Potential-Dependent Interaction of DOPC Liposomes with anOctadecanol-Covered Au(111) Surface Investigated UsingElectrochemical Methods Coupled with in Situ FluorescenceMicroscopyAmanda 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 lip-osomes (100 nm diameter) into an adsorbed octadecanol layer onAu(111) was studied using electrochemical and in situ fluorescencemicroscopy. The adsorbed layer of octadecanol included a smallamount of a lipophilic fluorophoreoctadecanol modified withBODIPYto enable fluorescence imaging. The deposited octadecanollayer was found not to allow liposomes to interact unless the potentialwas less than −0.4 V/SCE, which introduces defects into the adsorbedlayer. Small increases in the capacitance of the adsorbed layer weremeasured after introducing the defects, allowing the liposomes tointeract with the defects and then annealing the defects at 0 V/SCE. Achange in the adsorbed layer was also signified by a more positivedesorption potential for the liposome-modified adsorbed layer ascompared 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 moredirect measure of the interaction. The incorporation of liposomes should result in an increase in the fluorescence measuredbecause the fluorophore should become further separated from the gold surface, reducing the efficiency of fluorescencequenching. No significant increase in the fluorescence of the adsorbed layer was observed during the potential pulses used in theporation procedure in the absence of liposomes. In the presence of liposomes, the fluorescence intensity was found to depend onthe potential and time used for poration. At 0 V/SCE, no significant change in the fluorescence was observed for defect-freeadsorbed layers. Changing the poration potential to −0.4 V/SCE caused significant increases in the fluorescence and theappearance 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 ofporation through changes in the electrode potential.

■ INTRODUCTION

The creation of supported lipid bilayers is a promising platformfor developing biosensors.1 The immobilized lipid bilayer, ifproperly designed, has the potential to support membraneproteins 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 Thesebilayers are conveniently formed by self-assembly from asolution of liposomes;5,6 however, this method can lead toheterogeneous surface coverage, even including intact vesiclesadsorbed on the surface.7,8 The interaction of liposomes withmetal/solution interfaces has been studied as a function of thepotential or charge at the interface,9−14 revealing that thesurface energy plays a significant role in the adhesion andrupturing process that results in the coating of the metal surfacewith lipid.

The creation of supported lipid membranes in a controlledmanner that can be accomplished in situ and can be adapted toallow for spatial control of the formation is desirable. Theformation of defects in an organic-coated electrode surfacethrough the application of discrete potentials has beendemonstrated for lipid-modified electrode surfaces, rangingfrom DOPC-coated Hg15−27 to octadecanol-coatedAu(111).28−31 Control over the surface energy of the modifiedinterface enables the poration of these layers in a manner that issimilar to the initial stages of electroporation.32 Figure 1illustrates a possible method for modifying supported lipidbilayers covering part of an electrode surface through the

Received: January 6, 2013Revised: February 13, 2013Published: February 15, 2013

Article

pubs.acs.org/Langmuir

© 2013 American Chemical Society 3347 dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−3360

controlled introduction of liposomes into the adsorbed layer. Inthis approach, the electrode is first coated in a bilayer oflipidlike material that prevents liposomes from interacting withthe solid support (Figure 1a). Under the application of anappropriate potential, defects or pores are created in theadsorbed lipidlike layer (Figure 1b) through which liposomes insolution are able to interact with the solid support or the edgesof the defect in the organic layer (Figure 1c). When thepotential is removed or changed to a value that allows for adefect-free organic layer, the pores are removed or healed(Figure 1d) and the liposomes can interact with the lipidlikelayer. The thus-formed hybrid layer will have regions of anorganic-coated surface containing partially incorporated lip-osomes within this adsorbed layer. A schematic of thesepossible structures is shown with the possible redistribution ofthe fluorophore initially present in the adsorbed layer, withfluorophores far from the electrode surface. Moreover, thesesupported layers have the prerequisite bilayer with water onboth sides, enabling the incorporation of membrane proteins intheir natural environment.The model used in the work presented below is based on a

physisorbed layer of octadecanol on Au(111), which has beenshown to form defects reversibly within a well-characterizedpotential range.30,33 This creates an option for the simpleincorporation of the liposome into the octadecanol layer via theapplication of an appropriate electrical potential, therebycreating defects, and then relaxation or annealing of the defectsby altering the applied potential after allowing time forliposome interation. In this work, we show the ability to useelectrochemical control to facilitate the incorporation ofliposomes into a bilayer of octadecanol as a proof of conceptfor this method of forming solid-supported biomembranelayers. Capacitance changes of the interface during potentialperturbation and interaction with DOPC liposomes in solutionas a function of the potential and the length of time spent at theporation potential will be measured. The modified layer isfurther characterized by in situ fluorescence imaging with theintroduction of 3 mol % of a lipophilic fluorophore into the

initial adsorbed layer. Fluorescence from the adsorbedfluorophore is strongly quenched, and ideally no fluorescencewill be observed from the adsorbed layer. Changes incapacitance and fluorescence will indicate a change in thestructure of the adsorbed layer.29 If portions of the adsorbedlayer exist further from the electrode surface because of theincorporation of liposomes, then fluorescence quenching is lesseffective and a fluorescence signal will be measured because thefluorophore initially near the electrode surface will be foundfurther away in the new adsorbed structure. Performing thesemeasurements using microscopy will also detail the nature ofthe liposome interaction with the adsorbed layer and theinfluence of the layer properties on the extent of incorporationor interaction.

