Characterization of Mixed Alcohol Monolayers Adsorbed onto a Au

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Characterization of Mixed Alcohol Monolayers Adsorbed onto a Au(111) Electrode Using Electro-fluorescence Microscopy Jeff L. Shepherd† and Dan Bizzotto*,† Department of Chemistry, AdVanced Materials and Process Engineering Laboratory (AMPEL), UniVersity of British Columbia, 2355 East Mall, VancouVer BC, Canada V6T 1Z4 ReceiVed NoVember 7, 2005. In Final Form: March 9, 2006 A single-crystal Au(111) electrode modified with an adsorbed layer of 1-octadecanol (C18OH) or oleyl alcohol (OLA) in pure or mixed composition was characterized using electrochemical and in situ fluorescence microscopy. Cyclic voltammetry and differential capacitance measurements revealed a repeatable, potential-induced adsorption/ desorption process of the surfactant to/from the electrode surface while charge density and film pressure measurements indicated quasi-ideal mixing of the two adsorbed alcohols. A layer less defective than pure C18OH was created with incorporated OLA. Optical characterization was accomplished using epi-fluorescence microscopy combined with electrochemistry (electro-fluorescence microscopy) through the incorporation of two fluorescent probes into the adsorbed surfactant layer. Since molecular luminescence is quenched by a nearby metal, fluorescence was only observed when the fluorescent dye/alcohol layers were desorbed and therefore separated from the metal surface. When desorbed, the structure of the alcohol layers were similar in character, revealing aggregated features which did not change in morphology over numerous desorption/re-adsorption cycles. We have also used the electro-fluorescence technique to estimate the distance separating the metal and desorbed surfactant and believe that the molecules are displaced from the electrode surface by a distance not more than 40 nm.

Introduction Surfaces modified with organic molecules can result in materials having a variety of applications which has included corrosion resistance,1,2 lubrication,3,4 catalysis,5 optical and electronic devices,6 and both chemical and biochemical sensors.7,8 For noble metals, the most common form of surface modification is achieved through the self-assembly of alkanethiol molecules resulting in a robust chemical bond formed between the sulfur and metal. Organic molecules may also physisorb onto the metal surface, a process that is driven by a decrease in the interfacial tension. In either case, there are fundamental issues that should be addressed before the application of these materials are fully realized.9 For example, a charged substrate may alter the character of the adsorbed organic layer due to changes in the interfacial tension. As a result, the use of the organic-coated surface may be limited to specific chemical surroundings that do not induce a surface charge on the substrate. Therefore, a more comprehensive understanding of how the electrical variable can induce conformational changes in the adsorbed layer is required to clarify possible uses or limitations of such materials. Electrochemical techniques are suitable for this purpose and offer control over interfacial potential/charge. As such, electrochemistry is often used as a means of characterization; moreover, the electrical variable can control the orientation of an adsorbed surfactant * Corresponding author. E-mail: [email protected]. † Current address: Department of Chemistry, McGill University, Montreal QC, Canada H3A 2K6. (1) Ma, H. Y.; Yang, C.; Yin, B. S.; Li, G. Y.; Chen, S. H.; Luo, J. L. Appl. Surf. Sci. 2003, 218, 143-153. (2) Telegdi, J.; Rigo, T.; Kalman, E. Corros. Eng. Sci. Technol. 2004, 39, 65-70. (3) Nie, H. Y.; Miller, D. J.; Francis, J. T.; Walzak, M. J.; McIntyre, N. S. Langmuir 2005, 21, 2773-2778. (4) Zhang, Q.; Archer, L. A. J. Phys. Chem. B 2003, 107, 13123-13132. (5) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 45-52. (6) Swalen, J. D. J. Mol. Electron. 1986, 2, 155-181. (7) Chaki, N. K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17, 1-12. (8) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Sensors Update 2001, 8, 3-19. (9) Allara, D. L. Biosens. Bioelectron. 1995, 10, 771-783.

and may result in new application avenues for these unique interfaces. Electrochemical characterization of organic molecules physisorbed onto different metal surfaces have been previously described.10-12 These studies detailed a potential-induced adsorption/desorption process of a surfactant layer to/from the metal surface and are a first step toward understanding the process by which interfacial potential/charge can affect the character of the adsorbed layer. While these studies are fundamental, electrochemistry alone can only measure average properties of the interface and in situ methods are required for a more comprehensive investigation. We have recently combined electrochemistry with epi-fluorescence microscopy for this purpose. Historically, fluorescence microscopy has been used to analyze the components of biological tissues within a specimen using a fluorescent dye or stain as the contrast agent and only recently used in combination with electrochemical methods.13-16 The epi-fluorescent microscope is an effective interfacial probe for our unique systems because the surfactant exists only in the interfacial region or as a floating monolayer at the gas/solution interface, resulting in no fluorescence background from the bulk electrolyte. Moreover, the epi-fluorescence configuration uses the same objective for illumination and collection of fluorescence. Because of this, the in-focus region of the fluorescence image is optically aligned to obtain the optimal fluorescence signal. This is of great advantage when measuring fluorescence from molecules near a metal electrode surface since the intensity of (10) Bizzotto, D.; No¨el, J. J.; Lipkowski, J. Thin Solid Films 1994, 248, 6977. (11) Bizzotto, D.; No¨el, J. J.; Lipkowski, J. J. Electroanal. Chem. 1994, 369, 259-265. (12) Bizzotto, D.; Nelson, A. Langmuir 1998, 14, 6269-6273. (13) Chung, E.; Shepherd, J. L.; Bizzotto, D.; Wolf, M. O. Langmuir 2004, 20, 8270-8278. (14) Shepherd, J. L.; Kell, A.; Chung, E.; Sinclar, C. W.; Workentin, M. S.; Bizzotto, D. J. Am. Chem. Soc. 2004, 126, 8329-8335. (15) Stoodley, R.; Bizzotto, D. Analyst (Cambridge, United Kingdom) 2003, 128, 552-561. (16) Shepherd, J. L.; Bizzotto, D. J. Phys. Chem. B 2003, 107, 8524-8531.

