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Characterization of Micropatterned Lipid Membranes on a Gold Surface by Surface Plasmon Resonance Imaging and Electrochemical Signaling of a Pore-Forming Protein Zhuangzhi Wang, Thomas Wilkop, and Quan Cheng* Department of Chemistry, University of California, Riverside, California 92521 Received July 18, 2005 We report the fabrication and characterization of a micropatterned membrane electrode for electrochemical signaling of a bacterial pore-forming toxin, Streptolysin O (SLO) from S. pyogenes. Microcontact printing of an alkylthiol monolayer was used to fabricate an array template, onto which cholesterol-containing DMPC vesicles were fused to form lipid layer structures. The construction of the supported membranes, including pattern transfer and vesicle fusion, was characterized by in-situ surface plasmon resonance (SPR) imaging and electrochemistry. Quantitative analysis of the resulting membrane by using SPR angular shift measurements indicates that the membranes in the hydrophilic pockets have an average thickness of 8.2 ( 0.4 nm. Together with fluorescence microscopy studies, the results suggest that this could be a mixed lipid assembly that may consist of a bilayer, vesicle fragments, and lipid junctions. The voltammetric response of the redox probe ferrocene carboxylic acid (FCA) was measured to quantify the toxin action on the supported membrane. The electrochemical measurements indicate that fusion of vesicles on the template blocked the access of FCA, whereas the injection of SLO toxin restored the redox response. The anodic peak current of FCA was found to increase with toxin concentration until a plateau was reached at 40 HU/mL. The method is highly sensitive such that 0.1 HU/mL of SLO (1.25 pM) can yield a welldefined response. In addition, it eliminates the need for a highly insulating layer in membrane sensing, which opens up new avenues in developing novel sensing interfaces for membrane-targeting proteins and peptides.
Introduction Supported lipid membranes (SLMs) have received considerable attention in recent years because they preserve many biophysical properties of cellular membranes1,2 and offer a unique system for protein structurefunction studies3 and the development of biosensors.4 In the endeavor of sensor development, membrane-associated ligand-receptor interaction,3,5 ion channel gating,4,6 and permselectivity have been explored as important elements in the pursuit of specificity. An interesting development for the membrane-based sensors is stochastic sensing with the pore-forming toxin R-haemolysin (RHL).7 This method monitors the ionic current flow through transmembrane pores, which is modulated by the interaction of the target molecules with the pores, providing detection with remarkable sensitivity and selectivity.8,9 Highly insulating black lipid membranes (BLMs) have been employed in these studies to ensure the measurement of the ionic current in the picoampere range. To enable convenient recording of the sensing signal, use of supported lipid membranes as the hosting interface has been proposed10 * Corresponding author. E-mail:
[email protected]. Fax: (951) 827-4713. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651653. (3) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105-8. (4) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D.; Raguse, J.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580. (5) Martinez, K. L.; Meyer, B. H.; Hovius, R.; Lundstrom, K.; Vogel, H. Langmuir 2003, 19, 10925-10929. (6) Yin, P.; Burns, C. J.; Osman, P. D.; Cornell, B. A. Biosens. Bioelectron. 2003, 18, 389-97. (7) Bayley, H. Curr. Opin. Biotechnol. 1999, 10, 94-103. (8) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999, 398, 686-90. (9) Cheley, S.; Gu, L. Q.; Bayley, H. Chem. Biol. 2002, 9, 829-38. (10) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226-30.