■ EXPERIMENTAL METHODSMaterials. All electrochemical measurements were made on a

Au(111) electrode prepared as described previously.28,31 Theelectrolyte used was 0.1 M aqueous NaF (Sigma, SigmaUltra)prepared in Milli-Q (18.2 M Ω) water. A 3 mg/mL solution ofoctadecanol (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-octa-decanol)-4-bora-3a,4a-diaza-s-indacene (BODIPY-C19-OH), synthe-sized in-house (description given in Supporting Information) wasprepared 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. Liposomeswere formed using 13 mg/mL DOPC (dioleoyl phosphatidylcholine,Avanti Polar Lipids, Inc.) in the NaF electrolyte by extrusion34 using a100 nm filter (Nucleopore 0.1 μm, Costar Corp). The liposome sizewas confirmed to be monodisperse at approximately 130(20) nm usingdynamic light scattering (Coulter N4+ particle size analyzer), and thesolution was checked periodically for lipid degradation by thin-layerchromatography (method adapted from Marathe et al.35). Allglassware used was cleaned by heating for a minimum of 2 h in a1:1 H2SO4/HNO3 bath (Fisher, ACS Pur), followed by rinsing,storing, and filling with Milli-Q water overnight. The electrochemicalsystem was purged with Ar (Praxair) before and during measurements.

Electrode Preparation. A Au(111) electrode was flame-annealedwith a butane torch and quenched in Milli-Q water three times andthen flame-dried and inserted into the electrochemical cell in a hangingmeniscus configuration. Cyclic voltammetry and capacitance measure-ments were performed to ensure the cleanliness of the system, and theelectrode was removed from the cell. Monolayers of octadecanol wereprepared for Langmuir−Schaefer deposition onto the electrode bydepositing excess chloroform solution onto the gas−electrolyteinterface and allowing the solvent to evaporate. One such floatingmonolayer was prepared in a small beaker of electrolyte using the 3mol % BODIPY-C19-OH-containing octadecanol solution. A secondmonolayer of pure octadecanol was prepared directly in theelectrochemical cell. The electrode was once again flame-annealed,rinsed, and cooled in Ar for ∼1 min. The electrode was then gentlytouched to the gas−electrolyte interface of the beaker and withdrawn,depositing a monolayer of 3 mol % BODIPYC19-OH/octadecanolonto the electrode surface. Any remaining droplet of electrolyte waswicked away using the edge of a laboratory tissue, and the electrodewas inserted into the electrochemical cell. After being equilibrated inthe Ar atmosphere for approximately 30 s, the electrode was touchedto the octadecanol-covered gas−electrolyte interface in the cell andlifted to form a hanging meniscus. This so-called double-touch methodpresumably forms a bilayer of octadecanol, with the BODIPY-labeledleaflet closest to the electrode surface. This method results inreproducibly modified electrode interfaces as compared to the singledeposition approach, in addition to the improved matching of theorganic layer thickness with the liposome bilayer.

Electrochemical Methods. Electrochemical measurements weremade using a potentiostat (HEKA PG590 with PAR 175 scan

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

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603348

generator) and lock-in amplifier (EG&G 5210), and signals wererecorded using custom National Instruments LabView software. Thecounter electrode was a flame-annealed Au coil, and the referenceelectrode was a saturated calomel electrode (SCE) connected to theelectrochemical cell via a salt bridge. All electrochemical measurementswere performed in deoxygenated electrolyte maintained under ablanket of Ar. Scanning differential capacitance measurements wereobtained using an ac perturbation of 5 mV (rms) at 25 Hz with asweep 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 valuegreater than 1.08 μF/cm2 were discarded as flawed depositions. Whenrequired, liposome solution was then injected directly into theelectrolyte to an approximate concentration of 30 μg DOPC/mL ofelectrolyte, and the electrode potential was maintained at 0 V/SCE.The capacitance was measured during the hour that was allowed forthe dispersal of the liposomes via diffusion (in lieu of mechanicalstirring). 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 porationpotential, followed by a scan at 5 mV/s to −0.2 V/SCE where thepotential was held for 15 min and then returned to 0 V/SCE. A varietyof values and durations of the poration potential were tested withcapacitance measurements made throughout. After the application ofthe poration procedure, the adsorbed layer was further characterizedby 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 layeron the Au(111) surface. To form these layers, a flame-annealedAu(111) electrode was placed in a hanging meniscus arrangement inthe electrochemical cell and held at 0 V. Liposome solution wasinjected directly into the electrolyte and allowed to diffuse for 60 minwhile the electrode was maintained under potential control. Tocharacterize the layer, the capacitance was measured during a potentialscan from +0.150 to −1.00 V/SCE.In Situ Fluorescence Methods. The Au(111) surface was further