10.1021/la052994i CCC: $33.50 © 2006 American Chemical Society Published on Web 04/13/2006

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luminescence is related to the distance separating the chromophore and the metal.17-20 A fluorophore in close proximity to the metal results in fluorescence quenching for which the efficiency decreases with a cubic dependence on the separation distance.21 In this regard, weak fluorescence from molecules near but not on the metal surface can be measured, enabling further characterization of the re-adsorption process. Furthermore, due to the distant-dependent fluorescence intensity, electro-fluorescence may allow an estimate of the distance the molecules are displaced from the electrode surface at the desorption potential. Electro-fluorescence has been a powerful tool for describing the adsorption/desorption of octadecanol to/from a Au(111) surface,16,22 DOPC from a Hg electrode,15 and the selective reductive desorption of a thiol SAM from a polycrystalline Au bead.14 Recently, the developed technique has been used to confirm the electrochemically induced dimerization of 2-(2′thienyl)pyridine adsorbed on Au(111).13 Until now, we have used the electro-fluorescence method to study single-component monolayers adsorbed on the Au(111) surface with the incorporation of only one fluorescent dye probe. In our early investigations of octadecanol adsorbed onto Au(111), we focused on the structure and distribution of the desorbed alcohol molecules.22 In this report we describe the spectro-electrochemical characterization of the adsorption of octadecanol (C18OH) and oleyl alcohol (OLA) monolayers onto Au(111) in pure components or in mixed compositions in an attempt to determine the mixing characteristics of the two alcohols. The investigations consist of both electrochemical measurements and in situ epi-fluorescence microscopy. Electrochemistry will be used to describe the average behavior of the surfactant adsorbed onto the metal surface. Fluorescence microscopy using two fluorescent probes will detail the spatial distribution and arrangement of the desorbed surfactant. We will show how the monolayers of C18OH, OLA, and mixtures of C18OH/OLA behave in response to electric potential and describe the extent of mixing between monolayers of C18OH and OLA (which form solid- and liquidlike floating monolayers23) onto the electrode surface. Experimental Section Materials. Experiments were conducted in a standard threeelectrode cell consisting of a single-crystal Au(111) working electrode (WE), a Au coil counter electrode (CE), and a saturated calomel (SCE) reference electrode. The supporting electrolyte was 0.05 M KClO4 (Fluka suprapure, triply recrystallized) made with Millipore water (>18 MΩ cm) and purged with Ar (Praxair). The waterinsoluble surfactants used in the investigations were 1-octadecanol (C18OH) and cis-9-octadecen-1-ol (oleyl alcohol, or OLA) both acquired from SIGMA with HPLC-grade purity of 99% or better and were used as-received. At room temperature C18OH and OLA are solid and liquid, respectively. The alcohols were dissolved in chloroform (Fluka, HPLC grade) to a concentration of roughly 3 mg/mL. Because the alcohols are not fluorescent, they were mixed with two fluorescent dyes; 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiIC18(5)) and 5-octadecanoylaminofluorecein (5-C18-FL) when performing the electro-fluorescence measurements. Both fluorescent dye molecules were purchased from Molecular Probes and were used as-received. (17) Drexhage, K. H. Sci. Am. 1970, 222, 108-119. (18) Chance, R. R.; Prock, A.; Silbey, R. AdV. Chem. Phys. 1978, 37, 1-65. (19) Astilean, S.; Barnes, W. L. Appl. Phys. B 2002, 75, 591-594. (20) Barnes, W. L. J. Mod. Opt. 1998, 45, 661-699. (21) Chance, R. R.; Miller, A. H.; Prock, A.; Silbey, R. J. Chem. Phys. 1975, 63, 1589-1595. (22) Shepherd, J.; Yang, Y.; Bizzotto, D. J. Electroanal. Chem. 2002, 524525, 54-61. (23) Gains, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers: New York, 1966.

