What Happens to the Thiolates Created by Reductively Desorbing

(26, 27) For SAMs created with 1-nonanethiol, the electrolyte pH was found to have a significant .... as detailed previously(21)) in a 1:1 mixture of ...
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What Happens to the Thiolates Created by Reductively Desorbing SAMs? An in Situ Study Using Fluorescence Microscopy and Electrochemistry Jannu R. Casanova-Moreno and Dan Bizzotto* Department of Chemistry, AMPEL, University of British Columbia, Vancouver, Canada S Supporting Information *

ABSTRACT: In situ examination of the reductive desorption process for Au microelectrodes modified with a thiol selfassembled monolayer (SAM) using fluorescence microscopy enabled the study of the fate of the desorbed thiolate species. The Bodipy labeled alkyl-thiol SAM, when adsorbed, is not fluorescent due to quenching by the Au surface. Once reductively desorbed, the thiolate molecules fluoresce and their direction and speed are monitored. At moderately negative reduction potentials, the thiolate species hemispherically diffuse away from the microelectrode. Also observed is the influence of a closely positioned counter electrode on the direction of the desorbed thiolate movement. As the potential becomes more negative, the molecules move in an upward direction, with a speed that depends on the amount of dissolved H2 produced by water reduction. Shown is that this motion is controlled, in large part, by the change in the electrolyte density near the electrode due to dissolved H2. These results should help in explaining the extent of readsorption at oxidative potentials observed in cyclic voltammetry (CV) reductive desorption measurements, as well as improving the general understanding of the SAM removal process by reductive desorption. The electrogenerated H2 was also shown to be able to reductively remove the thiol SAM from the Pt/Ir particles that decorate the microelectrode glass sheath.

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length.18 A careful selection of reductive potential then enables the selective removal of either only one component of a multicomponent segregated layer19 or of all the thiols from a particular surface feature.20,21 Subsequent backfilling with another thiol enables the creation of surfaces otherwise inaccessible by traditional self-assembly. Although agreement exists in the literature regarding the nature of the reduction reaction,2,6,22,23 the fate of the desorbed molecules once in solution is speculatively described. Cyclic voltammetry experiments by Morin et al. have shown that the reductively desorbed thiolates can oxidize at more positive potentials to reform a SAM layer. The quality or coverage of this reformed monolayer strongly depends on the solubility of the desorbed thiols in the surrounding medium.7 Short alkyl thiols (e.g., 1-butanethiol) once desorbed redeposited only about 20% of the original SAM, whereas values of 70%24 and 90%25 have been reported for the reformation of 1hexadecanethiol SAMs. Furthermore, some authors claim negligible loss of thiol when using alkylthiols with 13, 17, and 18 carbon atoms, studied using chronoamperometry rather than cyclic voltammetry.26,27 For SAMs created with 1nonanethiol, the electrolyte pH was found to have a significant influence on the extent of reformation of the SAM, as higher pH favored the presence of soluble thiolates while acidic solutions favor the insoluble protonated form. This contribu-

INTRODUCTION Compounds containing thiol moieties are commonly employed as adsorbates for the fabrication of self-assembled monolayers (SAMs) on gold, platinum, silver, and copper, among other substrates, through the formation of a covalent metal−sulfur bond.1,2 The stability of a SAM depends on the thiol chosen and the metal−adsorbate, adsorbate−adsorbate, and solvent− adsorbate interactions.3,4 The application of reductive potentials to the metallic substrates reduces the sulfur−metal bond thereby effectively removing the SAM from the metal surface,2,5,6 creating thiolate molecules according to the following reaction:3 − RS(surf,M(hkl)) + x H 2O(aq) + ne(M( hkl)) − ⇌ RS(aq) + x H 2O(surf,M(hkl))

(1)

where “surf” refers to the species at the metallic surface (with a (hkl) orientation) and “aq” refers to the species in solution. This process has been studied for short and long alkyl-thiols,7−9 aromatic thiols,3,10,11 and thiolated DNA,3 among many other compounds. Electrochemical reduction has been used to regenerate the clean metal surface after using the SAM for sensing12 or controlled nanoparticle growth13 and for the controlled release of biomolecules, nanoparticles,14 and adhered cells.15,16 The potential required to remove the layer depends on parameters such as the adsorption site (terrace vs step edge),17 the metal surface crystallinity,7 and the alkyl chain © 2013 American Chemical Society

