Potential-Dependent Interaction of DOPC Liposomes with an

Article pubs.acs.org/Langmuir

Potential-Dependent Interaction of DOPC Liposomes with an Octadecanol-Covered Au(111) Surface Investigated Using Electrochemical Methods Coupled with in Situ Fluorescence Microscopy Amanda Musgrove, Colin R. Bridges, Glenn M. Sammis, and Dan Bizzotto* Advanced Materials and Process Engineering Laboratory (AMPEL), Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada S Supporting Information *

ABSTRACT: The potential-controlled incorporation of DOPC liposomes (100 nm diameter) into an adsorbed octadecanol layer on Au(111) was studied using electrochemical and in situ fluorescence microscopy. The adsorbed layer of octadecanol included a small amount of a lipophilic fluorophoreoctadecanol modified with BODIPYto enable fluorescence imaging. The deposited octadecanol layer was found not to allow liposomes to interact unless the potential was less than −0.4 V/SCE, which introduces defects into the adsorbed layer. Small increases in the capacitance of the adsorbed layer were measured after introducing the defects, allowing the liposomes to interact with the defects and then annealing the defects at 0 V/SCE. A change in the adsorbed layer was also signified by a more positive desorption potential for the liposome-modified adsorbed layer as compared to that for an adsorbed layer that was porated in a similar fashion but without liposomes present in the electrolyte. These subtle changes in capacitance are difficult to interpret, so an in situ spectroscopic study was performed to provide a more direct measure of the interaction. The incorporation of liposomes should result in an increase in the fluorescence measured because the fluorophore should become further separated from the gold surface, reducing the efficiency of fluorescence quenching. No significant increase in the fluorescence of the adsorbed layer was observed during the potential pulses used in the poration procedure in the absence of liposomes. In the presence of liposomes, the fluorescence intensity was found to depend on the potential and time used for poration. At 0 V/SCE, no significant change in the fluorescence was observed for defect-free adsorbed layers. Changing the poration potential to −0.4 V/SCE caused significant increases in the fluorescence and the appearance of new structural features in the adsorbed layers that were more easily observed during the desorption procedure. The extent of fluorescence changes was found to be strongly dependent on the nature of the adsorbed layer under investigation, which suggests that the poration and liposome interaction are dependent on the quality of the adsorbed layer and its ease of poration through changes in the electrode potential.

■

INTRODUCTION The creation of supported lipid bilayers is a promising platform for developing biosensors.1 The immobilized lipid bilayer, if properly designed, has the potential to support membrane proteins in an environment similar to that of a cell membrane, resulting in a wide range of potential applications for sensing, from environmental contaminants2 to drug discovery.3,4 These bilayers are conveniently formed by self-assembly from a solution of liposomes;5,6 however, this method can lead to heterogeneous surface coverage, even including intact vesicles adsorbed on the surface.7,8 The interaction of liposomes with metal/solution interfaces has been studied as a function of the potential or charge at the interface,9−14 revealing that the surface energy plays a significant role in the adhesion and rupturing process that results in the coating of the metal surface with lipid. © 2013 American Chemical Society

The creation of supported lipid membranes in a controlled manner that can be accomplished in situ and can be adapted to allow for spatial control of the formation is desirable. The formation of defects in an organic-coated electrode surface through the application of discrete potentials has been demonstrated for lipid-modified electrode surfaces, ranging from DOPC-coated Hg 1 5 −2 7 to octadecanol-coated Au(111).28−31 Control over the surface energy of the modified interface enables the poration of these layers in a manner that is similar to the initial stages of electroporation.32 Figure 1 illustrates a possible method for modifying supported lipid bilayers covering part of an electrode surface through the Received: January 6, 2013 Revised: February 13, 2013 Published: February 15, 2013 3347

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−3360

Langmuir

Article

initial adsorbed layer. Fluorescence from the adsorbed fluorophore is strongly quenched, and ideally no fluorescence will be observed from the adsorbed layer. Changes in capacitance and fluorescence will indicate a change in the structure of the adsorbed layer.29 If portions of the adsorbed layer exist further from the electrode surface because of the incorporation of liposomes, then fluorescence quenching is less effective and a fluorescence signal will be measured because the fluorophore initially near the electrode surface will be found further away in the new adsorbed structure. Performing these measurements using microscopy will also detail the nature of the liposome interaction with the adsorbed layer and the influence of the layer properties on the extent of incorporation or interaction.

■

EXPERIMENTAL METHODS

Materials. All electrochemical measurements were made on a Au(111) electrode prepared as described previously.28,31 The electrolyte used was 0.1 M aqueous NaF (Sigma, SigmaUltra) prepared in Milli-Q (18.2 M Ω) water. A 3 mg/mL solution of octadecanol (Fluka, SelectoPhore) was prepared in chloroform (Fisher, HPLC grade). An octadecanol solution containing 3 mol % of lipophilic fluorophore 4,4-difluoro-1,3,5,7-tetramethyl-8-(18-octadecanol)-4-bora-3a,4a-diaza-s-indacene (BODIPY-C19-OH), synthesized in-house (description given in Supporting Information) was prepared by the addition of a stock BODIPY-C19-OH solution (7 mg/ mL in chloroform) to a clean vial, evaporating the solvent under argon, and filling the vial with a 3 mg/mL octadecanol solution. Liposomes were formed using 13 mg/mL DOPC (dioleoyl phosphatidylcholine, Avanti Polar Lipids, Inc.) in the NaF electrolyte by extrusion34 using a 100 nm filter (Nucleopore 0.1 μm, Costar Corp). The liposome size was confirmed to be monodisperse at approximately 130(20) nm using dynamic light scattering (Coulter N4+ particle size analyzer), and the solution was checked periodically for lipid degradation by thin-layer chromatography (method adapted from Marathe et al.35). All glassware used was cleaned by heating for a minimum of 2 h in a 1:1 H2SO4/HNO3 bath (Fisher, ACS Pur), followed by rinsing, storing, and filling with Milli-Q water overnight. The electrochemical system was purged with Ar (Praxair) before and during measurements. Electrode Preparation. A Au(111) electrode was flame-annealed with a butane torch and quenched in Milli-Q water three times and then flame-dried and inserted into the electrochemical cell in a hanging meniscus configuration. Cyclic voltammetry and capacitance measurements were performed to ensure the cleanliness of the system, and the electrode was removed from the cell. Monolayers of octadecanol were prepared for Langmuir−Schaefer deposition onto the electrode by depositing excess chloroform solution onto the gas−electrolyte interface and allowing the solvent to evaporate. One such floating monolayer was prepared in a small beaker of electrolyte using the 3 mol % BODIPY-C19-OH-containing octadecanol solution. A second monolayer of pure octadecanol was prepared directly in the electrochemical cell. The electrode was once again flame-annealed, rinsed, and cooled in Ar for ∼1 min. The electrode was then gently touched to the gas−electrolyte interface of the beaker and withdrawn, depositing a monolayer of 3 mol % BODIPYC19-OH/octadecanol onto the electrode surface. Any remaining droplet of electrolyte was wicked away using the edge of a laboratory tissue, and the electrode was inserted into the electrochemical cell. After being equilibrated in the Ar atmosphere for approximately 30 s, the electrode was touched to the octadecanol-covered gas−electrolyte interface in the cell and lifted to form a hanging meniscus. This so-called double-touch method presumably forms a bilayer of octadecanol, with the BODIPY-labeled leaflet closest to the electrode surface. This method results in reproducibly modified electrode interfaces as compared to the single deposition approach, in addition to the improved matching of the organic layer thickness with the liposome bilayer. Electrochemical Methods. Electrochemical measurements were made using a potentiostat (HEKA PG590 with PAR 175 scan

Figure 1. Proposed model of liposome incorporation into a lipidlike layer into an electrode surface modified by an adsorbed lipidlike layer (a). The application of a negative potential creates defects in the adsorbed layer (b) where liposomes in solution can interact (c). Upon resetting the potential to values that favor an ordered adsorbed layer (d), liposomes may be incorporated into the adsorbed layer in a hemiliposomal structure that will contain fluorophores far from the electrode surface with increased fluorescence intensity.