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Figure 1. Schematic depiction of the electro-fluorescence setup. A 50× objective is able to focus on the electrode surface under potential control. The filters are mounted on a rotation device, allowing for interchange without changes to the experimental arrangement. The transmission spectra for the two filter cubes used are shown at the bottom. Electrochemistry. A detailed description of the electrochemical procedures can be found in the literature.24 Briefly, an aliquot of the surfactant/chloroform mixture was deposited onto the deaerated electrolyte surface, forming a monolayer at its equilibrium spreading pressure (ESP). After chloroform evaporation, the electrode was flame-annealed and cooled in the Ar-purged electrochemical cell. The clean, dry electrode was then touched to the floating monolayer under potential control (0 V/SCE), thereby transferring the surfactant from the gas/solution (G/S) interface to the metal/solution (M/S) interface. The electrode was then gently raised to form a hanging meniscus arrangement, ensuring electrochemistry was carried out only at the (111) surface. All electrochemical investigations of the C18OH/OLA mixtures were conducted in the absence of DiIC18(5) and 5-C18-FL. Cyclic voltammetry (CV) and differential capacitance measurements were primarily used for electrochemical characterization. In addition, the charge density on the metal was measured and the film pressure calculated following the back integration technique.25 Epi-fluorescence Microscopy. The in situ electro-fluorescence arrangement is schematically depicted in Figure 1. An inverted epifluorescence microscope (Olympus IX70) equipped with a 50× objective (numerical aperture (NA) ) 0.5, working distance (WD) ) 10 mm) enabled the surface of the electrode to be imaged without disturbing the hanging meniscus arrangement. All images of the electrode surface were captured using a monochromatic digital camera (SPOT RT, Diagnostic Instruments) with 2 × 2 binning and 256 gray levels (8-bit). The optical path is also outlined in Figure 1. Light from a Xe arc lamp (UXL-S75XE, 75 W) was directed toward a filter set which transmitted a band of wavelengths specific to the absorption wavelengths of the fluorophore. Measuring fluorescence from DiIC18(5) was achieved using a Chroma filter cube (Set 41008) consisting of three filters: excitation (590-650 nm), dichroic (640 nm), and emission (660-740 nm). An Olympus filter cube (UM(24) Bizzotto, D.; Wong, E.; Yang, Y. J. Electroanal. Chem. 2000, 480, 233240. (25) Lipkowski, J.; Stolberg, L. Molecular Adsorption at Gold and Silver Electrodes. In Adsorption of Molecules at Metal Electrodes; Lipkowski, J., Ross, P. N. Eds.; VCH: New York, 1992; pp 171-237.

Alcohol Monolayers Adsorbed on a Au(111) Electrode WIBA) was used to measure fluorescence from 5-C18-FL using a separate excitation (455-480 nm), dichroic (500 nm), and emission filter (515-550 nm). The transmission spectra of the two filter sets are shown at the bottom of Figure 1. Standard Imaging Routine. Imaging the organic-coated electrode surface was conducted immediately after the deposited layers were characterized with CV and capacitance measurements. Images of the interface were acquired every 30 s. The potential was incremented by 25 mV between each image (0.150 and -0.800 V/SCE). This resulted in images of the interface acquired at an effective potential scan rate of less than 1 mV s-1. Each image was acquired with an exposure time of 10 s and cropped to 0.1 mm wide × 0.15 mm in length with a resolution of 0.385 µm/pixel. These were the standard imaging conditions for all data reported. The incorporation of two fluorescent dye molecules into the adsorbed layer allowed for consecutive imaging of exactly the same area of the electrode/surfactant interface for both fluorophores. In this regard, the organic-modified surface was imaged twice through a separate collection of the fluorescence from each dye molecule from the same region of the electrode surface under identical conditions. For example, fluorescence from 5-C18-FL was measured first in the standard imaging routine followed by the same imaging sequence measuring the fluorescence from DiIC18(5) without changes in the position of the Au(111) surface. Image Analysis. All images collected in an imaging routine were analyzed using IMAGE PRO PLUS 4.5 and DIPimage26 for MATLAB. The average gray scale of the images was considered proportional to fluorescence intensity since a typical CCD response is linear with incident intensity.27 The raw images collected during an imaging routine were flatfield corrected by division with an Io image, creating images that were designated as I/Io. The Io image was obtained by collecting a fluorescence image of the electrode surface in the absence of any fluorophore. This image results from the small amount of light that leaks through the filters and is considered representative of the distribution of incident intensity across the imaging region. As such, nonuniform illumination is corrected by normalization with this image. An adsorption I/Io image (Iads/Io) was subtracted from all I/Io images in the sequence, yielding ∆I/Io images. In some cases, small intense features were observed for an adsorbed layer. In these cases, a mask was applied to the array of ∆I/Io images, eliminating the intense features in the calculations. The mask was created by thresholding the first and final ∆I/Io adsorption image in one imaging routine, creating a binary image. This mask was significantly expanded by a number of dilation steps, ensuring that the bright features were masked throughout the sequence of images. The average histogram of the masked ∆I/Io images were calculated excluding the zeros created from this masking procedure. The ∆I/Io images presented are pseudo-colored consistent with their fluorescence wavelengths (green for 5-C18-FL and red for DiIC18(5)) and then added together, creating one pseudo-colored fluorescence image.