and attempted.11 Despite the success in obtaining SLMs with ultrahigh impedance by tethered12 and nontethered approaches,13 the formation of transmembrane pores by toxins on these surfaces that allow subsequent sensing measurement proved to be rather difficult.11 Generation of such a sensing interface appears to require a delicate balance between stability (high insulation) and lateral fluidity (essential to pore formation) of the membranes.14 Recent advances on supported membranes indicate that micropatterning of lipid layers can provide a high degree of control over the membrane structure and thus its properties.15-18 This method is particularly useful for creating hydrophilic “patches” into which specific proteins or peptides can be inserted for functional analysis. The micropatterning process involves the fusion of lipid vesicles on an alkylthiol monolayer template, which leads to the formation of bilayer regions where the functional activity of a fluid membrane is largely retained.19,20 It is an attractive approach not only because of the improved stability of the lipid layers but also because of the applicability to various surfaces21 and the ease with which (11) Glazier, S. A.; Vanderah, D. J.; Plant, A. L.; Bayley, H.; Valincius, G.; Kasianovic, J. J. Langmuir 2000, 16, 10428-10435. (12) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew. Chem., Int. Ed. 2003, 42, 208-211. (13) Hillebrandt, H.; Wiegand, G.; Tanaka, M.; Sackmann, E. Langmuir 1999, 15, 8451-8459. (14) Plant, A. Langmuir 1993, 9, 2764-2767. (15) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (16) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751. (17) Plant, A. Langmuir 1999, 15, 5128-5135. (18) Jenkins, A. T. A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Scho¨nherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274-5280. (19) Jenkins, A. T. A.; Bushby, R. J.; Evans, S. D.; Knoll, W.; Offenhausser, A.; Ogier, S. D. Langmuir 2002, 18, 3176-3180. (20) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (21) Tero, R.; Takizawa, M.; Li, Y.-J.; Yamazaki, M.; Urisu, T. Langmuir 2004, 20, 7526-7531.
10.1021/la051937m CCC: $30.25 © 2005 American Chemical Society Published on Web 10/12/2005
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Figure 1. Schematic illustration of the fabrication of a micropatterned membrane on a gold substrate and the generation of an electrochemical signal by the pore-forming action of the toxin in the presence of a redox probe.
different functional moieties can be introduced in a spatially defined manner. Selective ion transport of K+ with valinomycin embedded in the supported membrane was reported using conductance measurements.18 In this work, we report the fabrication of a micropatterned membrane electrode with phosphocholine lipid and octadecanethiol and its use in the development of a sensing interface for pore-forming bacterial toxins. Figure 1 shows a schematic illustration of the membrane pattern on a gold substrate and the detection strategy. Microcontact printing of the alkylthiol monolayer was used to fabricate the template, and the construction steps, including pattern transfer and vesicle fusion, were characterized by surface plasmon resonance (SPR) imaging. Instead of probing the ionic current or membrane conductance, the voltammetric response of a redox probe in the buffer solution was measured to quantify the toxin action on the supported membrane. Voltammetric measurement is advantageous owing to its straightforward quantification relationship and nanoampere current magnitude, which virtually eliminate the need for a highly insulating membrane. Such a strategy has been employed in the study of a peptide nanotube incorporated into an alkanethiol SAM layer for probing the size-exclusive accessibility of redox molecules.22
Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids (Alabaster). Cholesterol, 1-octadecanethiol (ODT/C18SH), 2-mercaptoethanol, and ferrocenecarboxylic acid (FCA) were from Aldrich. Bovine serum albumin (BSA), fetal bovine serum (FBS), and Streptolysin O (from Streptococcus pyogenes) were obtained from Sigma. Silicone elastomer base and the curing agent were purchased from Dow Corning Corporation Midland. All chemicals were used as received without further purification. Preparation of Vesicles. The vesicles were prepared by probe sonication with a sonifier (Branson Ultrasonics). DMPC and cholesterol (2:1 molar ratio) were mixed in chloroform in a small vial. The organic solvent was removed with a N2 stream to form a thin lipid layer on the inner wall of the vial. Tris buffer (10 mM, pH 7.5, containing 0.15 M NaCl) was then added, and the