controlled introduction of liposomes into the adsorbed layer. In this approach, the electrode is first coated in a bilayer of lipidlike material that prevents liposomes from interacting with the solid support (Figure 1a). Under the application of an appropriate potential, defects or pores are created in the adsorbed lipidlike layer (Figure 1b) through which liposomes in solution are able to interact with the solid support or the edges of the defect in the organic layer (Figure 1c). When the potential is removed or changed to a value that allows for a defect-free organic layer, the pores are removed or healed (Figure 1d) and the liposomes can interact with the lipidlike layer. The thus-formed hybrid layer will have regions of an organic-coated surface containing partially incorporated liposomes within this adsorbed layer. A schematic of these possible structures is shown with the possible redistribution of the fluorophore initially present in the adsorbed layer, with fluorophores far from the electrode surface. Moreover, these supported layers have the prerequisite bilayer with water on both sides, enabling the incorporation of membrane proteins in their natural environment. The model used in the work presented below is based on a physisorbed layer of octadecanol on Au(111), which has been shown to form defects reversibly within a well-characterized potential range.30,33 This creates an option for the simple incorporation of the liposome into the octadecanol layer via the application of an appropriate electrical potential, thereby creating defects, and then relaxation or annealing of the defects by altering the applied potential after allowing time for liposome interation. In this work, we show the ability to use electrochemical control to facilitate the incorporation of liposomes into a bilayer of octadecanol as a proof of concept for this method of forming solid-supported biomembrane layers. Capacitance changes of the interface during potential perturbation and interaction with DOPC liposomes in solution as a function of the potential and the length of time spent at the poration potential will be measured. The modified layer is further characterized by in situ fluorescence imaging with the introduction of 3 mol % of a lipophilic fluorophore into the 3348


Langmuir

■

generator) and lock-in amplifier (EG&G 5210), and signals were recorded using custom National Instruments LabView software. The counter electrode was a flame-annealed Au coil, and the reference electrode was a saturated calomel electrode (SCE) connected to the electrochemical cell via a salt bridge. All electrochemical measurements were performed in deoxygenated electrolyte maintained under a blanket of Ar. Scanning differential capacitance measurements were obtained using an ac perturbation of 5 mV (rms) at 25 Hz with a sweep rate of 5 mV/s. Once the adsorbed octadecanol was formed, cyclic voltammetry and differential capacitance measurements were made between +0.150 and −0.150 V/SCE. Octadecanol layers with a minimum capacitance value greater than 1.08 μF/cm2 were discarded as flawed depositions. When required, liposome solution was then injected directly into the electrolyte to an approximate concentration of 30 μg DOPC/mL of electrolyte, and the electrode potential was maintained at 0 V/SCE. The capacitance was measured during the hour that was allowed for the dispersal of the liposomes via diffusion (in lieu of mechanical stirring). Afterwards, a series of potential steps was applied. Generally, the electrode was held at 0 V/SCE to establish a baseline capacitance, and then the potential was swept negatively at 5 mV/s to the poration potential, followed by a scan at 5 mV/s to −0.2 V/SCE where the potential was held for 15 min and then returned to 0 V/SCE. A variety of values and durations of the poration potential were tested with capacitance measurements made throughout. After the application of the poration procedure, the adsorbed layer was further characterized by capacitance measurements during a desorption potential scan (from +0.150 to −0.800 V/SCE). For comparison, measurements were also performed for DOPC liposomes in solution in the absence of a modifying octadecanol layer on the Au(111) surface. To form these layers, a flame-annealed Au(111) electrode was placed in a hanging meniscus arrangement in the electrochemical cell and held at 0 V. Liposome solution was injected directly into the electrolyte and allowed to diffuse for 60 min while the electrode was maintained under potential control. To characterize the layer, the capacitance was measured during a potential scan from +0.150 to −1.00 V/SCE. In Situ Fluorescence Methods. The Au(111) surface was further characterized using in situ fluorescence measurements performed using a spectroelectrochemical cell (built in-house with a 250 μm optical window as the base of the cell36) and an inverted/ epifluorescence microscope (Olympus IX70) fitted with an EMCCD (Evolve-512 by Photometrics) and a light source (EXFO Exacte) using a 50× objective (Olympus LMPlan-FL) and a customassembled fluorescence filter cube tailored for BODIPY monomer fluorescence.36 The microscope setup was housed in a light-tight box. Changes in potential, electrochemical measurements, and fluorescence images were triggered and recorded simultaneously, as controlled by a custom LabView program. The lamp shutter was closed between images to minimize photobleaching. Images during the 0 V hold time were taken at a 5 s exposure time with an electron multiplier (EM) gain of 400 and a 5 min image spacing, and images recorded during poration or desorption were taken using a 2.5 s exposure time, an EM gain of 200, and a 5 s spacing. For ease of comparison between image sets, all image intensity values have been converted to equivalent counts per second of exposure at the lower EM gain using an empirically determined calibration factor to correct for the differences in the camera gain settings. The field of view was 147 μm × 147 μm. Preparation of the modified electrode surface and electrochemical measurements were performed as described above. The electrochemical cell used for fluorescence imaging is significantly smaller (1/ 10) than the basic electrochemistry cell, which resulted in some changes in procedure detailed in the fluorescence section of the results. As in the basic electrochemical measurements, the electrolyte is initially purged of oxygen using argon and is maintained under a positive pressure of argon throughout the experiment. The capacitance measurement was performed using a 200 Hz 5 mV rms perturbation so as to allow for fast stabilization of the lock-in measurements (capacitance) after the potential steps.

Article

RESULTS AND DISCUSSION Electrochemical Characterization of Adsorbed Octadecanol and DOPC Liposomes onto Au(111). The electrochemical behavior of physisorbed octadecanol and spontaneously adsorbed DOPC from liposomes on Au(111) has been described thoroughly in the literature,30,33,37,38 and is reviewed here for clarity. The DOPC-coated Au(111) electrode was prepared by allowing a liposomal solution of DOPC to interact with the Au(111) surface at 0 V/SCE for 15 min. The resulting layer was then characterized using differential capacitance as shown in Figure 2a. The octadecanol-modified

Figure 2. Differential capacitance measurements of adsorbed layers onto Au(111) in 0.1 M NaF: (a) adsorbed DOPC layer from liposomes and (b) octadecanol formed as described in the text. Desorption (negative-going, solid line) and readsorption (positivegoing, dashed line) potential scans. The minimum capacitance region is expanded for desorption scans shown for the first and second scans. The capacitance was measured using 5 mV/s, 5 mV rms, and 25 Hz.

Au(111) created using the method described in the Experimental Methods section, which presumably creates two octadecanol monolayers on the electrode surface (with the layer closest to the electrode including 3 mol % BODIPYC19OH), was characterized by capacitance measurements (Figure 2b). From approximately +0.15 to −0.2 V/SCE, the layer is stably adsorbed onto the electrode surface. When the potential is scanned negatively to values more negative than −0.2 V/SCE, pores or defects form in the octadecanol layer, causing a small increase in capacitance. Beyond approximately −0.6 V/SCE, the layer begins to desorb from the electrode surface via a solvent displacement mechanism, resulting in a capacitance at −0.8 V/SCE that is similar to the value obtained for the water-covered Au surface (20 μF/cm2). The readsorption (positive-going) scan shows significant hysteresis, with a decrease in capacitance starting at −0.4 V/SCE, resulting in the octadecanol layer readsorbing with a capacitance slightly larger than the starting condition. The readsorbed layer is thought to be oriented differently than the initial layer.30 The layer has a greater increase in capacitance at −0.4 V/SCE, 3349