Results and Discussion CV and Differential Capacitance of OLA/C18OH. In the absence of incorporated fluorophore, the extent of mixing between OLA and C18OH when adsorbed on the electrode surface was determined using electrochemical methods. The adsorbed layers investigated resulted from the deposition of a floating monolayer consisting of 0, 25, 50, 70, and 100 mol % OLA in C18OH. Figure 2a shows a stack plot of the CVs (left column) and the corresponding differential capacitance measurements (right column) for the various mixtures. Pure C18OH is at the bottom of the columns with increasing OLA content going up. The dotted line represents the CV and capacitance measurement for (26) van Vliet, L. J. DIPLib, the Delft Image Processing Library, http:// www.ph.tn.tudelft.nl/DIPlib/, 2005. (27) Howell, S. B. Handbook of CCD Astronomy; Cambridge University Press: Cambridge, UK, 2000; p 153.

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Figure 2. (a) CV (left column) and differential capacitance measurements (right column) for the mixed monolayers of OLA/ C18OH adsorbed on the Au(111) electrode. The dotted line represents the measurement for the electrode in contact with the supporting electrolyte and the thin and bold lines represent the negative and positive potential scan directions for the organic-coated electrode. The capacitance was measured using 25 Hz, 5 mV rms potential perturbation and calculated assuming the interface can be modeled as a simple RC circuit. (b) The minimum capacitance measured at positive potentials with incorporated OLA.

Au(111) in the absence of adsorbed surfactant and the thin and bold lines represent the negative and positive potential scan directions measured in the presence of surfactant. The CV and capacitance for C18OH are very similar to those reported previously.22 At positive potentials near the pzc for the uncoated electrode (0.255 V/SCE), the capacitance (6 µF cm-2) is decreased as compared to the water-coated surface (30 µF cm-2), indicating the replacement of water molecules at the interface with a lower dielectric organic material. Scanning potential negatively results in a series of pseudo-capacitance peaks between -0.150 and -0.325 V/SCE These capacitive peaks are also present in the CV and arise from capacitive changes, which suggest modifications in the orientation or coverage of the adsorbed organic layer and not from a charge-transfer process. Given the rather high values of capacitance, the adsorbed layer is disorganized at these potentials, allowing water to penetrate the interface. Potentials more negative of the pseudo-capacitance peaks result in a relatively constant value of 13 µF cm-2 until -0.600 V/SCE where an increase in the capacitance indicates the onset of organic desorption. At the negative potential limit (-0.800 V/SCE) a coincidence between the capacitance in the presence of the C18OH and in the absence of the surfactant occurs (17 µF cm-2), demonstrating that the C18OH molecules are displaced from the metal surface by water. On the positive potential scan, capacitance remains coincident with the values for a water-covered electrode

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until values just positive of -0.400 V/SCE where a decrease in capacitance occurs from the contact adsorption of the C18OH aggregates during the initial stages of re-adsorption. At the contact adsorption potential, the CV displays a small peak followed by a sharper re-adsorption peak represented in the capacitance plot by a depression to the original adsorption value of 6 µF cm-2. This potential-controlled adsorption/desorption/re-adsorption process is repeatable over many cycles as long as slow potential scans are used. The CV and capacitance characteristics for pure OLA (top panel) are similar to C18OH in that an adsorbed state is established at positive potentials followed by defect formation at -0.200 V/SCE on the negative potential scan. Desorption is achieved at the negative limit; however, some slight differences between the electrochemical character of the two surfactants are noted. The pseudo-capacitance peaks present in the CV and the capacitance plots for adsorbed OLA occur at slightly negative shifted potentials when compared to C18OH. Also, the minimum capacitance measured at positive potentials for OLA is lower (3.5 µF cm-2) than that measured for C18OH (6 µF cm-2). This lower capacitance indicates that the OLA molecules are more effective at blocking the electrode from the supporting electrolyte. This may be achieved through the formation of a more wellordered adsorbed layer of OLA in comparison to C18OH, or simply a disordered layer that is more effective at blocking the electrode from the electrolyte (i.e., more liquid-like film). However, the negative shift in potentials where the pseudocapacitance peaks are observed indicates that the electrocapillary curve for OLA is less sharp than C18OH. Because of this, more work is required to create defects in the layer of OLA, pointing to a less defective layer in comparison to C18OH. The miscibility of the adsorbed surfactants making up the desorbed layer can be estimated by comparing the CV and capacitance plots containing various amounts of OLA in C18OH. With increasing OLA content, a slight negative shift occurs in the potentials of the pseudo-capacitance peaks during the negative scan, as well as where the capacitance diverges from that for the water-covered surface during the positive sweep, and at potentials where the depression in capacitance follows contact adsorption. The differences are slight in comparison to a previous electrochemical study of largely immiscible surfactants.24 The current system is characteristic of a more organized or less defective layer that is stable on the electrode surface with the inclusion of OLA into the C18OH layers. A slight deviation from ideal mixing is noted for the measured minimum capacitance at positive potentials with increasing OLA content (Figure 2b). The general decrease in Cmin with increasing OLA content is further evidence of a more compact layer of the liquid alcohol adsorbed onto the Au electrode. A deviation from ideal mixing behavior is noted for this mixed monolayer and further estimates of their miscibility can be determined from other electrochemical measurements. It should be noted that layers of C18OH, OLA, and 25 mol % OLA/C18OH were reproducibly measured as indicated by the error bars in the plot. Layers containing 50 and 70 mol % OLA/ C18OH were not created as reliably and required close inspection after the charge density measurements described next. Charge Density and Film Pressure. Measuring the charge density on the metal surface in the absence and in the presence of adsorbed surfactant is a method for determining film pressure,25 resulting in a more quantitative measure of the character of the adsorbed organic films. The measurement of charge density requires a potential stepping procedure that may disrupt the adsorbed layer. As a result, only those layers that were similar in minimum capacitance values before and after the potential