suspension was sonicated at low amplitude in an ice bath for 30 min. The resulting vesicle solution was then incubated at 4 °C for 1 h before use. For fluorescence experiments, 1% NBD-PC was added to the lipid mixture. Preparation of Substrate and Printed Arrays. The gold substrates were prepared by e-beam deposition on microscope slides. A 2-nm chromium sublayer was deposited to enhance adhesion, followed by a 47-nm layer of gold. Prior to array fabrication, the gold substrate was thoroughly cleaned in a piranha solution, extensively rinsed with deionized water and ethanol, and dried with N2. A poly(dimethylsiloxane) (PDMS) stamp was used for microcontact printing of the ODT patterns. After inking the stamp with 10 mM 1-octadecanethiol and allowing it to dry in air for 30 s, it was then gently applied to the gold substrate for 1 min. After washing with absolute ethanol, the patterned substrate was immersed in a 10 mM 2-mercaptoethanol solution for 60 min, followed by extensive washing with ethanol. Fabrication of Supported Lipid Membranes. The vesicles were carefully applied to the patterned gold substrates through injection into a flow cell or direct pipetting, followed by incubation for 1 h. Vesicle incubation has been tested at both room temperature and 37 °C. After incubation, the excess vesicles were removed, and the membrane was extensively rinsed with tris buffer. Electrochemical Procedure. Cyclic voltammetry and linear sweep voltammetry were performed using an Epsilon electrochemical station (Bioanalytical Systems Inc). The electrochemical cell was fabricated from Teflon and mounted onto a piece of gold electrode through an O-ring (i.d. ) 2 mm). The gold substrate was used as the working electrode, with Ag/AgCl as the reference and a platinum wire as the counter electrode. Surface Plasmon Resonance Imaging. SPR imaging was performed by using a home-built SPR imager with an LED light source. The instrument setup was described in detail in a previous publication.23 The patterned gold substrate, made from F2 glass (Schott Glass), was mounted in a flow cell on a prism that was staged on an optical turntable. To ensure better image contrast, mercaptosilane was used as the adhesion layer instead of Cr. A constant flow was maintained by a syringe pump, and vesicles were injected via an HPLC valve. The images were collected with a cooled CCD camera and streamed to a PC for grayscale analysis by lab-developed software. For the angular scans, the turntable was mechanically rotated while an image sequence was recorded to construct the partial angular spectrum. Fluorescence Microscopy. Fluorescence microscopy was carried out with a Meridian Insight confocal laser scanning
(22) Motesharei, K.; Ghadiri, M. R. J. Am. Chem. Soc. 1997, 119, 11306-11312.
(23) Wilkop, T.; Wang, Z.; Cheng, Q. Langmuir 2004, 20, 1114111148.
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Figure 2. SPR imaging analysis of the supported membrane on Au. (A) SPR image of the printed pattern of the C18SH monolayer on Au that defines 100 × 100 µm2 open pockets. The image was collected in air. (B) In-situ monitoring of vesicle fusion on the monolayer pattern by SPRi. Images a-d were collected 6 s apart in sequence starting with image a, showing the pattern just before vesicle injection. (C) Angular scans of the reflectivity near the SPR angle for all elements in the micropattern before and after vesicle fusion. The solid symbols show the experimental data, and the curves represent a third-order polynomial fit. microscope (CLSM) with a cooled CCD detector and a 40× objective. To prepare for the fluorescence analysis, the DMPC vesicles were doped with 1% NBD-PC.
Results and Discussion The fabrication of the supported membrane for toxin sensing was performed using a two-step procedure: (1) PDMS microcontact printing of a predetermined, octadecanethiol (C18SH) monolayer pattern and (2) fusion of cholesterol-containing DMPC vesicles on the pattern. Both steps were characterized and monitored by surface plasmon resonance (SPR) imaging, and the results are shown in Figure 2. SPR imaging is a powerful surface-sensitive technique that has been used in the characterization of a variety of surface structures and phenomena, providing subnanometer thickness resolution and detection sensitivity without the need for fluorescent labels.24 From Figure 2, the PDMS stamp features were transferred with high fidelity, generating a printed pattern of the C18SH monolayer on Au with open areas (“wells”) 100 µm × 100 µm in size and 200 µm apart. Despite the monolayer thickness of the alkyl film, the obtained image displays remarkable contrast, demonstrating excellent sensitivity of SPR imaging for thin film characterization. The gold (24) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41-63.