characterized using in situ fluorescence measurements performedusing a spectroelectrochemical cell (built in-house with a 250 μmoptical window as the base of the cell36) and an inverted/epifluorescence microscope (Olympus IX70) fitted with an EM-CCD (Evolve-512 by Photometrics) and a light source (EXFOExacte) using a 50× objective (Olympus LMPlan-FL) and a custom-assembled fluorescence filter cube tailored for BODIPY monomerfluorescence.36 The microscope setup was housed in a light-tight box.Changes in potential, electrochemical measurements, and fluorescenceimages were triggered and recorded simultaneously, as controlled by acustom LabView program. The lamp shutter was closed betweenimages to minimize photobleaching. Images during the 0 V hold timewere taken at a 5 s exposure time with an electron multiplier (EM)gain of 400 and a 5 min image spacing, and images recorded duringporation or desorption were taken using a 2.5 s exposure time, an EMgain of 200, and a 5 s spacing. For ease of comparison between imagesets, all image intensity values have been converted to equivalentcounts per second of exposure at the lower EM gain using anempirically determined calibration factor to correct for the differencesin 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 electro-chemical cell used for fluorescence imaging is significantly smaller (1/10) than the basic electrochemistry cell, which resulted in somechanges in procedure detailed in the fluorescence section of the results.As in the basic electrochemical measurements, the electrolyte isinitially purged of oxygen using argon and is maintained under apositive pressure of argon throughout the experiment. The capacitancemeasurement was performed using a 200 Hz 5 mV rms perturbationso as to allow for fast stabilization of the lock-in measurements(capacitance) after the potential steps.

■ RESULTS AND DISCUSSIONElectrochemical Characterization of Adsorbed Octa-

decanol and DOPC Liposomes onto Au(111). Theelectrochemical behavior of physisorbed octadecanol andspontaneously adsorbed DOPC from liposomes on Au(111)has been described thoroughly in the literature,30,33,37,38 and isreviewed here for clarity. The DOPC-coated Au(111) electrodewas prepared by allowing a liposomal solution of DOPC tointeract with the Au(111) surface at 0 V/SCE for 15 min. Theresulting layer was then characterized using differentialcapacitance as shown in Figure 2a. The octadecanol-modified

Au(111) created using the method described in theExperimental Methods section, which presumably creates twooctadecanol monolayers on the electrode surface (with thelayer closest to the electrode including 3 mol % BODIPY-C19OH), was characterized by capacitance measurements(Figure 2b). From approximately +0.15 to −0.2 V/SCE, thelayer is stably adsorbed onto the electrode surface. When thepotential 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 electrodesurface via a solvent displacement mechanism, resulting in acapacitance at −0.8 V/SCE that is similar to the value obtainedfor the water-covered Au surface (20 μF/cm2). The read-sorption (positive-going) scan shows significant hysteresis, witha decrease in capacitance starting at −0.4 V/SCE, resulting inthe octadecanol layer readsorbing with a capacitance slightlylarger than the starting condition. The readsorbed layer isthought to be oriented differently than the initial layer.30 Thelayer has a greater increase in capacitance at −0.4 V/SCE,

Figure 2. Differential capacitance measurements of adsorbed layersonto Au(111) in 0.1 M NaF: (a) adsorbed DOPC layer fromliposomes and (b) octadecanol formed as described in the text.Desorption (negative-going, solid line) and readsorption (positive-going, dashed line) potential scans. The minimum capacitance regionis 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.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603349

which can be explained through an increase in defects. A similarchange in capacitance is also observed for the initial adsorbedlayer as shown in the magnified region. The simple change inpotential allows defects to be created and the layer to bedesorbed and then readsorbed, which are consequences ofchanges in the surface energy of the interface as a function ofthe charge or potential.39,40 It is clear that potentials morenegative than −0.5 V/SCE will result in quite disrupted ordefective adsorbed octadecanol layers and that layers that wereexposed to these negative potentials may not be defect-freeupon readsorption.An adsorbed DOPC layer on Au(111), shown in Figure 2a,

has a minimum capacitance of 5 μF/cm2 (at positivepotentials), which is greater than the octadecanol layer,indicating a less-organized adsorbed layer. Such adsorbed layersof DOPC on Hg show a much lower capacitance (1.8 μF/cm2),indicating a better-organized layer that has been shown toimpede faradaic reduction processes significantly.18,19,22,26,41−44