Langmuir

Article

which can be explained through an increase in defects. A similar change in capacitance is also observed for the initial adsorbed layer as shown in the magnified region. The simple change in potential allows defects to be created and the layer to be desorbed and then readsorbed, which are consequences of changes in the surface energy of the interface as a function of the charge or potential.39,40 It is clear that potentials more negative than −0.5 V/SCE will result in quite disrupted or defective adsorbed octadecanol layers and that layers that were exposed to these negative potentials may not be defect-free upon readsorption. An adsorbed DOPC layer on Au(111), shown in Figure 2a, has a minimum capacitance of 5 μF/cm2 (at positive potentials), which is greater than the octadecanol layer, indicating a less-organized adsorbed layer. Such adsorbed layers of DOPC on Hg show a much lower capacitance (1.8 μF/cm2), indicating a better-organized layer that has been shown to impede faradaic reduction processes significantly.18,19,22,26,41−44 Scanning negatively, the DOPC layer begins to undergo significant changes in capacitance beginning at approximately −0.4 V/SCE, interpreted as a phase change prior to the desorption of the layer (fully desorbed at approximately −1.1 V/SCE37 as shown in Figure 2a). The DOPC layer is not significantly disrupted if the negative potential limit is restricted to −0.8 V/SCE. A higher minimum capacitance is observed for an equilibrium scan to the desorption potential. Comparing the capacitance−potential behaviors of the DOPC- and octadecanol-coated electrode shows that between −0.2 and −0.4 V/SCE a potential region exists in which defects are created in the adsorbed octadecanol layer, enabling the interaction of DOPC liposomes with the electrode surface. These defects are annealed when the potential is changed to −0.2 V/SCE and DOPC is adsorbed onto the electrode. To compare the effect of the potential-induced changes in the octadecanol layer on liposome interaction, a selection of potentials within this range were tested from 0 V, where few defects in the deposited layer exist, to −0.8 V/SCE, where the octadecanol layer is completely desorbed. Liposome Interaction with Octadecanol-Covered Au(111) at 0 V/SCE. The interaction of liposomes in electrolyte with an octadecanol-coated Au(111) substrate was investigated at 0 V/SCE to ensure that the adsorbed layer acts efficiently to preclude the incorporation or interaction with the Au surface. Investigations of the equilibrium surface pressure (ESP) of the floating octadecanol monolayer showed no change after a similar modification of the subphase with liposomes. Typically, the incorporation of a foreign species into a monolayer tends to decrease the ESP.45 This was not observed, which suggests that liposomes do not significantly interact with the unsupported monolayer. Changes in the adsorbed layer were monitored by capacitance while holding at 0 V/SCE for 1 h to allow time for the injected liposomes to diffuse throughout the electrolyte solution. This also provided an opportunity to monitor the interaction of the liposomes with a defect-free octadecanol layer before creating potentialinduced defects. The capacitance behavior shows two distinct types of responses, shown in Figure 3. The most common behavior, modeled by runs 1A and 1B, was an essentially unchanging capacitance during the measurement time. This behavior supports the hypothesis that liposomes will not incorporate into an octadecanol bilayer in the absence of significant defects. The change in the capacitance during these runs was less than 5%, which can be attributed to changes in the

Figure 3. Examples of the variety of capacitance responses observed for octadecanol-modified Au(111) exposed to a solution of DOPC liposomes (injected at t = 0 s) when the potential is held at 0 V/SCE.

electrode area due to wetting/dewetting of the electrode edges by the meniscus. Control runs of an octadecanol-coated electrode in the absence of liposomes exhibited the same type of behavior as runs 1A and 1B when held under these conditions. A minority of the experiments (3 of 23 data sets) showed an increase in capacitance, similar to that of run 2 in Figure 3. It appears that, despite screening the initial capacitance values to ensure reproducible layer quality, some did contain defects upon formation. Liposomes could then interact with these defects, producing the increase in capacitance observed. Because these types of layers are rare and obviously outside the typical behavior, they will not be considered in discussions of electrochemically induced liposome interaction to follow. Poration Potential and the Extent of Liposome Interaction. The interaction of liposomes with potentialinduced defects in the adsorbed octadecanol layer was probed using a series of potential steps applied to the adsorbed octadecanol layers with and without liposomes in the electrolyte solution. The capacitance behavior during these steps is shown in Figure 4. As described in the previous section, in the absence of the potential-induced poration of the octadecanol layer (0 V/SCE, Figure 4a) no significant change in capacitance in the presence of liposomes was observed. With the addition of a potential excursion to −0.2 V/SCE, only minor changes in capacitance behavior were observed. During the potential step to −0.2 V/SCE, the capacitance in both the control (liposome-free) and liposome-containing systems increased slightly because of changes in the adsorbed layer. When the potential is changed back to 0 V/SCE, the capacitance returns to nearly the initial value, again indicating that there are no significant structural changes to the adsorbed layer. When a potential step is applied to −0.4 V/SCE, a noticeable change in the capacitance is observed. A similar increase in capacitance is observed for adsorbed layers in the absence or presence of liposomes in the electrolyte as a result of the formation of defects in the adsorbed layer. Changing the potential to −0.2 V/SCE allows the layer to recover or anneal, as evidenced by the slow change in capacitance. This allows for a consistent comparison of the potential excursion effects. Returning to 0 V/SCE results in a capacitance similar to the initial value for the octadecanol layer in the absence of 3350


Langmuir

Article

Figure 4. Differential capacitance behavior of adsorbed octadecanol bilayers on Au(111) during potential perturbations with and without liposomes in the electrolyte. The potential perturbation applied during these measurements is shown in the inset for poration potentials of (a) 0, (b) −0.2, and (c) −0.4 V/SCE. Capacitance measured for potential scans to desorption potentials for adsorbed octadecanol layers on Au(111) with or without liposomes present in the electrolyte. The data shown are for different times spent at poration potentials of (d) 0, (e) −0.2, and (f) −0.4 V/SCE.

Figure 5. Capacitance behavior of the adsorbed octadecanol layer on Au(111) during the poration procedure at various times at the poration potential (−0.4 V/SCE). The insets show the potential perturbation applied in each case: (a) 1, (b) 15, and (c) 45 min. Insets show the potential profile applied. Capacitance measured for potential scans to desorption potentials for adsorbed octadecanol layers on Au(111) with or without liposomes present in the electrolyte. The data shown are for different times spent at a poration potential of −0.4 V/SCE: (d) 1, (e) 15, and (f) 45 min.

octadecanol (or the fluorophore) are able to exchange positions. This is difficult to determine by simple measurements of capacitance but should be observable as increases in the fluorescence intensity observed in the images as the fluorophore diffuses further from the electrode surface. The adsorbed layers modified by potential treatment were further characterized by measuring the capacitance during the potential-induced desorption of the adsorbed octadecanol or liposome-modified octadecanol layer. The electrode potential was scanned from +0.15 to −0.8 V/SCE while measuring the capacitance. As shown previously, the octadecanol layer is fully desorbed from the electrode surface at −0.8 V/SCE; that is, it is replaced or displaced by adsorbed water. Figure 3d−f shows the

liposomes, and the adsorbed layer exposed to a liposomecontaining electrolyte returns with an approximately 25% increase in capacitance over its initial value. This change in capacitance can be interpreted as being due to the interaction of liposomes with the defects formed at −0.4 V/SCE, incorporating into the octadecanol layer and increasing the average capacitance. As seen in Figure 2, the DOPC-coated surface has a higher capacitance than the octadecanol layer, suggesting that the increase seen after poration may be due to DOPC incorporation into the octadecanol layer. Strictly speaking, liposome incorporation will occur only if the newly formed adsorbed layer is characterized by molecules that are able to diffuse throughout the adsorbed layer, where lipid and 3351