surface in the open areas was subsequently functionalized with 2-mercaptoethanol (HSCH2CH2OH) to form hydrophilic pockets. Figure 2B shows the SPR images of the injection of DMPC vesicles in the pockets, separated by 6 s. The slightly more visible “distortion” of the pockets is due to in-situ SPR imaging in the buffer, which takes place at a higher image angle than in air (Figure 2A). The diminished dielectric contrast between the pattern and the buffer changes the excitation conditions to higher angles as compared to those in air, which leads to feature shrinkage along the x axis. Nevertheless, this slight image distortion does not affect SPR imaging characterization and quantification. The vesicle fusion occurred very rapidly, reaching an equilibrium state in about 18 s. Note that the increase in reflectivity occurs in both pockets and the confining matrix of the C18SH monolayer. An angular scan of the reflectivity was performed by tracing the minimal SPR angles for all of the features in the pattern. Data collected for the mercaptoethanol regions and the C18SH monolayer before and after vesicle injection are shown in Figure 2C. The raw data points were fitted to a high-order polynomial (solid lines) to determine the SPR angle. A 0.39° angular shift was obtained for the C18SH monolayer as compared to that of the mercaptoethanol-treated pockets. This was attributed to an effective well depth of 1.7 nm, which is obtained by subtracting the mercaptoethanol layer from the 2-nm thickness of the
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Figure 3. Cyclic voltammograms of 1 mM FCA on membrane array electrodes under different conditions. The scan rate was 20 mV/s. The electrode area defined by the O-ring is 3.14 mm2. The 1 mM FCA solution was prepared in 10 mM tris (pH 7.5) and 150 mM NaCl.
C18SH monolayer.25 The SPR angular shift observed after vesicle fusion was used for thickness quantification in the hydrophilic pockets. In comparison to the reference well depth, the angular shift yields a thickness value of 8.2 ( 0.4 nm for the membrane in the pockets after vesicle injection. This method was based on the accepted assumption that the refractive index for alkanethiol layers is in good agreement with that of PC lipids.26 The pore-forming toxin studied in this work is Streptolysin O (SLO), a 61-kD protein toxin produced by S. pyogenes.27 Upon binding to the cholesterol molecules in cell membranes or bilayer vesicles, SLO forms transmembrane pores up to 35 nm in diameter.28 The electrochemical measurements were carried out with the micropatterned Au as the working electrode and ferrocene carboxylic acid (FCA) as the redox probe. Figure 3 shows the voltammetric response under various conditions. On the C18SH monolayer-defined electrode where the wells had been functionalized with HSCH2CH2OH, a welldefined cyclic voltammogram of FCA was observed. Fusion of vesicles on the surface formed a hybrid lipid layer, which essentially blocked the access of the redox probe and suppressed the faradaic current, resulting in a flat background. Injection of 40 HU/mL SLO into the cell, however, restored the redox response, albeit at a smaller magnitude. The hemolytic unit for SLO toxin, HU, is defined as the amount of protein that causes 50% lysis of a 2% red blood cell suspension in phosphate-buffered saline at pH 7.4. To verify that the response is due to pore-forming action, 40 HU/mL denatured SLO (briefly treated with base and brought back to neutral pH) was tested, and no signal was detected. An additional control experiment was performed using 1 mg/mL BSA, which did not generate any redox response. For BSA, further reduction of the background current was observed. We believe that the redox response after SLO injection is a result of pore formation on the membrane, and this unique property observed on the supported lipid layer is possibly due to the fluid nature of the membrane “patches” made by the micropatterning method. Previously, we (25) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731-4740. (26) Jenkins, A. T. A.; Neumann, T.; Offenha¨usser, A. Langmuir 2001, 17, 265-267. (27) Bhakdi, S.; Bayley, H.; Valeva, A.; Walev, I.; Walker, B.; Kehoe, M.; Palmer, M. Arch. Microbiol. 1996, 165, 73. (28) Sekiya, K.; Danbara, H.; Yase, K.; Futaesaku, Y. J. Bacteriol. 1996, 178, 6998.