Shepherd and Bizzotto

Figure 3. Charge density measurements for the organic-coated and water-coated electrode surface. The surfactant mixtures are represented in the legend in the plot.

steps were used in the calculation of film pressure. The measured charge density curves for the various mixtures of OLA/C18OH are shown in Figure 3. The charge density for an adsorbed layer of C18OH (n) is similar to previous reports.22,28 A region of constant slope is obtained at positive potentials between 0.150 and -0.100 V/SCE. This represents a region of constant capacitance that was calculated to be 8 µF cm-2, similar to the value measured by ac voltammetry. At more negative potentials the charge density becomes more negative (between -0.150 and -0.250 V/SCE), coincident with the pseudo-capacitance peaks observed at these potentials. Negative of -0.250 V/SCE, the charge density decreases less rapidly and the slope remains fairly constant until -0.500 V/SCE, much like the constant capacitance measured over this potential range. The charge density becomes consistently more similar to the values for the water-coated electrode negative of -0.500 V/SCE, indicating the desorption of the organic molecules. Incorporation of OLA into the C18OH layers changes the region of constant slope at positive potentials, producing a decreasing value for the capacitance. The potential of zero charge (pzc, measured by extrapolating the constant capacitance to values of zero charge density) approaches the value for a water-covered surface as OLA content increases. This is an indication that a small dipole exists at the electrode surface, which can be explained by the headgroup of C18OH oriented normal to the electrode surface. This dipole would be oriented more perpendicular to the electrode surface for an adsorbed film of OLA. It is also possible that the incorporated water in the more defective C18OH layer is strongly organized, resulting in the shift in the pzc. Similar to C18OH, a steep decrease in charge density at potentials negative of -0.150 V/SCE is observed for adsorbed layers containing both C18OH and OLA, occurring at slightly negatively shifted potentials, consistent with the shift observed for the pseudocapacitance peaks. Furthermore, a change in the slope is observed between -0.300 V/SCE to -0.400 V/SCE for pure OLA, coincident with the peak observed in the CV. The film pressure or interfacial tension lowering was calculated as described in the literature24 and the values are shown in Figure 4. The expected parabolic shape with a change in the measured maximum film pressure near the pzc is observed. Shown in the inset of Figure 4 is the variation in the maximum film pressure with increasing OLA content. The data again indicate a deviation from ideal mixing (represented by the straight dotted line) also observed for the minimum capacitance. (28) Yang, Y.; Bizzotto, D. J. Electroanal. Chem. 2001, 500, 408-417.

Alcohol Monolayers Adsorbed on a Au(111) Electrode

Figure 4. Calculated film pressure for the various alcohol mixtures adsorbed on the electrode surface. The inset shows the variation of the maximum film pressure with OLA content.

The electrochemical characterization of the mixed monolayers indicates a deviation from ideal mixing, but when compared to the previous investigation of immiscible surfactants,24 C18OH and OLA appear more miscible. The difference in monolayer phase behavior (solid vs liquid) may result in possible phase segregation, accounting for the deviation from ideal mixing. In situ electro-fluorescence investigations is used to shed light on this issue by probing the local character of the layers. Electro-fluorescence characterization of a 3 mol % DiIC18(5)/C18OH layer was previously reported.16,22 Only a small disruption of the adsorbed C18OH monolayer due to the fluorophore was noted with the 1, 3, and 5 mol % ratios used in that investigation. We assume that DiIC18(5) mixes well into the lipid-like organic layers of OLA and C18OH since these carbocyanine dyes are well-known to partition into the hydro-