Scanning negatively, the DOPC layer begins to undergosignificant changes in capacitance beginning at approximately−0.4 V/SCE, interpreted as a phase change prior to thedesorption of the layer (fully desorbed at approximately −1.1V/SCE37 as shown in Figure 2a). The DOPC layer is notsignificantly disrupted if the negative potential limit is restrictedto −0.8 V/SCE. A higher minimum capacitance is observed foran 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 defectsare created in the adsorbed octadecanol layer, enabling theinteraction 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. Tocompare the effect of the potential-induced changes in theoctadecanol layer on liposome interaction, a selection ofpotentials within this range were tested from 0 V, where fewdefects in the deposited layer exist, to −0.8 V/SCE, where theoctadecanol layer is completely desorbed.Liposome Interaction with Octadecanol-Covered

Au(111) at 0 V/SCE. The interaction of liposomes inelectrolyte with an octadecanol-coated Au(111) substrate wasinvestigated at 0 V/SCE to ensure that the adsorbed layer actsefficiently to preclude the incorporation or interaction with theAu surface. Investigations of the equilibrium surface pressure(ESP) of the floating octadecanol monolayer showed nochange after a similar modification of the subphase withliposomes. Typically, the incorporation of a foreign species intoa monolayer tends to decrease the ESP.45 This was notobserved, which suggests that liposomes do not significantlyinteract with the unsupported monolayer. Changes in theadsorbed layer were monitored by capacitance while holding at0 V/SCE for 1 h to allow time for the injected liposomes todiffuse throughout the electrolyte solution. This also providedan opportunity to monitor the interaction of the liposomes witha defect-free octadecanol layer before creating potential-induced defects. The capacitance behavior shows two distincttypes of responses, shown in Figure 3. The most commonbehavior, modeled by runs 1A and 1B, was an essentiallyunchanging capacitance during the measurement time. Thisbehavior supports the hypothesis that liposomes will notincorporate into an octadecanol bilayer in the absence ofsignificant defects. The change in the capacitance during theseruns was less than 5%, which can be attributed to changes in the

electrode area due to wetting/dewetting of the electrode edgesby the meniscus. Control runs of an octadecanol-coatedelectrode in the absence of liposomes exhibited the sametype of behavior as runs 1A and 1B when held under theseconditions. A minority of the experiments (3 of 23 data sets)showed an increase in capacitance, similar to that of run 2 inFigure 3. It appears that, despite screening the initialcapacitance values to ensure reproducible layer quality, somedid contain defects upon formation. Liposomes could theninteract with these defects, producing the increase incapacitance observed. Because these types of layers are rareand obviously outside the typical behavior, they will not beconsidered in discussions of electrochemically induced lip-osome interaction to follow.

Poration Potential and the Extent of LiposomeInteraction. The interaction of liposomes with potential-induced defects in the adsorbed octadecanol layer was probedusing a series of potential steps applied to the adsorbedoctadecanol layers with and without liposomes in theelectrolyte solution. The capacitance behavior during thesesteps is shown in Figure 4. As described in the previous section,in the absence of the potential-induced poration of theoctadecanol layer (0 V/SCE, Figure 4a) no significant changein 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 inboth the control (liposome-free) and liposome-containingsystems increased slightly because of changes in the adsorbedlayer. When the potential is changed back to 0 V/SCE, thecapacitance returns to nearly the initial value, again indicatingthat there are no significant structural changes to the adsorbedlayer.When a potential step is applied to −0.4 V/SCE, a noticeable

change in the capacitance is observed. A similar increase incapacitance is observed for adsorbed layers in the absence orpresence of liposomes in the electrolyte as a result of theformation of defects in the adsorbed layer. Changing thepotential to −0.2 V/SCE allows the layer to recover or anneal,as evidenced by the slow change in capacitance. This allows fora consistent comparison of the potential excursion effects.Returning to 0 V/SCE results in a capacitance similar to theinitial value for the octadecanol layer in the absence of

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

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603350

liposomes, and the adsorbed layer exposed to a liposome-containing electrolyte returns with an approximately 25%increase in capacitance over its initial value. This change incapacitance can be interpreted as being due to the interaction ofliposomes with the defects formed at −0.4 V/SCE,incorporating into the octadecanol layer and increasing theaverage capacitance. As seen in Figure 2, the DOPC-coatedsurface has a higher capacitance than the octadecanol layer,suggesting that the increase seen after poration may be due toDOPC incorporation into the octadecanol layer. Strictlyspeaking, liposome incorporation will occur only if the newlyformed adsorbed layer is characterized by molecules that areable to diffuse throughout the adsorbed layer, where lipid and

octadecanol (or the fluorophore) are able to exchangepositions. This is difficult to determine by simple measure-ments of capacitance but should be observable as increases inthe fluorescence intensity observed in the images as thefluorophore diffuses further from the electrode surface.The adsorbed layers modified by potential treatment were

further characterized by measuring the capacitance during thepotential-induced desorption of the adsorbed octadecanol orliposome-modified octadecanol layer. The electrode potentialwas scanned from +0.15 to −0.8 V/SCE while measuring thecapacitance. As shown previously, the octadecanol layer is fullydesorbed from the electrode surface at −0.8 V/SCE; that is, it isreplaced or displaced by adsorbed water. Figure 3d−f shows the