Received: January 2, 2013 Published: January 14, 2013 2065

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movement or position of these desorbed molecules impacting the local stimulation of cells or in drug delivery studies.39 Recently, we have used in situ epifluorescence microscopy to observe the reductive desorption of SAMs created from fluorescently tagged alkylthiols. This in situ microscopy enabled monitoring of the differences in the electrodesorption process on different areas of the electrode surface.21,30,31,40 The technique relies on the fact that light emission from a fluorophore is quenched when placed in close proximity (∼50 nm) to a thick metallic surface.41,42 As a result, these fluorescent molecules adsorbed in a SAM do not fluoresce and will only become fluorescent once the separation from the electrode surface is significant (tens of nanometers). The observation of the desorption of a variety of fluorescently labeled SAMs has revealed significant movement in the desorbed thiol molecules. Desorbed alkylthiol SAMs appear as a diffuse ″cloud″,21,40 whereas very localized aggregates were observed to skate across the electrode surface for SAMs created from thiolated DNA.43 The origin of this movement has been speculated to involve the shape of the electrode surface (i.e., a bead or a planar polished surface) and electrophoretic attraction toward the counter electrode. In this work, microelectrodes (μEs) are used to separately address each of these possible causes for the movement of the desorbed thiolates. Microelectrodes offer the advantage of being small enough so that the complete electrode surface can fit in the field of view of an optical microscope. Furthermore, due to their polycrystalline nature, these electrodes do not possess crystalline domains large enough to be optically resolved. As a result, the fluorescence appears even across the surface of the electrode, ameliorating a difficulty encountered previously when we used larger multicrystalline electrodes which have many coexisting grains with a variety of surface crystallographies, each one possessing its characteristic desorption potential. Monitoring the movement of desorbed thiols is complicated due to the interference from the desorbed molecules from neighboring grains that may desorb at slightly less negative potential. This overlap precludes accurate analysis of the movement of the desorbed molecules. Similar spectroelectrochemical experiments have been reported by Ghaly et al. using microband electrodes.44 They employed different alkylthiol chain lengths and found that, while the behavior of short chains (C6) could be modeled by simple diffusion, longer chains (C11) deviated significantly. They speculated that this was primarily due to differences in solubility, but we will show that other factors should also be considered. In this contribution, we characterize reductive desorption of SAMs from gold microelectrodes using in situ fluorescence microscopy coupled with capacitance measurements. By adjusting the experimental conditions, we will demonstrate that the anisotropic movement of the desorbed thiolates is a result of two different forces. Understanding their influence will enable further control over the movement of species released when reductively desorbing the SAM.

tion explores the nature of reductive desorption and the fate of the desorbed molecules using in situ fluorescence microscopy during the electrochemical reduction process. The study of the structure of SAMs has benefited greatly from in situ spectroscopic techniques. Among them, infrared spectroscopy has been the most commonly employed for this purpose due to its ability to report on specific functional groups and the average molecular orientation with respect to the surface.23,28 Using these techniques, Morin’s group proposed a model to explain the presence of two cathodic peaks and two anodic peaks on the cyclic voltammograms of a hexadecanethiol SAM on Au(111).28 Their model suggests a two-step mechanism for the desorption of insoluble thiolates: first, the reduction of the Au−S bond creates a physisorbed layer of thiolates, similar to the original SAM. Then, at more negative potential, the solvent displaces these thiolates forming physisorbed micellar-type species with a large fraction of the electrode surface covered with solvent.28 This second step is similar to the mechanism previously reported for the desorption of a physisorbed octadecanol layer from Au(111).29−31 Neutron reflectivity studies in other insoluble films physisorbed onto Au show that, on average, when desorbed, the water content of the layers increases and, in some cases, the desorbed layer remains close to the electrode separated by a ∼10 Å layer of solvent.32,33 Some evidence of the presence of the above-mentioned aggregates during the reduction of thiol SAMs has been obtained by in situ scanning tunneling microscopy (STM) imaging of the desorption process of SAMs composed of 1propanethiol, 1-hexanethiol, 1-hexadecanethiol, as well as 3mercaptopropionic acid.17,24 However, the identity of the observed aggregates is still controversial, since they could also be gold adatom islands formed after the SAM desorption or even Au islands with adsorbed thiol aggregates.34 Recently, Cai and Baldelli proposed an alternate view of the structure of the desorbed layers employing electrochemical impedance measurements and sum frequency generation spectroscopy.35 They suggest that hexadecanethiol and octadecanethiol monolayers retain the two-dimensional order even at reductive potentials. However, their measurements were performed in situ at open circuit potential or ex situ, preventing the conclusive use of this data to infer the state of the desorbed thiols. SAMs created from thiol compounds are dynamic, as evidenced by the potential independent transformations of the structure of the SAM switching between the well-known √3 × √3R30° and the c(4 × 2) superlattice in a matter of seconds, as well as the continuous creation and disappearance of defects.17,36 Initially, the desorption process was proposed to involve a nucleation and growth of these defects,17,37,38 but more recent analysis suggests a better fit to a model in which domains of thiol shrink from the edges.9 As seen above, most often the study of the desorbed molecules rely upon indirect evidence of their behavior as they change the electrochemical response during desorption or readsorption. Only a few studies have probed the nature of the desorbed molecules. These primarily used in situ techniques which provide an average picture of the molecular state. In other words, with the exception of STM, the employed techniques are limited and cannot characterize the fate of the desorbed molecules once reductively released from the electrode surface. An in-depth understanding of this reductive desorption process would potentially enable control of the