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demonstrated that phosphatidylcholine (PC) vesicles can offer the needed fluidity for pore formation by SLO, and the encapsulation of redox probes in the PC vesicles immobilized on a gold surface allowed electrochemical sensing of the toxin.29 A similar strategy was recently utilized by Jenkins et al. in a vesicle-based phospholipase A2 sensor.30 Indeed, the lipid assembly in the hydrophilic pocket demonstrates fluid-layer characteristics. It could be largely removed by a 0.5% tween 20 solution (a lipiddisrupting agent), whereas the lipid structures on the C18SH monolayer could not. The ultimate proof for a fluid membrane would be a fluorescence recovery after photobleaching (FRAP) experiment, which was unfortunately nonconclusive in this case because of heavy fluorescence quenching in the proximity of a metal surface.31 Nevertheless, the observed fluorescence intensity in the hydrophilic pocket was very low. To investigate if nonfused vesicles are present in a large quantity in the lipid assembly, we immobilized intact vesicles containing 1% NBD-PC and 1% octadecanethiol on a bare gold surface. A considerably high fluorescence signal was obtained for the intact vesicles because a large portion of the vesicle is out of the quenching region. From this experiment, we concluded that the lipid assembly in the hydrophilic pocket has a primarily planar morphology. It should be pointed out that the notion of obtaining a pure bilayer membrane, typically expected for vesicle fusion on a planar substrate, is not supported by the experimental data. The SPR imaging measurements yield a membrane thickness of 8.2 nm, which is higher than the predicted thickness for a planar bilayer (roughly 5 nm thick). On the basis of these results, we propose that the fluid membrane in the pocket has a mixed morphology that may consist of a bilayer, vesicle fragments, and lipid junctions,32 as illustrated in Figure 1. It is important to emphasize that this mixed membrane is highly fluidic, which makes it suitable for studying protein-membrane interactions. Additionally, it should be noted that the DMPC lipid membrane fabricated by this method is not highly insulating. The charging current observed around the formal potential of the redox probe is ca. 120 nA, indicating high ion flux across the membrane. What we believe is critically important about this membrane is that the flux of FCA across the membrane is largely suppressed, which provides an excellent means for studying transmembrane properties by voltammetry. The injection of BSA reduces the background current to ca. 18 nA, an 85% reduction that is likely a result of enhanced insulation due to the blocking of any defect sites. From Figure 3, the kinetics of FCA was significantly slowed in the presence of the lipid membrane on the electrode. However, once the pores were formed, the kinetics of the response improved substantially, and the peak-to-peak separation was comparable to that on the patterned electrode before vesicle injection. For the membrane electrode treated with 40 HU/mL SLO toxin, ∆Ep ) 64 mV, strongly suggesting a diffusion-controlled process by transmembrane pores, as similarly observed for peptide nanotubes on the alkanethiol monolayer.22 The sensing performance of the membrane electrode was assessed by studying the concentration dependence of the redox signal. Figure 4A shows a working relationship between the anodic peak currents of FCA and SLO concentrations. With increasing toxin concentration, the (29) Xu, D.; Cheng, Q. J. Am. Chem. Soc. 2002, 124, 14314-14315. (30) Jenkins, A. T. A.; Olds, J. A. Chem. Commun. 2004, 2106-2107. (31) Lakowicz, J. R. Anal. Biochem. 2001. 298, 1-24. (32) Wong, A. P.; Groves, J. T. J. Am. Chem. Soc. 2001, 123, 1241412415.