Langmuir

Article

liposomes became incorporated into the defects or adsorbed onto the electrode surface, thus stabilizing the adsorbed layer as expected from the interaction of DOPC with Au(111) (Figure 2). The adsorbed layers were further characterized via potential-induced desorption using capacitance (Figure 5d−f). The shift in the desorption potential gives an indication of the incorporation of liposomes in the adsorbed layer, and coupled with a stable, low capacitance, it will identify the best conditions for the creation of this hybrid layer. The desorption of the layers created after poration at −0.4 V/SCE for 45 min indicates a strongly defective layer in the absence of liposomes but less so when liposomes are present. The layers created using 1 and 15 min poration times show similar characteristics, with the 1 min poration showing a larger shift in the desorption potential that may indicate a greater liposome incorporation, though this is difficult to prove conclusively using only capacitance. Although capacitance is a useful measure of the average change in the nature of the adsorbed layer, it is not able to determine changes in the adsorbed layer due to interaction with liposomes. It is clear that the potential-induced defects create opportunities for liposome interaction/incorporation into the adsorbed octadecanol layer, and a more detailed characterization of these adsorbed layers requires additional in situ methods. Fluorescence Imaging of the Adsorbed Layer during and after Poration. The interpretation of changes in capacitance due to potential-induced poration and the resulting liposome interaction suggest only liposome incorporation. From these electrochemical measurements, it is clear that introducing defects into the adsorbed layer with appropriate potentials results in a change in the capacitance of the layer only if liposomes are present in the electrolyte. These observed changes may be due to a simple occupation of the potentialinduced defect or the incorporation of liposomes into the adsorbed layer, which would result in subtle changes in the capacitance minimum observed at 0 V/SCE. These changes are not specific enough to provide information about the physical nature of the liposome interaction. Fluorescence imaging of the electrode−electrolyte interface during the poration process and the desorption process can be used to characterize further the changes occurring at the surface and provide evidence of incorporation. It is important to note that the octadecanolmodified electrode is composed of two layers; the layer closest to the electrode surface (the layer most efficiently quenched) is octadecanol with 3 mol % of a BODIPY-C19-OH fluorophore, and the second layer is only octadecanol. Liposome incorporation is expected to be observed as an increase in the fluorescence signal. Therefore, characterization by in situ fluorescence microscopy during the poration process and the subsequent desorption of the layer were performed. In transitioning to the in situ fluorescence measurements, changes in the technique were needed to accommodate the much smaller spectroelectrochemical cell. To decrease the time needed for liposomes to interact with the adsorbed layer, the concentration of liposomes in the electrolyte was increased by 50% to 45 μg/L DOPC. In addition, we found more variability in the capacitance of the deposited octadecanol layer, which led to a higher variability in the interactions of liposomes with octadecanol as well as a potentially higher degree of incorporation when compared to the basic electrochemistry trials. A number of poration studies were performed, and representative data is presented. We found a much greater variability in the fluorescence imaging results on the basis of the

capacitance during the desorption (negative potential scan) of layers previously exposed to the poration potentials described above. With no poration or only mild poration (0 or −0.2 V/ SCE, respectively), no significant shift in the potential for the onset of desorption was noted within the reproducibility of the measurements. However, after exposure to the −0.4 V/SCE poration potential (Figure 3f), the desorption of the octadecanol layer occurs at a less-negative potential in the presence of liposomes. The introduction of defects due to the interaction with liposomes would have a destabilizing influence on the adsorbed layer and thereby facilitate the desorption process, indicated by the shift to less-negative desorption potentials. The effect of applying more negative potentials during the poration phase of liposome incorporation was also attempted. Using −0.6 V/SCE as the poration potential results in some liposome incorporation (as interpreted by the increase in capacitance) but was not as reproducible as when using −0.4 V/SCE. Although this potential (−0.6 V/SCE) should cause more defects to form in the initial octadecanol layer and therefore should be more effective at facilitating liposome interaction, the reproducibility of the experiment was poor. This is likely because −0.6 V/SCE is very close to the potential where the octadecanol layer begins to desorb from the electrode surface (typically between −0.6 and −0.65 V/SCE). Small variations in layer properties will shift the desorption potential and may cause the layer to be in an intermediate state (partially desorbed) during the poration step, hampering reproducibility. The complete desorption of the octadecanol layer results in a gold surface that may be free for DOPC interaction, but the stability of the octadecanol layer at the desorption potential is limited, which affects the readsorption process. Moreover, DOPC was shown to interact weakly with the gold surface at these potentials (Figure 2 top). On the basis of the tests of liposome incorporation at varying potentials, it is clear that liposomes do not interact with octadecanol layers that are free of defects. However, the application of a negative potential, creating defects, will allow the incorporation of liposomes into the adsorbed layer. The application of too negative a potential significantly changes the adsorbed octadecanol layer by causing total or partial layer desorption, creating an interface that does not reproducibly interact with the liposomes. Of the potential values tested, −0.4 V/SCE showed the most reliable interaction of liposomes with the octadecanol layer and was therefore chosen for further study. Effect of Time at the Poration Potential of −0.4 V/SCE. Shown in Figure 5 are the changes in the capacitance of the octadecanol layer in the presence and absence of liposomes as a function of the time (1, 15, and 45 min) spent at the poration potential (−0.4 V/SCE). The increase in capacitance after poration in the presence of liposomes was observed for the 1 and 15 min poration times. In the absence of liposomes, the capacitance did not change significantly. As before, this increase is interpreted as being due to the incorporation of the DOPC liposomes into the adsorbed layer. Using a poration time of 45 min showed very different behavior. The octadecanol layer in the absence of liposomes showed large changes in the capacitance that resulted in a significantly more defective film that could not be recovered by waiting at 0 V/SCE. In the presence of liposomes, the layer’s capacitance did not increase beyond 2.5 μF cm−2 during poration, returning to reasonable values when the potential changed to 0 V/SCE. It seems that 3352


Langmuir

Article

images shows either a general increase in fluorescence evenly distributed across the image or the creation of small fluorescently intense regions. In the latter case, these layers were abandoned as defective. The layers showing a uniform increase in fluorescence were found to behave similarly to the majority of the data with regard to changes due to potential perturbation and were further analyzed. In a few cases, the fluorescence and capacitance increased during the waiting time, clearly indicating liposome incorporation with existing defects in the layer. These layers were also not analyzed further. The layers that demonstrated a stable capacitance during this waiting time were porated and studied. The increase in fluorescence can be explained as a reorganization of the adsorbed layer decreasing the quenching efficiency by increasing the separation from the gold surface. The initial fluorescence intensity for the adsorbed layers also displayed a wide range of values (2.5−4.5 kcnts/s). The apparent insensitivity of capacitance to the changes in the adsorbed layer as seen by fluorescence deserves comment, as does the range of initial fluorescence intensities. Previous fluorescence imaging studies36 have shown that floating octadecanol monolayers with 3 mol % of a lipophillic fluorophore, as used in this experiment, have a variety of structures and intensities that are retained when the layer is transferred to the electrode surface. Fluorescent images of an octadecanol-modified electrode showed a variety of intensities and structures. The difference in intensity is a result of the amount of fluorophore present in the region analyzed, resulting in regions that were dark (devoid of fluorophore) and fluorescently intense regions. Other regions in the same layer showed greater inhomogeneity in the distribution of the fluorophore, yielding speckles or stripes of fluorescence. The exact nature of these fluorescent structures as possible multilayers or aggregates is not yet understood, but their presence suggests that the adsorbed octadecanol layers are not completely uniform, possibly having defect sites with which the liposomes may interact. The electrode surface, at approximately 0.26 cm2, will have deposited onto it several varieties of these regions for a given deposition. Though all of these layers had a similar initial capacitance, this is an average measure across the entire surface and may be insensitive to these types of imperfections. The fluorescent images presented (about 2.2 × 10−4 cm2) will reveal only changes specific to the imaged area, resulting in a more varied behavior across experiments as compared to the capacitance. It is probable that all layers investigated have these regions of fluorescence behavior, indistinguishable by capacitance, but what is seen in fluorescence depends on the region imaged. Because the fluorescence in the initially deposited layer is quenched, little information on the layer structure is available when choosing the imaging area. Liposomes may interact with any defects present by either slightly perturbing the octadecanol layer or leaching fluorophore into the phospholipid bilayer, which would result in a change in fluorescence dependent on the structure of the region imaged. Any change in the adsorbed octadecanol layer at 0 V/SCE is not large enough to alter the capacitance value significantly, so incorporating the vesicles into the octadecanol bilayer is unlikely, at least on a large scale. On the basis of this evidence, we believe that for most of the adsorbed layers analyzed the liposomes do not incorporate into the octadecanol bilayers in the absence of defects and that the interaction between the liposomes and adsorbed bilayer is minimal.

area chosen for study when compared to the variation in the average capacitance measured for the total electrode surface. Liposome Interaction during Dispersion at 0 V/SCE. The adsorbed layer was exposed to liposomes in the electrolyte for 60 min while at 0 V/SCE, allowing the injected liposomes to diffuse throughout the solution. As seen previously, the capacitance of the interface did not change significantly during this time. In addition to capacitance, the interface was also monitored using fluorescence imaging. Trends in the average fluorescence intensity and capacitance are presented in Figure 6. In the control experiments (without liposomes), a slight