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EXPERIMENTAL SECTION

Microelectrode Fabrication and Characterization. Microelectrodes were fabricated by sealing a 25-μm-diameter gold wire (World Precision Instruments) inside a borosilicate pipet (Sutter) using a Sutter P-87 pipet puller equipped with a Pt/Ir heating filament. While applying vacuum, the filament was heated enough to soften the glass but not to melt the Au. Once a good seal was attained between 2066

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In situ fluorescence imaging was performed using an Olympus IX70 inverted microscope equipped with a Photometrics Evolve 512 camera. The camera and the light source (X-Cite eXacte) were controlled using μManager46 software. The dichroic mirror and excitation and emission filters employed were a T495LPXR 233302, ET470/40X 233523, and ET525/50 M 231230, respectively, all fabricated by Chroma. The exposure time was either 25 or 100 ms. The working microelectrode was positioned from the top of the cell using a polytetrafluoroethylene (PTFE) 14/23 joint. The tilt angle was measured optically. A Narishige ONW-131 micromanipulator which allows movements as small as 1 μm was employed to control the position of the counter electrode in the case where the small CE was used. The optimized deposition procedure is as follows: Before every desorption, the microelectrode was cleaned by submerging it into piranha solution (hydrogen peroxide and sulfuric acid) for 5 min, followed by potential cycling in 0.5 M H2SO4 (between −0.4 and 1.4 V/Ag|AgCl). The electrode was then rinsed in ultrapure water (MilliQ, Millipore), and then HPLC-grade methanol (Fisher Scientific). The surface was then modified by introducing the electrode tip into an approximately 1 mM solution of 4,4-difluoro-1,3,5,7-tetramethyl-8[(10-mercapto)]-4-bora-3a,4a-diaza-s-indacene (BodipyC10SH, synthesized as detailed previously21) in a 1:1 mixture of CHCl3 and MeOH for 10 min. This fluorescently labeled alkylthiol has an octanol/water partition coefficient (determination in Supporting Information) comparable to 1-heptanethiol, facilitating comparisons with unlabeled alkylthiol SAMs. The electrode was then successively rinsed with a mixture of HPLC-grade chloroform and methanol, methanol, and finally ultrapure water. The potential perturbation program consisted of a series of potential steps starting at −0.4 V sequentially decreasing in 25 mV steps to a given final potential (Ef). The time between images was either 180 ± 8 ms or 25 ± 33 ms (for exposures times of 25 and 100 ms) which results in an effective sweep rate of 140 and 100 mV/s, respectively. One image was acquired at every potential step. The images were analyzed with the ImageJ software.47 All the images corresponding to a single desorption experiment were collected into an image stack. Correction for drift in the position of the electrode was performed using the Image Stabilizer plugin for ImageJ.48 A Gaussian blur filter (radius = 2 px) was applied; the minimum values for each pixel in a stack (minimum projection) was used to create an image which was subtracted from all the other images. In this way, a new stack representing the difference in fluorescence intensity with respect to the minimum was created. An anisotropic diffusion filter (a1 = 0.5, a2 = 0.9, 20 iterations)49 and another Gaussian blur filter were applied to decrease the noise in the image. Selected desorption sequences experiments (as noted in the figure captions) are included as videos in the Supporting Information.