Figure 4. Differential capacitance behavior of adsorbed octadecanol bilayers on Au(111) during potential perturbations with and without liposomesin 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 withoutliposomes 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 porationpotential (−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 potentialprofile applied. Capacitance measured for potential scans to desorption potentials for adsorbed octadecanol layers on Au(111) with or withoutliposomes 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) 45min.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603351

capacitance during the desorption (negative potential scan) oflayers previously exposed to the poration potentials describedabove. With no poration or only mild poration (0 or −0.2 V/SCE, respectively), no significant shift in the potential for theonset of desorption was noted within the reproducibility of themeasurements. However, after exposure to the −0.4 V/SCEporation potential (Figure 3f), the desorption of theoctadecanol layer occurs at a less-negative potential in thepresence of liposomes. The introduction of defects due to theinteraction with liposomes would have a destabilizing influenceon the adsorbed layer and thereby facilitate the desorptionprocess, indicated by the shift to less-negative desorptionpotentials.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 someliposome incorporation (as interpreted by the increase incapacitance) but was not as reproducible as when using −0.4V/SCE. Although this potential (−0.6 V/SCE) should causemore defects to form in the initial octadecanol layer andtherefore should be more effective at facilitating liposomeinteraction, the reproducibility of the experiment was poor.This is likely because −0.6 V/SCE is very close to the potentialwhere the octadecanol layer begins to desorb from theelectrode surface (typically between −0.6 and −0.65 V/SCE).Small variations in layer properties will shift the desorptionpotential and may cause the layer to be in an intermediate state(partially desorbed) during the poration step, hamperingreproducibility. The complete desorption of the octadecanollayer results in a gold surface that may be free for DOPCinteraction, but the stability of the octadecanol layer at thedesorption potential is limited, which affects the readsorptionprocess. Moreover, DOPC was shown to interact weakly withthe 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 withoctadecanol layers that are free of defects. However, theapplication of a negative potential, creating defects, will allowthe incorporation of liposomes into the adsorbed layer. Theapplication of too negative a potential significantly changes theadsorbed octadecanol layer by causing total or partial layerdesorption, creating an interface that does not reproduciblyinteract with the liposomes. Of the potential values tested, −0.4V/SCE showed the most reliable interaction of liposomes withthe octadecanol layer and was therefore chosen for furtherstudy.Effect of Time at the Poration Potential of −0.4 V/SCE.

Shown in Figure 5 are the changes in the capacitance of theoctadecanol layer in the presence and absence of liposomes as afunction of the time (1, 15, and 45 min) spent at the porationpotential (−0.4 V/SCE). The increase in capacitance afterporation in the presence of liposomes was observed for the 1and 15 min poration times. In the absence of liposomes, thecapacitance did not change significantly. As before, this increaseis interpreted as being due to the incorporation of the DOPCliposomes into the adsorbed layer. Using a poration time of 45min showed very different behavior. The octadecanol layer inthe absence of liposomes showed large changes in thecapacitance that resulted in a significantly more defective filmthat could not be recovered by waiting at 0 V/SCE. In thepresence of liposomes, the layer’s capacitance did not increasebeyond 2.5 μF cm−2 during poration, returning to reasonablevalues when the potential changed to 0 V/SCE. It seems that

liposomes became incorporated into the defects or adsorbedonto the electrode surface, thus stabilizing the adsorbed layer asexpected from the interaction of DOPC with Au(111) (Figure2). The adsorbed layers were further characterized viapotential-induced desorption using capacitance (Figure 5d−f).The shift in the desorption potential gives an indication of theincorporation of liposomes in the adsorbed layer, and coupledwith a stable, low capacitance, it will identify the best conditionsfor the creation of this hybrid layer. The desorption of thelayers created after poration at −0.4 V/SCE for 45 minindicates a strongly defective layer in the absence of liposomesbut less so when liposomes are present. The layers createdusing 1 and 15 min poration times show similar characteristics,with the 1 min poration showing a larger shift in the desorptionpotential that may indicate a greater liposome incorporation,though this is difficult to prove conclusively using onlycapacitance. Although capacitance is a useful measure of theaverage change in the nature of the adsorbed layer, it is not ableto determine changes in the adsorbed layer due to interactionwith liposomes. It is clear that the potential-induced defectscreate opportunities for liposome interaction/incorporationinto the adsorbed octadecanol layer, and a more detailedcharacterization of these adsorbed layers requires additional insitu methods.