Figure 6. Average fluorescence image intensity (a) and capacitance (b) of adsorbed octadecanol layers on Au(111) during the waiting time at 0 V/SCE in the presence of DOPC liposomes (curves 1−3) or in the absence of liposomes (curves 4 and 5) in the electrolyte. These curves illustrate representative examples of the behavior observed.

increase (curve 4) or no increase (curve 5) in fluorescence is seen over the hold time (less than 10% of the initial intensity) as explained by the possible reorganization of the adsorbed layer during this waiting time that redistributes the fluorophore from the layer closest to the electrode surface to the layer farthest from the electrode. Photobleaching is not evident, likely because the octadecanol layer remains adsorbed onto the electrode surface and most fluorescence is quenched, reducing its effect, in addition to the low duty cycle used for imaging. Also noteworthy is the difference in the fluorescence intensity measured for these two seemingly similar adsorbed layers as determined from the capacitance. Included in the intensity is a background signal that comes from a portion of the excitation light that is reflected from the electrode surface and transmits through the filters (an extremely small fraction of the incident light). In the presence of liposomes, a majority (about 60%) of the adsorbed layers studied show a modest (10−15%) increase in fluorescence over time (curves 1 and 3 in Figure 6) without significant changes in capacitance. The combination of a steady capacitance and weak fluorescence increase indicates that liposomes are not interacting significantly with the adsorbed octadecanol layer at this potential. In some cases (∼20% of experiments), even with a stable capacitance, the fluorescence was found to increase (curve 2 in Figure 6). An analysis of the 3353


Langmuir

Article

Poration at −0.4 V/SCE for 1 Minute. As seen in the electrochemical studies, a poration potential of −0.4 V/SCE for either 1 or 15 min resulted in adsorbed layers that were slightly higher in capacitance and showed significant shifts in the potential for the onset of desorption. These subtle changes were interpreted as being due to the interaction of liposomes with the potential-created defects in the adsorbed layer, modifying the adsorbed layer. To confirm the incorporation of the liposomes into the defective adsorbed layer, fluorescence images during the poration and subsequent annealing at −0.2 and 0 V/SCE were performed. Many regions analyzed contained fluorescent features that we have shown to be due to the preferential segregation of the fluorophore into specific phases of the octadecanol layer.36 In addition, the presence of small but highly intense regions of fluorescence were also observed during adsorption. We believe that these are multilayers of adsorbate that are present in the floating layer that then become deposited onto the electrode surface.28,31,36 The areas around these regions are more characteristic of adsorbed layers with dim, essentially quenched fluorescence when adsorbed. These intensely fluorescent regions of interest (ROIs) are analyzed separately and are shown as outlined features in the fluorescence images that follow. Figure 7 shows the capacitance and fluorescence determined for three separate experiments where the adsorbed layer was treated to a 1 min poration at −0.4 V/SCE procedure. In two of these experiments, liposomes were added to the electrolyte. Also presented is an example of the control measurement made in the absence of liposomes, highlighting the variety of structures of the adsorbed layer. In many cases, the fluorescent images observed were more uniform than this, with little fluorescence variation across the image. The capacitance in the 15 min before the potential excursion is similar for all three examples. The magnitude of the fluorescence intensity varies depending on the region chosen for imaging, a consequence of the structure of the adsorbed layer discussed above. The intense ROIs were separated from the analysis, and the results are shown as dotted lines in Figure 7. During the first 15 min spent at 0 V/SCE (after the initial hour at 0 V/SCE during liposome dispersion), the fluorescence decreases because of photobleaching and is more significant for the intense ROIs. Because these ROIs are thought to be structures in the adsorbed layer that are not efficiently quenched (e.g., multilayers sitting higher or away from the surface), they therefore would have a greater chance of photodegrading. This overall decrease in intensity is in contrast to the results presented in Figure 5 where the fluorescence was found to increase slightly. During the 15 min described here, the layer was subjected to more frequent periods of illumination (2.5 s of every 5 s) compared to that occurring in the first hour (5 s of every 60 s), so photobleaching effects are stronger in these data sets. A change in potential to the poration value (−0.4 V/SCE) causes a significant increase in capacitance in all three examples because of a change in layer structure, as seen in the electrochemical studies above. In the absence of liposomes, the increase was small, and the capacitance returned to values slightly higher than the initial value after annealing for 15 min each at −0.2 V and 0 V/SCE. A very similar behavior is also observed in the presence of liposomes, but the adsorbed layer after poration has a larger capacitance. Larger changes in capacitance were also observed in the presence of liposomes (curve 2), though the layer did return to capacitance values similar to those expected after liposome interaction. The

Figure 7. Poration of the adsorbed octadecanol/3 mol % layer using 1 min at −0.4 V/SCE and annealing at −0.2 V/SCE for 15 min and 0 V/ SCE for 15 min, monitored with capacitance and with fluorescence imaging. (a) Capacitance and fluorescence intensity for adsorbed layers undergoing poration without and with DOPC liposomes in the electrolyte. The accompanying fluorescent images in part b show the ROIs used in the analysis. Shown are the changes in fluorescence intensity for the ROIs (thin lines) and the other parts of the images (thick lines). (b) Fluorescence images for the three experiments taken at the times indicated in part a. Top row: with liposomes (1). Middle row: with liposomes (2). Bottom row: without liposomes. The scale bar is 20 μm.

changes in fluorescence during this poration procedure show that the liposome-free control essentially displayed a constant decrease in intensity (more quickly for the intense ROIs) and very little potential dependence. The two examples in the presence of liposomes show different responses, illustrating that the changes observed can be significantly different depending on the region chosen for analysis. The increase in fluorescence observed when the potential changes to −0.4 V/SCE is small but significant, indicating the quick interaction of liposomes with the adsorbed layer (the scan to −0.4 V/SCE takes 20 s). The decrease in fluorescence when the potential is changed to −0.2 V/SCE is slight and in both cases continues to increase while the potential remains at −0.2 V/SCE. Changing again to 0 V/SCE results in an immediate decrease in fluorescence intensity, followed by a smaller increase over 15 min. These changes in fluorescence suggest that the liposomes have disrupted the adsorbed layer in such a way as to increase the separation of fluorophore from the electrode surface. We have shown that changes of 10−20 nm in this separation can result in a significant increase in fluorescence due to decreased quenching efficiency.29 The changes observed during poration are small and indicate that the liposomes may not be fully incorporated into the annealed adsorbed layer or that few 3354


Langmuir

Article

Figure 8. Capacitance and fluorescence characterization of an adsorbed layer during a potential desorption step experiment after poration for 1 min at −0.4 V/SCE in the absence or the presence of liposomes in the electrolyte. (a) Capacitance and fluorescence changes for two octadecanol-coated electrodes after poration for 1 min at −0.4 V/SCE in the presence of liposomes in the electrolyte and without liposomes. The thin lines represent the change in intensity of the ROIs shown in part b, and the thick lines are for the rest of the image. A selection of fluorescent images for potentials during desorption are also shown for the three experiments. Images in the bottom row are for a layer porated without liposomes in solution; the top two rows are for layers that were porated in the presence of liposomes. The images are falsely colored. The scale bar is 20 μm. (b) Fluorescence images in part a after rolling ball background subtraction. The images are falsely colored with a scale that is proportional to the net change in intensity due to desorption.

perturbation are not near the regions of high fluorescence outlined even though these regions would be considered to be a nonideally organized region or a possible defect. As demonstrated in the electrochemical studies, the interaction of liposomes with the adsorbed layer influences the potential-induced desorption, moving the onset of desorption to less-negative values. This process was also studied with in situ fluorescence microscopy and reveals more about the adsorbed layer structure because the fluorescence intensity is higher as a result of the displacement of the organic layer away from the gold surface. In the absence of liposomes, the capacitance (Figure 10a) increases at −0.6 V/SCE to a value characteristic of a water-covered electrode surface at −0.8 V/SCE, indicating a complete displacement of the layer off of

fluorophores from the adsorbed layer have diffused into the adsorbed lipid regions. A selection of fluorescent images during the poration procedure are shown for all three layers in Figure 7b. The changes in fluorescence are not uniform across the surface area imaged but are located in particular regions, illustrating that the adsorbed layer may have specific structures that are more amenable to poration and liposome interaction. The increase in fluorescence clearly shows that the adsorbed layer is changing its structure and supports the interaction of liposomes with the adsorbed layer because these changes are not solely due to the potential perturbation of the adsorbed octadecanol layer. The increase in fluorescence is also not located around the ROIs, which suggest that the defects created through the potential 3355