the gold and the glass (as inspected with a microscope), the construct was snapped in the middle forming two relatively symmetrical electrodes. An electrical connection was achieved by melting a small amount of fluxless solder (Kester) to the exposed core of a coaxial wire (Precision Instruments) and soldering to the gold wire using a heat gun. The use of fluxless solder was essential to achieve a low fluorescence background. A mono audio connector was used to connect both the core and the shield of the coaxial wire to the working electrode (WE) and ground connections, respectively, to reduce noise (Figure S1 in the Supporting Information (SI)). The tip of the electrode was polished with a Narishige EG-40 microgrinder. The ratio of the outside diameter to Au diameter (RG value) ranges from 5 to 17 (Figure S2). Brightfield imaging of the sides of the electrode shows a darkened area near the tip of the electrode assembly (Figure S3), which upon closer examination reveals the presence of particles deposited on the glass. Scanning electron microscopy (SEM) images were obtained with a Hitachi S-2300 electron microscope equipped with an Advanced Analysis Technologies energy-dispersive X-ray spectrometer (EDX) after a very thin film (less than 10 nm) of Au was vapor-deposited on the electrode construct to avoid charging of the glass (Figure 1). Furthermore, EDX

Figure 1. Surface of the electrode glass sheath at three different magnifications. (a) Optical microscopy image composed of 16 images taken at different focal positions stitched together with the ImageJ plugin “Extended depth of field”.45 (b) Scanning electron microscopy image. (c) Energy-dispersive X-ray spectroscopy mapping analysis.

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mapping analysis revealed the presence of Pt and Ir particles on the glass surface suggesting that the origin of these deposits is the heating filament in the pipet puller. Electrochemical and in Situ Fluorescence Methods. The spectroelectrochemical measurements were performed in a custommade glass cell with a 250-μm-thick glass-bottom optical window and two 1 in. lateral ports (see Figure S4). In most experiments, the counter electrode (CE) and reference electrode (RE) were a Pt coil and a Ag|AgCl reference electrode (BASi RE-6), respectively. For some other experiments, as noted in the text, a smaller CE was employed, which was fabricated in the same way as the WE but with a Au wire protruding from the glass surface by approximately 150 μm. The potential was controlled through a LabView program and applied using a FHI ELAB potentiostat. The electrolyte was 1 mM KOH (Sigma-Aldrich, 99.99% purity). The differential capacitance was measured using a 200 Hz 5 mV RMS AC potential superimposed onto the DC potential. The resulting current response was analyzed by a lock-in amplifier (EG&G 5208). The in-phase and out-of-phase amplitudes were then used to calculate the capacitance assuming a series RC circuit.31 All potentials reported are with respect to Ag|AgCl; they were not corrected for the iR drop in solution since it was not significant except at the most negative potentials.

RESULTS

Fluorescence Imaging of Reductive Desorption from a Au Μicroelectrode. The Au microelectrode surface was modified with BodipyC10SH and placed in the spectroelectrochemical cell, so that its surface was parallel to the focal plane of the inverted microscope. The changes in fluorescence and capacitance were measured as the potential was stepped to more negative potentials. The data presented were from layers that displayed capacitance values commensurate with highcoverage SAMs which resulted in significant fluorescence signals. Not all layers prepared were of sufficient quality for data analysis, but the trends observed are consistent. Figure 2a shows the typical changes in the electrode capacitance upon the application of a potential perturbation that progressively becomes more negative (Figure 2c). Initially, the capacitance of the electrode was 0.10 nF as compared to 0.26 nF for the bare uncoated Au (filled circles) indicating the presence of the low dielectric SAM on the electrode surface. It 2067

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Figure 2. Variation of the capacitance (a) and fluorescence intensity (b) upon the application of the potential perturbation shown in (c) also shown in the axis at the top of the figure. (d) Fluorescence images at selected potentials (shown in the images) during the reductive desorption. The dashed outlined region corresponds to the Au surface, while the continuous line represents the resulting ellipse after applying a threshold (0.35). Video 1 in the Supporting Information shows this desorption experiment. The length of the major and minor axis (e) as well as the displacement of the center of the ellipse (f) are plotted for a smaller subset of employed potentials (g). Symbols in (f) represent raw data, while the dotted line results from smoothing with a Savitzky-Golay filter. Error bars on the displacement are smaller than the symbol size.