Fluorescence Imaging of the Adsorbed Layer duringand after Poration. The interpretation of changes incapacitance due to potential-induced poration and the resultingliposome interaction suggest only liposome incorporation.From these electrochemical measurements, it is clear thatintroducing defects into the adsorbed layer with appropriatepotentials results in a change in the capacitance of the layeronly if liposomes are present in the electrolyte. These observedchanges may be due to a simple occupation of the potential-induced defect or the incorporation of liposomes into theadsorbed layer, which would result in subtle changes in thecapacitance minimum observed at 0 V/SCE. These changes arenot specific enough to provide information about the physicalnature of the liposome interaction. Fluorescence imaging of theelectrode−electrolyte interface during the poration process andthe desorption process can be used to characterize further thechanges occurring at the surface and provide evidence ofincorporation. It is important to note that the octadecanol-modified electrode is composed of two layers; the layer closestto the electrode surface (the layer most efficiently quenched) isoctadecanol with 3 mol % of a BODIPY-C19-OH fluorophore,and the second layer is only octadecanol. Liposomeincorporation is expected to be observed as an increase in thefluorescence signal. Therefore, characterization by in situfluorescence microscopy during the poration process and thesubsequent desorption of the layer were performed. Intransitioning to the in situ fluorescence measurements, changesin the technique were needed to accommodate the muchsmaller spectroelectrochemical cell. To decrease the timeneeded for liposomes to interact with the adsorbed layer, theconcentration of liposomes in the electrolyte was increased by50% to 45 μg/L DOPC. In addition, we found more variabilityin the capacitance of the deposited octadecanol layer, which ledto a higher variability in the interactions of liposomes withoctadecanol as well as a potentially higher degree ofincorporation when compared to the basic electrochemistrytrials. A number of poration studies were performed, andrepresentative data is presented. We found a much greatervariability in the fluorescence imaging results on the basis of the

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603352

area chosen for study when compared to the variation in theaverage 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 for60 min while at 0 V/SCE, allowing the injected liposomes todiffuse throughout the solution. As seen previously, thecapacitance of the interface did not change significantly duringthis time. In addition to capacitance, the interface was alsomonitored using fluorescence imaging. Trends in the averagefluorescence intensity and capacitance are presented in Figure6. In the control experiments (without liposomes), a slight

increase (curve 4) or no increase (curve 5) in fluorescence isseen over the hold time (less than 10% of the initial intensity)as explained by the possible reorganization of the adsorbedlayer during this waiting time that redistributes the fluorophorefrom the layer closest to the electrode surface to the layerfarthest from the electrode. Photobleaching is not evident,likely because the octadecanol layer remains adsorbed onto theelectrode surface and most fluorescence is quenched, reducingits effect, in addition to the low duty cycle used for imaging.Also noteworthy is the difference in the fluorescence intensitymeasured for these two seemingly similar adsorbed layers asdetermined from the capacitance. Included in the intensity is abackground signal that comes from a portion of the excitationlight that is reflected from the electrode surface and transmitsthrough the filters (an extremely small fraction of the incidentlight).In the presence of liposomes, a majority (about 60%) of the

adsorbed layers studied show a modest (10−15%) increase influorescence over time (curves 1 and 3 in Figure 6) withoutsignificant changes in capacitance. The combination of a steadycapacitance and weak fluorescence increase indicates thatliposomes are not interacting significantly with the adsorbedoctadecanol layer at this potential. In some cases (∼20% ofexperiments), even with a stable capacitance, the fluorescencewas found to increase (curve 2 in Figure 6). An analysis of the

images shows either a general increase in fluorescence evenlydistributed across the image or the creation of smallfluorescently intense regions. In the latter case, these layerswere abandoned as defective. The layers showing a uniformincrease in fluorescence were found to behave similarly to themajority of the data with regard to changes due to potentialperturbation and were further analyzed. In a few cases, thefluorescence and capacitance increased during the waiting time,clearly indicating liposome incorporation with existing defectsin the layer. These layers were also not analyzed further. Thelayers that demonstrated a stable capacitance during thiswaiting time were porated and studied. The increase influorescence can be explained as a reorganization of theadsorbed layer decreasing the quenching efficiency byincreasing the separation from the gold surface. The initialfluorescence intensity for the adsorbed layers also displayed awide 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. Previousfluorescence imaging studies36 have shown that floatingoctadecanol monolayers with 3 mol % of a lipophillicfluorophore, as used in this experiment, have a variety ofstructures and intensities that are retained when the layer istransferred to the electrode surface. Fluorescent images of anoctadecanol-modified electrode showed a variety of intensitiesand structures. The difference in intensity is a result of theamount of fluorophore present in the region analyzed, resultingin regions that were dark (devoid of fluorophore) andfluorescently intense regions. Other regions in the same layershowed greater inhomogeneity in the distribution of thefluorophore, yielding speckles or stripes of fluorescence. Theexact nature of these fluorescent structures as possiblemultilayers or aggregates is not yet understood, but theirpresence suggests that the adsorbed octadecanol layers are notcompletely uniform, possibly having defect sites with which theliposomes may interact. The electrode surface, at approximately0.26 cm2, will have deposited onto it several varieties of theseregions for a given deposition. Though all of these layers had asimilar initial capacitance, this is an average measure across theentire surface and may be insensitive to these types ofimperfections. 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 ascompared to the capacitance. It is probable that all layersinvestigated have these regions of fluorescence behavior,indistinguishable by capacitance, but what is seen influorescence depends on the region imaged. Because thefluorescence in the initially deposited layer is quenched, littleinformation on the layer structure is available when choosingthe imaging area. Liposomes may interact with any defectspresent by either slightly perturbing the octadecanol layer orleaching fluorophore into the phospholipid bilayer, whichwould result in a change in fluorescence dependent on thestructure of the region imaged. Any change in the adsorbedoctadecanol layer at 0 V/SCE is not large enough to alter thecapacitance value significantly, so incorporating the vesicles intothe octadecanol bilayer is unlikely, at least on a large scale. Onthe basis of this evidence, we believe that for most of theadsorbed layers analyzed the liposomes do not incorporate intothe octadecanol bilayers in the absence of defects and that theinteraction between the liposomes and adsorbed bilayer isminimal.