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phobic regions of biomembranes.29,30 In this report we mixed the C18OH and OLA layers with two dyes (5-C18-FL and DiIC18(5)) and imaged one adsorbed layer on the same region of the electrode/surfactant interface twice (once for each dye) and compared the desorption structures. This method relies upon the dye molecules faithfully reporting on the potential-induced behavior of the organic material and not segregating into a preferred local region such as a gel- or liquid-like phase of the alcohol monolayer. In the previous work on single-component monolayers, no such evidence of this segregation was observed. If the adsorbed mixed monolayers are segregated, we expect that the dye will report on this as well, either through an increase or decrease in concentration within a particular phase. Electro-fluorescence Investigation of C18OH Adsorbed onto Au(111). The electro-fluorescence imaging investigation of an adsorbed C18OH layer modified with 2 mol % DiIC18(5) and 2 mol % 5-C18-FL is described and compared to the previous work that included only the DiIC18(5) dye in the C18OH monolayer.22 The pseudo-colored images used to represent fluorescence from both dye molecules and are shown in Figure 5a. These images correspond to the letter legend in the measured capacitance plot (b). Images A and B, recorded at positive potentials, are featureless due to the quenching of fluorescence as a result of adsorption onto the metal at these potentials. Even through the pseudo-capacitance peaks on the negative potential scan (image B), the images are featureless, indicating that the rise in capacitance at these potentials results from defects forming in the adsorbed layer rather than by partial desorption of the surfactant. At potentials nearing -0.600 V/SCE, a slight increase in the measured intensity of 5-C18-FL is noticed in images C and D. This increase in fluorescence intensity occurs without a significant contribution from the DiIC18(5) dye, which will be shown in a later section to be due to a difference in metalmediated quenching profiles for these two dye molecules. The

Figure 5. (a) Images acquired during an electro-fluorescence imaging routine for a C18OH layer containing 2 mol % DiIC18(5) (red) and 2 mol % 5-C18-FL (green). The images are pseudo-colored to match the fluorescence wavelengths of the two dye molecules and summed together showing a combination from both. The images correspond to the letter legend on the capacitance plot in (b). (b) Measured capacitance during the imaging investigation along with the calculated fluorescence intensity from (c) DiIC18(5) and (d) 5-C18-FL. Open and closed symbols represent the negative and positive potential scan directions, respectively.

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fluorescence images at more negative potentials depict a clustered arrangement of the desorbed C18OH molecules (images E-G), indicating the increased separation between the surfactant and metal. Image G shows an aggregated and heterogeneous structure that contained fluorescent features from both dyes molecules. The desorbed structure does not change in morphology over the time spent at desorption potentials while the intensity increases to a maximum at the negative potential limit (image H). The pseudo-colored desorption image results from a contribution from both dye molecules, demonstrating that the dyes do not preferentially concentrate within a given region of the octadecanol matrix and therefore reliably report on the potential-induced behavior of the alcohol surfactant. On the positive (re-adsorption) potential scan, the image intensity remains large during the initial stage of the re-adsorption (images I-L), supporting the hysteresis observed in the electrochemical measurement. This observation indicates that the re-adsorption process occurs via a mechanism different from the desorption process. Even over a large positive potential variation, the structure of the desorbed molecules remains similar, indicating a lack of diffusion. This is most clearly observed when tracking the clusters in images E-L, which are contained within the oval. This lack of diffusion is contrasted to previous work on a thiol-modified electrode surface in which the desorbed molecules diffuse at desorption potentials,14 producing images which lose clarity and become smeared out even over a time scale of 2 s. Fluorescence is largely quenched when nearing the potential region where contact adsorption (images M and N) presumably occurs and is nearly fully quenched when the potential moves through the re-adsorption peak potential (image O). Also shown in Figure 5 is the measured fluorescence intensity of (c) DiIC18(5) and (d) 5-C18-Fl for the negative (open symbols) and positive (closed symbols) potential scan. When adsorbed, the fluorescence intensity of both dye molecules is at a minimum, even at the potentials where defects occur in the adsorbed layer. Negative of -0.400 V/SCE, the intensity of 5-C18-FL increases while the fluorescence from DiIC18(5) is small. For 5-C18-FL, the intensity continues to increase, reaching a maximum at the negative potential limit. On the positive scan, the fluorescence intensity remains significant due to the hysteresis between desorption and re-adsorption. Interestingly, near the potentials of contact adsorption (just positive of -0.400 V/SCE) fluorescence intensity is still observed, which is clear evidence of a contact adsorbed state preceding full re-adsorption. When the aggregates are in the contact adsorbed state, some of the molecules remain far enough away from the metal surface to avoid complete fluorescence quenching. The measured fluorescence intensity for DiIC18(5) begins to increase negative of -0.600 V/SCE. In contrast to the trend for 5-C18-FL, the maximum intensity for DiIC18(5) is observed at -0.700 V/SCE, which is more positive than the desorption limit. A slight decrease in fluorescence intensity is observed between -0.700 and -0.800 V/SCE, which can be attributed to the formation of nonfluorescent aggregates of the DiIC18(5) molecules when desorbed from the solid electrode.16 On the positive potential scan, the fluorescence intensity is similar in character to 5-C18-FL, again maintaining appreciable fluorescence at the potentials of contact adsorption. Analysis of the number of aggregates and their relative sizes was performed on these images following the methodology described in a previous publication where we studied only C18OH/ DiIC18(5) monolayers.22 The image analysis quantitatively agrees with the previous work, demonstrating that the inclusion of the second fluorophore does not disrupt the adsorbed layers. (29) Krasne, S. Biophys. J. 1980, 30, 441-462. (30) Krasne, S. Biophys. J. 1980, 30, 415-439.