the more negative potentials, the decrease in fluorescence results from diffusion and the accompanying dilution of fluorophore in addition to photobleaching. The reductive desorption potentials appear to be more negative than typically reported in the literature3,21 possibily due to the uncompensated iR drop in the 1 mM electrolyte solution used or the slow diffusion of fluorescent thiolate from the electrode surface. Low electrolyte concentrations are necessary to study the effect of the migration of the negatively charged thiolates (see next section). The iR drop remains small (≪25 mV) because of the use of microelectrodes and the small currents to potentials as negative as −1.5 V. At potentials more negative than this, the current flow increases and the iR drop can be more significant, though the reductive desorption process is complete. In addition, the rate at which the thiolates diffuse away from the metal electrode is slow compared to the changes in potential which results in a lag of the fluorescence signal compared to the changes in the coverage. Separate measurements done in higher electrolyte concentrations (not shown) on larger electrodes with slower potential changes show a good correspondence with the literature reported values for desorption. Fluorescence images of the interface during the reductive desorption are shown in Figure 2d. It is clear that in this case the fluorescence and therefore the thiolate molecules become radially distributed away from the gold electrode. Image analysis of this process was done by using a constant intensity threshold for all images, which defines the edge of the diffusing plume of thiolates. Fitting an ellipse to this thresholded area (shown as a white continuous line in the images) enables characterization of the shape of the plume via the length of the

is worth mentioning that the expected capacitance for a bare Au electrode with this exposed geometric area (4.9 × 10−6 cm2) is 0.09 nF. This discrepancy can be partially explained via surface roughness. However, another very important factor is the stray capacitance created by the Au wire inside the glass immersed in the electrolyte. This becomes important for microelectrodes due to the small ratio of Au surface area exposed to the solution to the total Au/glass surface in the electrolyte solution. The capacitance of a SAM covered surface is at least 1 order of magnitude smaller than that of the bare Au surface, suggesting that the capacitance measured for the thiol-coated surface is dominated by the stray capacitance. As the electrode potential becomes more negative (Figure 2c), both the capacitance (Figure 2a) and the fluorescence intensity (Figure 2b) from the microelectrode increase as a consequence of the reductive desorption of the BodipyC10SH SAM, indicating its removal. Once desorption is complete, the capacitance is similar to that for the uncoated Au surface. It is important to note that, for potentials more negative than −1.4 V, the capacitance determined can yield incorrect values because of the faradaic current due to water reduction. The capacitance is calculated assuming that the electrochemical system can be represented as a series RC circuit, which is no longer accurate once faradaic currents are present. In spite of this artifact, the comparison between the SAM covered and uncoated surfaces clearly shows the loss of the layer at potentials between −1.05 V and −1.4 V. Coincident with the capacitance increase is an increase in fluorescence intensity, a result of the increasing separation between the reductively desorbed fluorescent thiolate and the metal so that fluorescence quenching is no longer efficient. At 2068

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(WE) creating an asymmetric electric field enhanced by the low concentration of electrolyte (1 mM) used. Figure 3c shows the fluorescence images at two selected values of potential during two independent experiments. When examining the top row, the first image at −1.5 V is comparable to the image at −1.5 V shown in Figure 2d revealing an essentially symmetric fluorescence distribution centered at the gold electrode. Closer inspection of the fluorescence images reveals an imperfect ellipse with a small deformation directed toward the counter electrode, something not observed in Figure 2d where the counter electrode was positioned more than 10 mm away. However, at more negative potentials, the fluorescent plume of desorbed molecules reversed direction. This was quantified by measurement of the displacement and speed of the center of the thresholded fluorescent signal (Figure 4a,b,c, filled circles) which shows an initial increase in the displacement at a speed of approximately 1 μm/s directed toward the CE (indicated as an angle between 0° and 90°), and then at a later time, the fluorescent plume reverses direction (∼−150°) and moves away from the counter electrode at a speed of about 4 μm/s. The same experiment (on a newly created SAM layer) was repeated using the same orientation of the CE (bottom row of Figure 3c). Once again, at small negative values of potential, the fluorescent plume initially moved toward the CE. Unlike the previous case, the direction of this movement was not reversed, but became constant at ∼35° at more negative potentials. These measurements clearly indicate that the position of the CE somewhat influences the direction of the desorbed molecule movement, but is not the major controlling factor. Moreover, these results clearly show that there is another influence that dominates the direction of the desorbed thiolate movement. Tilting the Microelectrode. The movement of the desorbed thiolates was again investigated using the same procedure as in Figure 2, but with the electrode surface purposely tilted in different directions, approximately 3° with respect to the focal plane of the microscope (same as the horizontal plane; see Figure 5a). The counter electrode was positioned far away (more than 10 mm) and remained in a fixed position during these experiments. In all cases, the fluorescent plume of the desorbed thiolates was found to move quickly (when compared to the previous measurements) toward the portion of the electrode that was situated higher, independent of the position of the counter electrode (Figure 5b). Furthermore, the shape of the fluorescence plume was initially circular, but became elliptical. This clearly illustrates that the main contribution toward the movement of the desorbed molecules is somehow related to gravity. Possible explanations for this observation can be the intrinsic buoyancy of the thiolate molecules or aggregates, or differences in the density of the electrolyte near the electrode produced by, e.g., resistive heating or gas evolution. Further experiments were performed using the microelectrode tilted at 8.8° from level. The potential was stepped negatively from −0.4 V to different final negative values (−1.3, −1.5, and −1.7 V) at an effective sweep rate of 140 mV/s. The final negative potential was held for 5.4 s and fluorescence images collected. The fluorescence images were analyzed as described previously and the speed of the center of the plume as a function of potential is shown in Figure 6a. The experiment terminating at the least negative potential (−1.3 V) showed the smallest movement of the plume of