Figure 6. Average fluorescence image intensity (a) and capacitance (b)of adsorbed octadecanol layers on Au(111) during the waiting time at0 V/SCE in the presence of DOPC liposomes (curves 1−3) or in theabsence of liposomes (curves 4 and 5) in the electrolyte. These curvesillustrate representative examples of the behavior observed.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603353

Poration at −0.4 V/SCE for 1 Minute. As seen in theelectrochemical studies, a poration potential of −0.4 V/SCE foreither 1 or 15 min resulted in adsorbed layers that were slightlyhigher in capacitance and showed significant shifts in thepotential for the onset of desorption. These subtle changeswere interpreted as being due to the interaction of liposomeswith the potential-created defects in the adsorbed layer,modifying the adsorbed layer. To confirm the incorporationof the liposomes into the defective adsorbed layer, fluorescenceimages during the poration and subsequent annealing at −0.2and 0 V/SCE were performed. Many regions analyzedcontained fluorescent features that we have shown to be dueto the preferential segregation of the fluorophore into specificphases of the octadecanol layer.36 In addition, the presence ofsmall but highly intense regions of fluorescence were alsoobserved during adsorption. We believe that these aremultilayers of adsorbate that are present in the floating layerthat then become deposited onto the electrode surface.28,31,36

The areas around these regions are more characteristic ofadsorbed layers with dim, essentially quenched fluorescencewhen adsorbed. These intensely fluorescent regions of interest(ROIs) are analyzed separately and are shown as outlinedfeatures in the fluorescence images that follow.Figure 7 shows the capacitance and fluorescence determined

for three separate experiments where the adsorbed layer wastreated to a 1 min poration at −0.4 V/SCE procedure. In twoof these experiments, liposomes were added to the electrolyte.Also presented is an example of the control measurement madein the absence of liposomes, highlighting the variety ofstructures of the adsorbed layer. In many cases, the fluorescentimages observed were more uniform than this, with littlefluorescence variation across the image. The capacitance in the15 min before the potential excursion is similar for all threeexamples. The magnitude of the fluorescence intensity variesdepending on the region chosen for imaging, a consequence ofthe structure of the adsorbed layer discussed above. The intenseROIs were separated from the analysis, and the results areshown as dotted lines in Figure 7. During the first 15 min spentat 0 V/SCE (after the initial hour at 0 V/SCE during liposomedispersion), the fluorescence decreases because of photo-bleaching and is more significant for the intense ROIs. Becausethese ROIs are thought to be structures in the adsorbed layerthat are not efficiently quenched (e.g., multilayers sitting higheror away from the surface), they therefore would have a greaterchance of photodegrading. This overall decrease in intensity isin contrast to the results presented in Figure 5 where thefluorescence was found to increase slightly. During the 15 mindescribed here, the layer was subjected to more frequentperiods of illumination (2.5 s of every 5 s) compared to thatoccurring in the first hour (5 s of every 60 s), sophotobleaching 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 examplesbecause of a change in layer structure, as seen in theelectrochemical studies above. In the absence of liposomes,the increase was small, and the capacitance returned to valuesslightly higher than the initial value after annealing for 15 mineach at −0.2 V and 0 V/SCE. A very similar behavior is alsoobserved in the presence of liposomes, but the adsorbed layerafter poration has a larger capacitance. Larger changes incapacitance were also observed in the presence of liposomes(curve 2), though the layer did return to capacitance valuessimilar to those expected after liposome interaction. The

changes in fluorescence during this poration procedure showthat the liposome-free control essentially displayed a constantdecrease in intensity (more quickly for the intense ROIs) andvery little potential dependence. The two examples in thepresence of liposomes show different responses, illustrating thatthe changes observed can be significantly different dependingon the region chosen for analysis. The increase in fluorescenceobserved when the potential changes to −0.4 V/SCE is smallbut significant, indicating the quick interaction of liposomeswith 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 increasewhile the potential remains at −0.2 V/SCE. Changing again to0 V/SCE results in an immediate decrease in fluorescenceintensity, followed by a smaller increase over 15 min. Thesechanges in fluorescence suggest that the liposomes havedisrupted the adsorbed layer in such a way as to increase theseparation of fluorophore from the electrode surface. We haveshown that changes of 10−20 nm in this separation can resultin a significant increase in fluorescence due to decreasedquenching efficiency.29 The changes observed during porationare small and indicate that the liposomes may not be fullyincorporated into the annealed adsorbed layer or that few