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Figure 4. (A) Plot of the anodic peak current of the redox probe FCA as a function of SLO toxin concentration. (B) Comparison of the linear sweep voltammogram of 1 mM FCA on a toxin-permeated membrane electrode treated with FBS to those without FBS treatment. The scan rate in all cases was 20 mV/s.
redox current increases and reaches a plateau at 40 HU/ mL. The electrochemical measurement appears to be highly sensitive for detection of low concentrations of the toxin. Even at 0.1 HU/mL SLO, a well-defined voltammetric response could be readily obtained (0.1 HU/mL corresponds to roughly 1.25 × 10-15 moles of toxin in 1 mL of solution, or 1.25 pM). It is worth noting that for all of the concentrations studied in this work the current response showed diffusion-controlled characteristics with well-defined cyclic voltammograms and an average peakto-peak separation of 64 ( 5 mV between the cathodic and anodic scans. To further investigate the pore-forming activity of SLO on the supported membrane, we studied the “repairing” process of the toxin pores on the supported membranes. It has been demonstrated that permeation of cells by SLO toxin pores can be repaired or sealed by using 5% fetal bovine serum (FBS),33 though this function on the supported membrane has not been tested. An aliquot of 10 HU/mL SLO toxin was applied to the supported lipid membrane to generate pores, followed by the injection of FBS medium. After 30 min, FBS medium was removed, and 1 mM FCA solution was injected for voltammetric analysis. Figure 4B shows the anodic scans of FCA, along (33) Fawcett, J. M.; Harrison, S. M.; Orchard, C. H. Exp. Physiol. 1998, 83, 293-303.
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with the responses of 0.1 and 1 HU/mL SLO toxin without FBS treatment. Surprisingly, the injection of FBS medium after pore formation turned the peak-shaped response into a sigmoidal-shaped curve, characteristic of a steady-state response. For comparison, the replacement of FBS medium with BSA did not alter the peak-shaped response. The steady-state response at this slow scan rate (20 mV/s) is typically obtained with ultramicroelectrodes (UME) as a result of enhanced (spherical) mass transport. On an array electrode, however, the semi-infinite linear behavior (peaked response) prevails because of the overlap of the diffusion layer. The diffusion layer overlap is also the reason that a large current was obtained on the microcontact printed electrode where the electrode was about 89% passivated (Figure 3). Compared to the response on the membrane electrode treated with 10 HU/mL SLO, the steady-state response after FBS incubation is much smaller (Figure 4B). Quantitative analysis of the currentpotential relationship for this array electrode requires the consideration of a mixed diffusion process and is thus complex. Girault et al. showed that for a microdisk electrode array an increase in the ratio of the interelectrode center-to-center distance to the radius of the active site (d/r) leads to the transition from peak to sigmoidal response.34 Admittedly, a precise definition of the activesite radius (r) in this case is difficult. From the peakshaped response observed before the addition of FBS, the value of the d/r ratio appears to fall in the linear or mixed diffusion region in the Girault model.34 Incubation with FBS apparently reduces the number of pores in the membrane and thus the value of r, resulting in an increase in the d/r ratio that leads to a steady-state response. Interestingly, the steady-state response could not be reached by simply decreasing the SLO concentration further. In conclusion, we have successfully developed an electrochemical method for toxin analysis by using a micropatterned membrane electrode containing hydrophilic lipid patches. The fluid membrane characteristics demonstrated on this electrode allow the formation of transmembrane pores by the toxin. SPR imaging analysis and angular shift measurements show that the membranes in the hydrophilic pockets have an average thickness of 8.2 ( 0.4 nm, indicating a mixed morphology of the lipid structures. Combined with electrochemical measurements using a redox probe, the method offers highly sensitive detection for the bacterial pore-forming SLO toxin. The voltammetric approach reported here eliminates the need for a highly insulating membrane, which bypasses the problem of reduced functionality typically observed with tightly packed membranes. This opens up new avenues in developing supported sensing interfaces for membrane-targeting proteins and peptides. Acknowledgment. This work is supported in part by the National Science Foundation (BES-0428908), an Eli Lilly Analytical Chemist grant, and UC Riverside. Supporting Information Available: Fluorescence images and intensity profiles of the micropatterned membrane after vesicle fusion and intact vesicles assembled on a gold surface. This material is available free of charge via the Internet at http://pubs.acs.org. LA051937M (34) Lee, H. J.; Beriet, C.; Ferrigno, R.; Girault, H. H. J. Electroanal. Chem. 2001, 502, 138.