major and minor axes. In this case, the thresholded fluorescence image has circular symmetry since the major and minor axes are of equal length throughout the potential steps. The initial increase in the size of the plume corresponds to an increasing number of fluorophores desorbing from the electrode. As the fluorescently labeled thiolates start diffusing away, their local concentration decreases, thereby causing a subsequent reduction of fluorescence intensity. The analysis method used will reflect this as a decrease in the length of the axes (modeling of this process is detailed in the Supporting Information). Moreover, the center of the ellipse does not change position (within 1.5 μm) from its initial location. These results suggest that hemispherical diffusion is the dominant mass transport mechanism as expected for a microelectrode under these conditions. Influence of the Counter Electrode Position. Previous experiments using fluorescence imaging to monitor reductive desorption have suggested a correlation between the direction of movement of the desorbed species with the position of the counter electrode.40,43 Using a microelectrode enabled a more controlled experiment in which another Au wire of approximately 150 μm in length was used as the counter electrode. As shown in Figure 3a,b, the counter electrode was positioned ∼30 μm away from the edge of the microelectrode

Figure 3. Desorption experiments performed with the CE in close proximity (30 m). Lateral view schematic (a) and bottom view micrograph (b) of the system. (c) Fluorescence images at two selected values of potential, for two different desorption experiments. The black circle represents the edge of the gold electrode, the white line represents the outline of the CE. Dashed lines are given as reference for the angular coordinate used. 2069

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Figure 5. (a) Scheme of the system employed to investigate the effect of gravity. (b) Fluorescence images at E = −2.0 V of two independent desorption experiments. The direction of the arrow points toward the higher point of the glass sheath. A video corresponding to the desorption experiment shown in (b) bottom is included as Video 2 in the Supporting Information.

Figure 4. Displacement (a), speed (b), and angle of movement (c) during the desorption for the two experiments shown in Figure 3c. The filled and empty symbols correspond to the top and bottom series of images, respectively. The potential perturbation is shown in (d) and in the axis at the top of the figure. Symbols represent raw data, while lines result from smoothing with a Savitzky-Golay filter. Error bars on the displacement are smaller than the symbol size; error bars on the speed (shown in the figure) were estimated by determining the sensitivity of the center of the ellipse on the threshold values used in image analysis.

Figure 6. (a) Speed of the center of the ellipse fitted to the fluorescent plume on three desorption experiments following the potential perturbations shown in (b). Symbols represent raw data, while lines result from smoothing using Savitzky-Golay filters. Error bars on the displacement are smaller than the symbol size; error bars on the velocity were calculated from the difference of the center of the ellipse with a change in intensity value used for threshold.

desorbed thiolates, with the center of the plume moving less than 1 m during the experiment. The speed of the center of the plume is small (less than 1 μm/s) and in no particular direction. The lengths of the major and minor axes which define the edge of the plume undergo much larger changes (displayed in Figure S5). The start of the desorption of the fluorescent molecules results in a predictable increase in the fluorescence intensity and therefore the size of the plume. At longer times, the length of the axes decrease as the molecules diffuse away into the electrolyte. Most importantly, the major and minor axes change in the same manner which clearly shows

that no distortion in the shape of the plume was observed; it is circular throughout the measurements and is clearly characteristic of the symmetrical diffusion of the desorbed molecules from the microelectrode at this low potential of −1.3 V even though the electrode surface was tilted. The measurements done at the two more negative potentials are clearly different as the plume moves consistently upward (i.e., up the inclined glass surface). The position of the center of the plume shifts with time/potential, moving approximately 20 μm. The speed of the center of the plume reaches values of 5− 10 μm/s. In both cases, the major and minor axes are similar 2070