Langmuir

Article

the electrode surface. The fluorescence also increases to a maximum at this potential, somewhat following the capacitance change. The change in the fluorescence with potential is evenly distributed across the image, with the fluorescence from the more intense ROIs behaving in a similar fashion. The fluorescence intensity is a complex function of the amount of dye present in the adsorbed layer, the distribution of dye away from the electrode surface, and any other processes that could change the fluorescence intensity (e.g., BODIPY is known to form dimers36,46−48). The rapid increase in fluorescence over a small range of potential indicates that the layer responds uniformly to potential and that the adsorbed layer does not show strong heterogeneity in its structure normal to the electrode surface (except possibly the ROIs). The desorption of the modified layers after exposure to liposomes and the poration procedure shows significant differences in the potential dependence of fluorescence and its increase in intensity. In both examples, the onset of desorption occurs at −0.45 V/SCE, coincident with an increase in fluorescence. The adsorbed layer at 0 V/SCE is characteristic of a well-formed layer with a low capacitance and a small fluorescence signal as seen in Figure 8. The increase in capacitance and fluorescence at a less negative potential than the unmodified layer suggests that the layer has interacted with liposomes, changing the layer organization and enabling desorption at less-negative potentials. The increased intensity at desorption is indicative of a modified layer structure where more of the fluorophore is further from the electrode surface, reducing quenching and increasing the signal. These fluorescent images also show a more nonuniform structure, highlighting the regions furthest from the electrode surface. The layer is desorbed at the negative scan limit, with fluorescence significantly increasing and displaying a maximum at −0.65 V/SCE in one case and a constant increase until −0.8 V/SCE in the other. The intensity increases observed for these modified layers are significantly larger than the control layers. This large difference can be explained by variations in the initial amount of fluorophore present in the adsorbed layer or is due to a significant change in the thickness/structure of the adsorbed layer, suggesting the incorporation of liposomes creating adsorbed layers with structure normal to the electrode plane (e.g., 3-D structures). The maximum in fluorescence during desorption has been observed in our previous work, resulting from a change in the fluorophore organization such as dimer formation.49 The large changes in fluorescence intensity observed during desorption can mask smaller details that are important in this analysis because the liposomes may incorporate into the layer in particular regions and the influence in structure may remain localized. Analyzing for these small features can be accomplished by removing the featureless background through a rolling ball background subtraction (with a 50 pixel (14 μm) radius). This was performed on the fluorescent images presented in Figure 8b and shown in Figure 8c. For the layer that was not exposed to liposomes, the number of features at the adsorption potential are the same as at the desorption potential, although they increase in size. Some subtle changes in the underlying structure are also noted. These features were initially present in the adsorbed layer (shown as outlined regions in the images) and become easily observable when the layer is desorbed because of the decrease in quenching experienced by the fluorophore. In contrast, most of the features seen in the fluorescent image of the two layers after

interaction with the liposomes come not from the initially deposited structure but are a result of interaction with the liposomes. Also present in this analysis are large regions that also show increased fluorescence after liposome interaction, which correlate well with the larger changes in fluorescence observed in the unprocessed images. In both of the examples that were exposed to liposomes and the poration procedure, the number of small fluorescent regions also changed with the applied potential. This is expected because any regions that are further from the electrode will increase more rapidly in fluorescence as a result of the nonlinear quenching with distance29,50 when the layers are farther away from the electrode surface. These small features (∼5 pixels in diameter, ∼2 μm) are much larger than an individual liposome and so cannot be assigned to one specific interaction event. Importantly, a large majority of these features are not present in the initially adsorbed layer before interaction with liposomes (outlined ROIs), and features of this type are not observed in the analysis of the layers that were not exposed to liposomes during the poration procedure. Moreover, a distinct difference in the number of these features is evident when comparing the two layers that were exposed to liposomes, indicating that the potential-controlled liposome interaction is dependent on the structure of the adsorbed layer, which is not uniform across the electrode surface. Poration at −0.4 V/SCE for 15 Minutes. The changes observed in the adsorbed layer due to the interaction of liposomes should depend on the time spent at the poration potential (−0.4 V/SCE), resulting in an increased possibility of liposome interaction. From the electrochemical measurements, increasing the time from 1 to 15 min resulted in adsorbed layers that were still intact, with capacitance values that did not change significantly after the poration process unless liposomes were present in the subphase. Also, the desorption of the adsorbed layer showed a shift in the potential of the onset of desorption that suggested a change in the adsorbed layer after interaction with liposomes. The capacitance, fluorescence intensity, and images of the adsorbed layer during the 15 min poration process in the absence and presence of liposomes are shown in Figure 9. The capacitance changes due to poration are larger than observed in the electrochemical measurements, but the trends are similar. In the absence of liposomes, the adsorbed layer capacitance increases when the potential is changed to −0.4 V/SCE, and a sharp increase in capacitance is also observed about 7 min after the move to −0.4 V/SCE. This can be explained as a change in the layer, but because no change in the fluorescence intensity was seen (for the visible region), it is more likely due to a change in the wetting of the sides of the electrode held in a hanging meniscus. The capacitance decreases during the time at −0.2 V/SCE and when returning to 0 V/SCE to a value that is slightly above its starting value. In the absence of liposomes in the electrolyte, the adsorbed layer did not experience significant changes during this extended poration process. The fluorescence intensity decreases because of photobleaching, with the moreintense ROIs photobleaching more quickly. Small increases are observed at each change in potential, which is expected because the layer will slightly change its organization at these potentials, similar to the behavior in Figure 8. The origin of these small changes could be a displacement of the fluorophore to the outer surface of the adsorbed layer, but the change in fluorescence intensity is much smaller than in the presence of liposomes. 3356


Langmuir

Article

similar increase was seen in the 1 min poration experiments, although not as large as that seen here. The changes in the adsorbed layer during the desorption process are shown in Figure 10. The adsorbed layer that did not interact with liposomes was very similar to that shown for the 1 min poration at −0.4 V/SCE (Figure 8). Similarly, this indicates that the poration process did not significantly perturb the adsorbed layer in the absence of liposomes, even though the capacitance increased slightly in the poration process. The desorption of the modified layer in the presence of liposomes was very different. The capacitance shows the onset of desorption at −0.45 V/SCE, with desorption attained by −0.8 V/SCE, as signified by the final capacitance value, similar to that for a water-covered electrode. The fluorescence also increased at less-negative potentials than did the capacitance, and it increased continuously, with a rapid increase at around −0.5 V/SCE. The change in fluorescence is most sensitive to the features that are furthest from the electrode surface because these features produce the largest fluorescence signals: small changes in the distance from the electrode result in large changes in the fluorescence. This indicates that features of the modified layer are indeed farther from the electrode surface. Capacitance increases are slight at this potential and do not mirror the large increase in fluorescence because the capacitance is most sensitive to the dielectric changes on the surface and less so to changes further from the surface. A further increase in fluorescence is also observed up to the desorption potential. It is important to note that the changes in fluorescence observed are dependent on the area chosen to study, so a direct comparison between the 1 and 15 min results is not entirely appropriate but the general trends are clear. Furthermore, these increases in fluorescence result from large general changes in the fluorescence as well as changes on a smaller scale, which can be revealed by treating the images to a background subtraction routine as described above for the 1 min poration case. A comparison of the changes in the small features observed in the absence of liposome interaction and in the presence of liposomes is shown in Figure 10. The outlined features are regions of higher fluorescence that were observed at the start of the poration experiment (e.g., at −15 min). The layer that was not exposed to liposomes shows very few increases in the features after poration, annealing, and desorption. In contrast, the layer that was exposed to liposomes through the poration procedure displays a significant number of these features, far above that observed before the poration process. When compared to the 1 min poration studies, these features are similar, with a density that is between that of the two 1 min poration examples. Even though it was expected that the time allowed for liposome incorporation should increase the number of features observed as well as the fluorescence intensity, the small region chosen for analysis has a large influence on the extent of liposome interaction and incorporation observed, making a comparative analysis difficult.

