is parallel with the electrode surface. Repeated injection of additional viologen into the bathing solution resulted in repeated momentary thickness increases which were followed again by equilibration to lower values, demonstrating the sensitivity of the adsorbed layer to external perturbation, and the likely molecular exchange among surface and solution species. Any variation of this equilibriumseither by injection, which increases solution viologen concentration, or by potential scan, which attracts more positively charged molecules to the surface, induces a thickness increase to vertical, fully extended orientation (∼25 Å). These thicker (less dense) films could be more suitable for reduction because of the easier Cl- movement in and out of the film. Such reasoning is supported by our optical spectroscopy results and can account for the loss of electroactivity in adsorbed C14MV. The movement of the solution anion was noted25 as essential during viologen reduction and could account for the observed diffusional limitation in the reduction of the surface-confined C14MV in decanethiolate, as was found from the characteristic peak current dependence on the scan rate (Figure 3). Due to its 18 Å length in the extended position, the decanethiolate layer can completely encompass the C14MV bipyridyl groups if these groups are attached at their methyl end, thereby extending only ∼11 Å from the electrode surface. Extending above these groups is an outer layer formed by the C14MV tetradecyl substituents. As seen from the solution looking down on the electrode, the tetradecyl chains have a significantly smaller molecular cross section (20 Å2) versus the bipyridinium groups (40-45 Å2)25. This suggests there is a rather disordered outer film which free Cl- anions must permeate. The local microenvironment at an electrode surface could result in different stability of the V2+ and V+ moieties and account for the 70-mV redox potential shift relative to the solution value (Figure 2). Andreu et al.27 have shown considerable potential shifts for redox couples that are embedded in alkanethiol layers due to ion pairing. The easier anion (Cl-) access to the bipyridinium group oriented along the normal to the electrode surface when coadsorbed with decanethiol thus could play a major role in the improved electroactivity of surfaceconfined C14MV. (27) Andreu, R.; Calvente, J. J.; Fawcett, R. W.; Molero, M. J. Phys. Chem B 1997, 101, 2884.
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Figure 8. Cyclic voltammograms (200 mV/s) for a gold electrode with sequentially adsorbed C14MV + C10T in 0.1-KCl + 0.1-M KHPO4, pH 7.4 buffer with 200-µM NaNO3: a, no enzyme present, and b, with 0.2 U/mL E. coli nitrate reductase. All solutions were thoroughly deaerated before and after mixing.
C14MV-Mediated Electron Transfer to Nitrate Reductase. The simple procedure of sequential viologen followed by decanethiol adsorption could be used for the preparation of an electrode interface capable of electrical communication with solution redox enzymes. Although insulating alkyl chains surround the bipyridinium groups, the outer film appears sufficiently flexible or defective to allow redox proteins access for electron transfer. We have tested the gold electrode prepared as described above, but without SERS activation, for electroactivity toward soluble nitrate reductase (NR) from Escherichia coli. Figure 8 demonstrates the effect of substrate (NO3-) addition on cyclic voltammetry curves recorded for such a gold electrode in the presence of NR at 0.2 U/mL. The catalytic cathodic current increase corresponds to electroenzymatic nitrate reduction mediated by C14MV. Rapid reoxidation of surface-confined viologen by enzymes also results in the expected decrease in the anodic peak. These observations are indicative of electron transfer to the soluble NR through mediation by intercalated C14MV. Conclusions This in situ optical study of electrochemically active tetradecylmethyl viologen adsorbed on a gold electrode describes the loss of initial electroactivity of the adsorbed C14MV film in the absence of unadsorbed species free in solution. The predominant orientation of the adsorbed molecules on gold was with the bipyridinium rings oriented parallel to the electrode plane. This configuration is thought to be unfavorable for the anion (Cl-) movement in and out of the film, which is essential for the redox reaction. The presence of the viologen in solution enables a continuous dynamic exchange between surface and bulk molecules, thus supporting the anion transport process, thereby sustaining the oxidation/reduction cycle. The electroactivity in the adsorbed film was restored by coadsorbing decanethiol (C10T) with C14MV. This gave an intercalated film with the end-on, bipyridinium ring oriented vertically relative to the electrode surface. In this film, the smaller methyl group is positioned closer to the electrode surface and the bipyridinium electroactive groups are surrounded by longer decanethiol molecules. Both SERS and spectroscopic ellipsometry measurements show the presence of radical dimers associated with solution anions in reduced surface films. Intercalated C14MV is stable for several thousand voltammetry scans and
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was found to be an efficient electron-transfer mediator to soluble nitrate reductase, despite being embedded in a decanethiolate layer. Acknowledgment. The authors express their gratitude to Dr. Anne Plant for making time available for their
Reipa et al.
work on the spectroellipsometer. This project was supported by the National Science Foundation (Award No. BES-9400523) and by the National Institute of Standards and Technology (Cooperative Agreement 70NANB7H0009). LA981371K