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within the time of the experiment, but if the potential of −1.7 V is maintained for 10 s, the plume becomes elliptical (aspect ratio of 1.2). Overall for conditions where the CE is separated far from the WE, the shape of the plume can be described as a sum of three distinct effects: (i) hemispherical diffusion that, similar to the images in Figure 2d, causes a radially symmetric change in the diameter of the plume; (ii) buoyancy type of influence that displaces the plume toward higher “elevations” (which is seen as lateral movement); and (iii) desorption which may occur over a range of potentials introducing newly desorbed thiolates into the plume closest to the electrode making it appear elongated. The Au/glass boundary, for example, tends to be a region which desorbs at slightly more negative potentials.

temperature of the solution near the electrode surface are found to occur with the passage of large AC currents.55,56 However, the small DC currents produced here (less than 0.2 μA) are shown to produce differences in temperature of less than 1 mK both through finite element method simulations (as shown in Figure S9) and by using the calculation given by Baranski.56 These calculations show that the maximum density change is only 1.15 × 10−5 kg/m3, more than 2000× smaller than in the case of dissolved H2 resulting in velocities of at least 1 order of magnitude smaller than observed. Furthermore, the observed movement of the plume of thiolates does not scale with the calculated difference in temperature, as the velocity plateaus at potentials more negative than −1.7 V (Figure S6). It is more likely that the concentration of dissolved hydrogen is close to saturation resulting in a constant velocity, while the temperature should be able to significantly increase56 resulting in a continuously increasing plume speed, which is not observed. Finally, the idea of intrinsically buoyant molecules or small aggregates is also not likely, since changes in potential to values more negative than the reduction potential should not influence their speed. In summary, a difference in density due to dissolved hydrogen is the source for the buoyant movement observed. The upward movement of electrolyte containing dissolved H2 is further supported by an unusual set of measurements detailed below which show the removal of the SAM from particles on the glass surface. Unusual Fluorescence from the μE Glass Sheath. Electrochemical generation of dissolved H2 produced a rather unexpected result. As shown in Figure 1, the μEs used have Pt/ Ir particles on the walls of the insulating glass sheath. The microelectrode tip is placed into the SAM deposition solution, thereby exposing and modifying both the Au surface and the Pt/Ir metallic features on the glass wall. The reductive desorption scan (Figure 7a left and center) shows the appearance of a fluorescent plume from the gold surface and at more negative potentials a ring of fluorescence which started from the glass walls (Video 4 in SI). This fluorescence ring is clearly due to the removal of BodipyC10SH SAM from the electrically isolated Pt/Ir particles resulting in an increase in fluorescence as quenching by the Pt/Ir metal surface is no longer efficient. Their removal must be due to a reductive process,57 but clearly not an electrochemically controlled one. The electrogenerated dissolved H2 is postulated to be the reducing agent which moves upward due to its buoyancy. The dissolved H2 polarizes the Pt/Ir particles negatively, reducing the metal-S bond and releasing the fluorophore from the surface. The presence of dissolved H2 without creation of H2 bubbles is certainly enhanced with microelectrodes due to the hemispherical diffusion of the H2 away from the surface, delaying the creation of a critical concentration needed to nucleate bubble formation. As shown previously (vide supra), this dissolved H2 creates a locally buoyant electrolyte region which then moves upward, reducing the thiol SAM formed on the Pt/Ir particles. Interestingly, the fluorescence was not observed if the glass surface was coated in gold via vapor deposition. This can be explained by monitoring the open circuit potential (OCP) for a Pt/Ir wire and a Au wire immersed in electrolyte solution initially saturated with Ar and during bubbling with H2 (Figure 7b). The Au electrode potential did not change significantly, but the Pt/Ir surface adopted a very negative potential (∼−800 mV/Ag|AgCl) in agreement with literature values reported for Pt electrodes.58