Figure 9. Poration of the adsorbed octadecanol/3 mol % layer using 15 min at −0.4 V/SCE and annealing at −0.2 and 0 V/SCE for 15 min each, as monitored with capacitance and fluorescence imaging. (a) Capacitance and fluorescence intensity for adsorbed layers undergoing poration without and with DOPC liposomes in the electrolyte. The accompanying fluorescent images in part b show the ROIs used in the analysis. Shown are the changes in fluorescence intensity for the ROIs (thin lines) and the other parts of the images (thick lines). (b) Fluorescent images for the three experiments taken at the times indicated in part a. Top row: with liposomes. Bottom row: without liposomes. The scale bar is 20 μm.

In the presence of liposomes, the adsorbed layer showed more distinctive changes, similar to the layers exposed to the 1 min poration time. On decreasing the potential to −0.4 V/SCE, the capacitance increases dramatically and significant jumps in capacitance are observed during the 15 min at the poration potential. Similar events were observed for organic droplet adsorption and bursting onto a bare Hg drop11 but cannot be used to explain these changes because the electrode surface is coated with an organic layer. The capacitance decreases when the potential is changed to −0.2 and 0 V/SCE, resulting in an adsorbed layer that has a capacitance of 5 μF/cm2, a larger change than in the electrochemical measurements. It must be remembered that the concentration of liposomes was ∼50% higher than that used in the electrochemical measurements; therefore, a larger extent of interaction is expected. The changes in the fluorescence of the adsorbed layer were much greater than the 1 min poration results in the presence of liposomes. The initial time spent at 0 V/SCE showed typical fluorescence bleaching (images in columns A and B in Figure 9). Changing to −0.4 V/SCE, the fluorescence intensity increased uniformly across the image. The fluorescence continues to increase when the potential changes to −0.2 and 0 V/SCE, again distributed mostly evenly throughout the image but with some larger increases seen on the right side of the last image. After the liposomes have had sufficient time to interact with the adsorbed layer, fluorophore may diffuse into their structures, resulting in an increase in fluorescence with time. A

■

CONCLUSIONS The interaction of liposomes with a lipid-coated metal surface was studied using electrochemical and in situ fluorescence microscopy. It was demonstrated that control over the liposome interaction with a physisorbed layer on Au(111) was achieved using the electrical potential for adsorbed layers that contain few initial defects. The liposome interaction with the lipid-modified electrode surface depends on the potentialcontrolled creation of defects in the adsorbed layer. These 3357


Langmuir

Article

Figure 10. Capacitance and fluorescence characterization during a potential desorption step experiment of an adsorbed layer after poration for 15 min at −0.4 V/SCE in the absence or the presence of liposomes in the electrolyte. (a) Capacitance and fluorescence changes for two octadecanolcoated electrodes after poration for 15 min at −0.4 V/SCE with liposomes in the electrolyte (solid) and without liposomes (dashed). The thin lines represent the change in intensity of the ROIs shown in part b, and the thick lines are for the rest of the image. A selection of fluorescent images for potentials during the desorption of the adsorbed layer are also shown for the three experiments. The bottom row shows images from the layer that was porated without liposomes in solution, and the top two rows show images for a layer that was porated in the presence of liposomes. The images are falsely colored, and the color scale used is shown. The scale bar is 20 μm. (b) Fluorescence images in part a after rolling ball background subtraction. The images are falsely colored using the same lookup table and a scale that is proportional to the net change in intensity due to desorption.

structures are strongly influenced by the initial quality or nature of the layer deposited, which was found to be neither uniform nor homogeneous. Further work will require the use of a more homogeneous adsorbed layer so as to interact with liposomes more uniformly across the interface. In progress are in situ AFM studies of the nature of the features observed after liposome interaction.

defects cannot be so extensive as to render the adsorbed layer unstable, and a limited potential range was found where the interaction is stable and sufficient so as to be observed. The potential-dependent incorporation of liposomes is shown using a combined electrochemical and fluorescence method. In the cases where liposomes are present in the electrolyte, the small increases in the adsorbed layer capacitance after the poration procedure coincide with increases in fluorescence due to a redistribution of fluorophore after liposome interaction. The incorporation of liposomes into the adsorbed layer was found to change the desorption potential of the adsorbed layer, desorbing at less negative potentials because of the defects created through liposome interaction. Fluorescence imaging of the desorption process reveals the presence of small structures that may be regions where liposomes are incorporated. These

■

ASSOCIATED CONTENT

S Supporting Information *

Synthesis of 4,4-difluoro-1,3,5,7-tetramethyl-8-(18-octadecanol)-4-bora-3a,4a-diaza-s-indacene (BODIPY-C19-OH). This material is available free of charge via the Internet at http://pubs.acs.org/. 3358


Langmuir

■

Article

(18) Nelson, A.; Bizzotto, D. Chronoamperometric study of Tl(I) reduction at gramicidin-modified phospholipid-coated mercury electrodes. Langmuir 1999, 15, 7031−7039. (19) Bizzotto, D.; Nelson, A. Continuing electrochemical studies of phospholipid monolayers of dioleoyl phosphatidylcholine at the mercury-electrolyte interface. Langmuir 1998, 14, 6269−6273. (20) Rueda, M.; Navarro, I.; Ramirez, G.; Prieto, F.; Nelson, A. Impedance measurements with phospholipid-coated mercury electrodes. J. Electroanal. Chem. 1998, 454, 155−160. (21) Rueda, M.; Navarro, I.; Ramirez, G.; Prieto, F.; Prado, C.; Nelson, A. Electrochemical impedance study of Tl+ reduction through gramicidin channels in self-assembled gramicidinmodi fied dioleoylphosphatidylcholine monolayers on mercury electrodes. Langmuir 1999, 15, 3672−3678. (22) Becucci, L.; Moncelli, M. R.; Herrero, R.; Guidelli, R. Dipole potentials of monolayers of phosphatidylcholine, phosphatidylserine, and phosphatidic acid on mercury. Langmuir 2000, 16, 7694−7700. (23) Buoninsegni, F. T.; Becucci, L.; Moncelli, M. R.; Guidelli, R. Total and free charge densities on mercury coated with self-assembled phosphatidylcholine and octadecanethiol monolayers and octadecanethiol/phosphatidylcholine bilayers. J. Electroanal. Chem. 2001, 500, 395−407. (24) Guidelli, R.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Buoninsegni, F. T. Some bioelectrochemical applications of self-assembled films on mercury. Solid State Ionics 2002, 150, 13−26. (25) Becucci, L.; Moncelli, M. R.; Guidelli, R. Ion carriers and channels in metal-supported lipid bilayers as probes of transmembrane and dipole potentials. Langmuir 2003, 19, 3386−3392. (26) Stoodley, R.; Bizzotto, D. Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop. Analyst 2003, 128, 552−561. (27) Agak, J. O.; Stoodley, R.; Retter, U.; Bizzotto, D. On the impedance of a lipid-modified Hg-electrolyte interface. J. Electroanal. Chem. 2004, 562, 135−144. (28) Shepherd, J.; Yang, Y.; Bizzotto, D. Visualization of potential induced formation of water insoluble surfactant aggregates by epifiuorescence microscopy. J. Electroanal. Chem. 2002, 524−525, 54−61. (29) Bizzotto, D.; Yang, Y.; Shepherd, J. L.; Stoodley, R.; Agak, J.; Stauffer, V.; Lathuilliere, M.; Akhtar, A. S.; Chung, E. Electrochemical and spectroelectrochemical characterization of lipid organization in an electricfield. J. Electroanal. Chem. 2004, 574, 167−184. (30) Zawisza, I.; Lipkowski, J. Layer by Layer characterization of 1octadecanol films on a Au(111) electrode surface. in situ polarization modulation infrared reflection absorption spectroscopy and electrochemical studies. Langmuir 2004, 20, 4579−4589. (31) Shepherd, J. L.; Bizzotto, D. Characterization of mixed alcohol monolayers adsorbed onto a Au(111) electrode using electrofluorescence microscopy. Langmuir 2006, 22, 4869−4876. (32) Weaver, J. C.; Chizmadzhev, Y. A. Theory of electroporation: a review. Bioelectrochem. Bioenerg. 1996, 41, 135−160. (33) Bizzotto, D.; Pettinger, B. Fluorescence imaging studies of the electrochemical adsorption/desorption of octadecanol. Langmuir 1999, 15, 8309−8314. (34) Mayer, L.; Hope, M.; Cullis, P. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 1986, 858, 161−168. (35) Marathe, D.; Mishra, K. P. Radiation-induced changes in permeability in unilamellar phospholipid liposomes. Radiat. Res. 2002, 157, 685−692. (36) Casanova-Moreno, J.; Bizzotto, D. Electrochemistry and in situ fluorescence microscopy of octadecanol layers doped with a BODIPYlabeled phospholipid: Investigating an adsorbed heterogeneous layer. J. Electroanal. Chem. 2010, 649, 126−135. (37) Zawisza, I.; Bin, X.; Lipkowski, J. Spectroelectrochemical studies of bilayers of phospholipids in gel and liquid state on Au(111) electrode surface. Bioelectrochemistry 2004, 63, 137−147. (38) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S.; Goddard, J.; Lipkowski, J. Electrochemical and photon polarization modulation infrared reflection absorption spectroscopy study of the