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The epi-fluorescence investigation of the 2 dye/C18OH layer confirms that the desorbed aggregates remain near but not on the electrode surface and are available for re-adsorption through a contact adsorbed state. Since the desorbed molecules do not laterally diffuse within the interfacial region limited by the spatial resolution of our technique (roughly 1 µm), it is possible that the desorbed layer is held in place by a well-ordered adsorbed water layer on the electrode. Neutron reflectometry studies have shown that a thin water layer exists between the metal and desorbed surfactant31 and impedance measurements for a DOPC-coated Hg electrode has proposed that the adsorbed water layer between the electrode and the desorbed organic molecules are in an organized rigid ice-like structure.32 The combination of fluorescence imaging and fluorescence intensity measurements confirm the potential control over the desorption and re-adsorption process of C18OH to and from the electrode surface. The same procedure is used for the adsorbed layer of OLA and the 25 mol % mixture. The electrochemical characterization of adsorbed OLA is similar to that of C18OH, showing only small differences presumably due to the difference in fluidity of the two surfactants. Electro-fluorescence Investigation of OLA Adsorbed onto Au(111). The electro-fluorescence imaging routine was performed for an adsorbed layer of OLA containing 2 mol % DiIC18(5) and 2 mol % 5-C18-FL in the same fashion as for C18OH. The results are shown in Figure 6. The data presented are similar to those measured for adsorbed C18OH showing fluorescence quenching at positive potentials and negative of the pseudocapacitance peaks (images A and B). Also consistent with the data for C18OH is the slight increase in fluorescence intensity measured from 5-C18-FL while DiIC18(5) remains quenched at the same potential -0.400 V/SCE (images C and D). The fluorescence contribution from both dye molecules is noticed at more negative potentials, showing an aggregated-type morphology of the OLA molecules during displacement from the metal surface. At the negative potential limit, however, a clear decrease in the fluorescence intensity of DiIC18(5) is observed while the fluorescence from 5-C18-FL remains intense (images G-I). This is also clearly represented in the calculated fluorescence intensity/ potential plot (b) and is again attributed to the formation of a nonfluorescent aggregate of the dye molecules when desorbed. Since the fluorescence intensity of DiIC18(5) decreases more rapidly in OLA than in C18OH, the dye must be able to form these nonfluorescent aggregates more rapidly. This is expected for the more fluid OLA phase and suggests that the desorbed OLA molecules retain fluidity similar to the floating monolayer. While the fluorescence from DiIC18(5) is largely decreased at the negative limit, the intensity of 5-C18-FL reaches a maximum. Inspection of the desorbed images (G-J) display a similar morphology when compared to C18OH, indicating that the desorption and re-adsorption process for the two alcohols proceeds by a similar mechanism. Therefore, it is not difficult to expect that the OLA molecules undergo re-adsorption through a similar contact adsorbed state preceding complete re-adsorption. Since the desorbed structures of C18OH and OLA are very similar, an imaging investigation containing a mixed concentration of 25 mol % OLA and 75 mol % C18OH was unable to unambiguously separate which region of the desorbed layer consisted of the solid or liquid phase. To demonstrate this, an image at the desorption potential -0.700 V/SCE for this (31) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763-1776. (32) Agak, J. O.; Stoodley, R.; Retter, U.; Bizzotto, D. J. Electroanal. Chem. 2004, 562, 135-144.

Alcohol Monolayers Adsorbed on a Au(111) Electrode

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Figure 6. (a) Images acquired during an electro-fluorescence imaging routing for an OLA layer containing 2 mol % DiIC18(5) (red) and 2 mol % 5-C18-FL (green). The images correspond to the letter legend on the capacitance plot in (b). (b) Measured capacitance during the imaging investigation along with the calculated fluorescence intensity from (c) DiIC18(5) and (d) 5-C18-FL. Open and closed symbols represent the negative and positive potential scan directions, respectively.

Figure 8. (a) Schematic depiction of a Au film with known thicknesses of SiO2 deposited. (b) The average fluorescence intensity measured for each 10 nm step.