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DISCUSSION Movement of the plume of reductively desorbed thiolates is directed upward. The potential dependence of the plume velocity can help identify the driving force behind this process. As mentioned before, at very negative potentials, the reduction of water produces molecular hydrogen which is somewhat soluble in the electrolyte (1.6 × 10−3 g H2/kg).50 The change in electrolyte composition near the electrode surface can produce differences in density due to the substitution of Ar for H2. This difference in density was employed to calculate the upward or buoyant force on the H2 saturated solution. This upward velocity was then used to calculate the velocity of solution on the surface of the tilted electrode. In this analysis, friction losses due to movement along the glass surface were not considered (details for these calculations are provided in the SI). The difference in density between water saturated with Ar and that saturated with H2 (2.81 × 10−2 kg/m3)51 is sufficient to drive advective movement at speeds of approximately 90 μm/s along the plane of the electrode surface. This velocity is much larger than what was measured, but it is still reasonable to conclude that the gradient in density due to the H2 content of the electrolyte (clearly not saturated) near the electrode surface causes the upward movement of electrolyte which as a consequence influences the motion of the desorbed thiolates. It is also possible that small H2 bubbles, which are inherently buoyant, may be responsible for the observed movement. The first appearance of a clearly visible bubble in these images occurs at −1.9 V, while the onset of the fast movement across the electrode is located at −1.7 V. Optically resolvable bubbles are approximately 1 μm in diameter. The presence of smaller bubbles is not likely as they are unstable due to the higher solubility of the gas due to the higher pressures created by the curvature of the bubble surface. Calculations of bubble dissolution time following the work of Duncan52 show that even in the case of continuous formation of H2 (saturation condition) it would require less than 0.01 s to completely dissolve a 1-m-diameter H2 bubble. Although the presence of smaller bubbles (nanobubbles) has been reported by the atomic force microscopy community, their stability seems to be conditional on the presence of the surface.53,54 While these nanobubbles may be active in the mechanism of hydrogen evolution, their existence is not the cause of the observed longrange (100 s of micrometers) movement away from the surface. Two other alternative explanations include resistive (Joule) heating and the intrinsic buoyancy of the desorbed molecules/ aggregates. Both possibilities can be addressed considering the terminal velocities observed for the −1.5 and −1.7 V measurements shown in Figure 6. Significant changes in the 2071

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when desorbed from the electrode surface, the thiolates radially diffuse away from the microelectrode. For more negative potentials, the creation of soluble H2 changes the density of the electrolyte near the electrode surface and carries the thiolate away from the electrode surface, upward. The velocity of the plume of thiolate was found to depend on the amount of H2 created, and therefore on the value of the potential. These observations clearly explain the extent of oxidative readsorption after desorption typically observed in CV experiments. The creation of dissolved H2 was also found to be sufficiently reductive for removing thiol SAMs from a Pt or Pt/Ir surface. Understanding the forces that determine the movement of these thiolates open the possibility of controlling their motion under specific electrochemical conditions.

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ASSOCIATED CONTENT

S Supporting Information *

Nine extra figures describing the experimental system as well as modeling and desorption results, four videos of the fluorescence process, and discussion of modeling. This material is available free of charge via the Internet at http://pubs.acs. org/.

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Figure 7. (a) Fluorescence images from a desorption experiment on a Au microelectrode (left and center) and from an experiment in which H2 was bubbled though an empty glass pipet treated in the same way the microelectrode was. (b) Open circuit potential of two macroscopic wires of Au and Pt/Ir in a 1 mM KOH solution. Initially, the solution is degassed with Ar; at the time indicated with the vertical line, H2 is bubbled into the solution.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.C.M. acknowledges the support of the Mexican National Science and Technology Council (CONACYT). The authors thank Brian Ditchburn, UBC chemistry glassblower for the fabrication of the electrochemical cell, Janine Mauzeroll for advice in the microelectrode fabrication, Arnold Kell and Mark Workentin for the synthesis of the BodipyC10SH and Andrzej Baranski for advice on resistive heating. This work was supported by a NSERC Discovery Grant.

Such potential values have been shown to be sufficient to at least partially remove the thiol from Pt surfaces.57 Further proof of these phenomena was achieved with the creation of a micropipet in the same fashion as the microelectrodes, but with a hollow interior. Fluorescence imaging of the micropipet after creating the fluorescent SAM on the Pt/Ir particles was performed while H2 was injected through the bore in the pipet. Figure 7a (right) shows the increase in fluorescence due to the increase in the dissolved H2 content in solution and the reductive removal of the thiol from the Pt/Ir particles on the glass surface. In addition to detailing another method for chemically removing thiol from Pt surfaces, this also implies the movement of dissolved H2 upward, suggesting that the electrolyte saturated with H2 would be more buoyant than electrolyte saturated with Ar, further supporting the mechanism proposed for the movement of the desorbed thiolates. This mechanism explains recent observations by Shepherd et al. in which they notice a difference in the fraction of oxidatively readsorbed thiolate depending on the experimental setup.59 A greater fraction of readsorbed thiolates was observed if the electrode surface was in a hanging meniscus configuration as compared to a submerged electrode. The movement of the thiolates from the submerged surface is facilitated by the advective movement described here (in part), while this upward movement is not possible in the hanging meniscus configuration.

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REFERENCES

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CONCLUSIONS Reductive desorption of thiol SAMs from a Au microelectrode was characterized using in situ fluorescence microscopy revealing the fate of the desorbed thiolates once departed from the electrode surface. At moderately negative potentials, 2072

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