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The spectroelectrochemical cell was created by Brian Ditchburn (UBC, chemistry, glassblowing), for which we are indebted. A.M. acknowledges support from the Agnes and Gilbert Hooley Scholarship in Chemistry (UBC). This research was funded by NSERC (Canada).

■

REFERENCES

(1) Castellana, E. T.; Cremer, P. S. Solid supported lipid bilayers: from biophysical studies to sensor design. Surf. Sci. Rep. 2006, 61, 429−444. (2) Xia, W.; Li, Y.; Wan, Y.; Chen, T.; Wei, J.; Lin, Y.; Xu, S. Electrochemical biosensor for estrogenic substance using lipid bilayers modified by Au nanoparticles. Biosens. Bioelectron. 2010, 25, 2253− 2258. (3) Merz, C.; Knoll, W.; Textor, M.; Reimhult, E. Formation of supported bacterial lipid membrane mimics. Biointerphases 2008, 3, FA41−FA50. (4) Ye Fang, B. W.; Hong, Y.; Lahiri, J. Applications of biomembranes in drug discovery. MRS Bull. 2006, 31, 541−545. (5) McConnell, H.; Watts, T.; Weis, R.; Brian, A. Supported planar membranes in studies of cellcell recognition in the immune system. Biochim. Biophys. Acta, Rev. Biomembr. 1986, 864, 95−106. (6) Xu, S.; Szymanski, G.; Lipkowski, J. Self-assembly of phospholipid molecules at a Au(111) electrode surface. J. Am. Chem. Soc. 2004, 126, 12276−12277. (7) Reviakine, I.; Brisson, A. Formation of supported phospholipid bilayers from unilamellar vesicles investigated by atomic force microscopy. Langmuir 2000, 16, 1806−1815. (8) Schönherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S. G. Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy. Langmuir 2004, 20, 11600−11606. (9) Ivoševic, N.; Tomaic, J.; Zutic, V. Organic droplets at an electrified interface: critical potentials of wetting measured by polarography. Langmuir 1994, 10, 2415−2418. (10) Ivoševic, N.; Zutic, V. Spreading and detachment of organic droplets at an electrified interface. Langmuir 1998, 14, 231−234. (11) Denardis, N. I.; Zutić, V.; Svetličcić, V.; Frkanec, R. Adhesion signals of phospholipid vesicles at an electrified interface. J. Membr. Biol. 2012, 245, 573−582. (12) Hellberg, D.; Scholz, F.; Schauer, F.; Weitschies, W. Bursting and spreading of liposomes on the surface of a static mercury drop electrode. Electrochem. Commun. 2002, 4, 305−309. (13) Hellberg, D.; Scholz, F.; Schubert, F.; Lovrić, M.; Omanović, D.; Hernández, V. A.; Thede, R. Kinetics of liposome adhesion on a mercury electrode. J. Phys. Chem. B 2005, 109, 14715−14726. (14) Agmo Hernandez, V.; Scholz, F. Kinetics of the adhesion of DMPC liposomes on a mercury electrode. effect of lamellarity, phase composition, size and curvature of liposomes, and presence of the pore forming peptide mastoparan. Langmuir 2006, 22, 10723−10731. (15) Nelson, A.; Auffret, N.; Borlakoglu, J. Interaction of hydrophobic organic compounds with mercury adsorbed dioleoylphosphatidylcholine monolayers. Biochim. Biophys. Acta 1990, 1021, 205−216. (16) Nelson, A. Electrochemical studies of thallium(I) transport across gramicidin modified electrode-adsorbed phospholipid monolayers. J. Electroanal. Chem. Interfacial Electrochem. 1991, 303, 221− 236. (17) Moncelli, M. R.; Becucci, L.; Nelson, A.; Guidelli, R. Electrochemical modeling of electron and proton transfer to ubiquinone-10 in a self-assembled phospholipid monolayer. Biophys. J. 1996, 70, 2716−2726. 3359


Langmuir

Article

electric field driven transformations of a phospholipid bilayer supported at a gold electrode surface. Biophys. J. 2003, 85, 4055−4075. (39) Buess-Herman, C. Non-faradaic phase transition in adsorbed organic layers part I. General theory. J. Electroanal. Chem. 1985, 186, 27−40. (40) Buess-Herman, C. Non-faradaic phase transition in adsorbed organic layers part II. Experiemental aspects. J. Electroanal. Chem. 1985, 186, 41−50. (41) Moncelli, M.; Guidelli, R. Test of Gouy-Chapman theory on phosphatidylcholine-coated mercury from differential capacity measurements. J. Electroanal. Chem. 1992, 326, 331−338. (42) Becucci, L.; Moncelli, M. R.; Guidelli, R.; Kelley, D. Surface charge density measurements on mercury electrodes covered by phospholipid monolayers. J. Electroanal. Chem. 1998, 413, 187−193. (43) Stauffer, V.; Stoodley, R.; Agak, J. O.; Bizzotto, D. Adsorption of DOPC onto Hg from the G S interface and from a liposomal suspension. J. Electroanal. Chem. 2001, 516, 73−82. (44) Becucci, L.; Moncelli, M. R.; Guidelli, R. Thallous ion movements through gramicidin channels incorporated in lipid monolayers supported by mercury. Biophys. J. 2002, 82, 852−864. (45) Bizzotto, D.; Wong, E.; Yang, Y. Adsorption of octadecanol/1pyrenenonanol mixed monolayers of insoluble surfactant onto Au(111): an electrochemical study. J. Electroanal. Chem. 2000, 480, 233−240. (46) Tleugabulova, D.; Zhang, Z.; Brennan, J. D. Characterization of bodipy dimers formed in a molecularly confined environment. J. Phys. Chem. B 2002, 106, 13133−13138. (47) Bergstrom, F.; Mikhalyov, I.; Hagglof, P.; Wortmann, R.; Ny, T.; Johansson, L. B.-A. Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. J. Am. Chem. Soc. 2002, 124, 196−204. (48) Musgrove, A.; Kell, A.; Bizzotto, D. Fluorescence imaging of the oxidative desorption of a BODIPY-alkyl-thiol monolayer coated Au bead. Langmuir 2008, 24, 7881−7888. (49) Shepherd, J. L.; Bizzotto, D. On the apparent fluorescence recovery due to electrosorption. J. Phys. Chem. B 2003, 107, 8524− 8531. (50) Chance, R. R.; Miller, A. H.; Prock, A.; Silbey, R. Fluorescence and energy transfer near interfaces: the complete and quantitative description of the Eu+3/mirror systems. J. Chem. Phys. 1975, 63, 1589− 1595.

3360


Potential-Dependent Interaction of DOPC Liposomes with an

Recommend Documents