Figure 7. A desorption image from an electro-fluorescence investigation of a mixed monolayer of 25 mol % OLA and 75 mol % C18OH containing both DiIC18(5) (red) and 5-C18-FL (green). The image does not show any clear indication of separate OLA or C18OH character.

investigation is shown in Figure 7. This evidence suggests that the two alcohols form a quasi-ideal mixed monolayer when adsorbed on the electrode surface. Metal-Mediated Quenching Profiles. The increase in fluorescence intensity of 5-C18-FL was observed at significantly less negative potentials than DiIC18(5) for both C18OH and OLA adsorbed layers. This can be explained through a difference in the metal-mediated quenching profiles for the two dye molecules. Figure 8a shows a schematic of a template composed

of 300 nm Au deposited onto an adhesive layer of Cr on a glass substrate. Onto the Au layer, four depositions of evaporated SiO2 were applied in steps each with a thickness of 10 nm. A 2 mol % DiIC18(5), 2 mol % 5-C18-FL, C18OH layer was deposited onto the SiO2 layer by the horizontal touching technique similar to the electrochemical technique used. This produced a controlled separation between the dye containing octadecanol layer from a gold film. At each step of SiO2, five fluorescence images of the interface were acquired within different regions of the step with various exposure times. After correction for the variation in the exposure time, the average fluorescence intensity was calculated for the flat-field corrected (I/Io) images and plotted against distance as shown in Figure 8b. Comparison of the fluorescence intensity with separation from the Au surface clearly illustrates the difference in the metal-mediated quenching of the two dyes. At the same distance from the Au surface, the

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fluorescence from DiIC18(5) remains small while a significant increase occurs for 5-C18-FL, which suggests that this dye reports more effectively on small variations in the separation from the electrode surface and therefore on the initial stages of surfactant desorption. This template can also be used to provide a rough estimation of the distance separating the desorbed surfactant and the Au(111) electrode. Following the description by Kuhn and coworkers,33,34 the intensity of fluorescence increases with separation between the chromophore (sensitizer) and metal (acceptor) according to

( ) ( ( )) Id do ) 1+ I∞ d

4 -1

where do is the Fo¨rster critical distance where 50% of the fluorescence is quenched by the metal. When the intensity is fitted with separation data for DiIC18(5) to this general equation, the parameter do can be extracted. Through a least-squares analysis, this value was found to be 37 nm. The fluorescence images presented in Figure 5 are at least 1/10 of the intensity for that recorded from the air/solution interface prior to deposition onto the electrode surface. A rough estimate for the upper limit of the distance separating the desorbed surfactant from the gold surface is certainly less than do or 37 nm. This distance is further than the edge of the diffuse layer but still near the electrode surface. A more accurate estimate may be obtained using lifetime measurements where the desorbed molecules would not be subject to the same extent of photobleaching or photodestruction as is this case with these long image exposure times. Development of this technique and lifetime measurements are planned in future experiments.

Conclusions A Au(111) electrode modified with adsorbed monolayers of C18OH and/or OLA was successfully characterized with (33) Drexhage, K. H.; Zwick, M. M.; Kuhn, H. Ber. Bunsen-Ges. Phys. Chem. 1963, 67, 67-70. (34) Kuhn, H.; Mo¨bius, D.; Bu¨cher, H. Spectroscopy of Monolayer Assemblies. In Physical Methods of Chemistry Part IIIB Optical, Spectroscopic, and RadioactiVity Methods; Weissberger, A., Rossiter, B. W., Eds.; Wiley-Interscience: New York, 1972; pp 577-702.

Shepherd and Bizzotto

electrochemistry in combination with epi-fluorescence microscopy. Electrochemical results for the mixed monolayers of OLA/ C18OH point to quasi-ideal mixing of the two surfactants when adsorbed onto the electrode surface. The minimum capacitance and maximum film pressure were measured with increasing OLA content, demonstrating that the incorporation of OLA created layers that are less defective than C18OH. Epi-fluorescence imaging of the desorption/re-adsorption process was conducted for adsorbed layers containing two fluorescent dyes having different metal-mediated quenching profiles. The double labeling approach using two fluorescent dyes results in an extra level of detail for analyzing the potentialinduced changes in the adsorbed layer and for the estimation of the distance separating the desorbed molecules and the metal surface. When desorbed, the aggregated structure of both alcohols were similar in nature, and the re-adsorption process proceeded through a contact adsorbed state followed by potential-driven phase changes, resulting in a low capacitance layer. The fluorescence intensity showed a decrease at the negative limit for one of the fluorophores. This was attributed to the formation of nonfluorescent dye aggregates when the layer was desorbed. OLA was shown to have a more rapid decrease in fluorescence intensity, suggesting that the fluidity of the desorbed OLA is similar to the floating monolayer. When mixed in a ratio of 25 mol % OLA and 75 mol % C18OH, no significant phase separation was observed, which confirms that the two molecules do not separate into domains of solid or liquid character. In an attempt to estimate the distance separating the metal and desorbed surfactant, a unique stepped surface of SiO2 on Au was created. Preliminary investigations indicate that the molecules become separated from the electrode not more than 40 nm. Acknowledgment. Financial support from the Natural Sciences and Engineering Research Council (NSERC) is greatly appreciated. J.S. was supported by an NSERC PGS B. Mike Lathuillie`re and Anisa S. Akhtar are gratefully acknowledged for their initial studies of OLA adsorbed on Au(111). The mechanical and glass blowing shops at the University of British Columbia are also gratefully acknowledged as are the efforts of Dr. Mario Beaudoin for creating the stepped surface of SiO2 deposited on the Au substrate. LA052994I