Figure 7. Poration of the adsorbed octadecanol/3 mol % layer using 1min 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 fluorescenceimaging. (a) Capacitance and fluorescence intensity for adsorbedlayers undergoing poration without and with DOPC liposomes in theelectrolyte. The accompanying fluorescent images in part b show theROIs used in the analysis. Shown are the changes in fluorescenceintensity for the ROIs (thin lines) and the other parts of the images(thick lines). (b) Fluorescence images for the three experiments takenat the times indicated in part a. Top row: with liposomes (1). Middlerow: with liposomes (2). Bottom row: without liposomes. The scalebar is 20 μm.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603354

fluorophores from the adsorbed layer have diffused into theadsorbed lipid regions.A selection of fluorescent images during the poration

procedure are shown for all three layers in Figure 7b. Thechanges in fluorescence are not uniform across the surface areaimaged but are located in particular regions, illustrating that theadsorbed layer may have specific structures that are moreamenable to poration and liposome interaction. The increase influorescence clearly shows that the adsorbed layer is changingits structure and supports the interaction of liposomes with theadsorbed layer because these changes are not solely due to thepotential perturbation of the adsorbed octadecanol layer. Theincrease in fluorescence is also not located around the ROIs,which suggest that the defects created through the potential

perturbation are not near the regions of high fluorescenceoutlined even though these regions would be considered to be anonideally organized region or a possible defect.As demonstrated in the electrochemical studies, the

interaction of liposomes with the adsorbed layer influencesthe potential-induced desorption, moving the onset ofdesorption to less-negative values. This process was alsostudied with in situ fluorescence microscopy and reveals moreabout the adsorbed layer structure because the fluorescenceintensity is higher as a result of the displacement of the organiclayer away from the gold surface. In the absence of liposomes,the capacitance (Figure 10a) increases at −0.6 V/SCE to avalue characteristic of a water-covered electrode surface at −0.8V/SCE, indicating a complete displacement of the layer off of

Figure 8. Capacitance and fluorescence characterization of an adsorbed layer during a potential desorption step experiment after poration for 1 minat −0.4 V/SCE in the absence or the presence of liposomes in the electrolyte. (a) Capacitance and fluorescence changes for two octadecanol-coatedelectrodes 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 thechange 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 potentialsduring desorption are also shown for the three experiments. Images in the bottom row are for a layer porated without liposomes in solution; the toptwo rows are for layers that were porated in the presence of liposomes. The images are falsely colored. The scale bar is 20 μm. (b) Fluorescenceimages in part a after rolling ball background subtraction. The images are falsely colored with a scale that is proportional to the net change inintensity due to desorption.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603355

the electrode surface. The fluorescence also increases to amaximum at this potential, somewhat following the capacitancechange. The change in the fluorescence with potential is evenlydistributed across the image, with the fluorescence from themore intense ROIs behaving in a similar fashion. Thefluorescence intensity is a complex function of the amount ofdye present in the adsorbed layer, the distribution of dye awayfrom the electrode surface, and any other processes that couldchange the fluorescence intensity (e.g., BODIPY is known toform dimers36,46−48). The rapid increase in fluorescence over asmall range of potential indicates that the layer respondsuniformly to potential and that the adsorbed layer does notshow strong heterogeneity in its structure normal to theelectrode surface (except possibly the ROIs).The desorption of the modified layers after exposure to

liposomes and the poration procedure shows significantdifferences in the potential dependence of fluorescence andits increase in intensity. In both examples, the onset ofdesorption occurs at −0.45 V/SCE, coincident with an increasein fluorescence. The adsorbed layer at 0 V/SCE is characteristicof a well-formed layer with a low capacitance and a smallfluorescence signal as seen in Figure 8. The increase incapacitance and fluorescence at a less negative potential thanthe unmodified layer suggests that the layer has interacted withliposomes, changing the layer organization and enablingdesorption at less-negative potentials. The increased intensityat desorption is indicative of a modified layer structure wheremore of the fluorophore is further from the electrode surface,reducing quenching and increasing the signal. These fluorescentimages also show a more nonuniform structure, highlighting theregions furthest from the electrode surface.The layer is desorbed at the negative scan limit, with

fluorescence significantly increasing and displaying a maximumat −0.65 V/SCE in one case and a constant increase until −0.8V/SCE in the other. The intensity increases observed for thesemodified layers are significantly larger than the control layers.This large difference can be explained by variations in the initialamount of fluorophore present in the adsorbed layer or is dueto a significant change in the thickness/structure of theadsorbed layer, suggesting the incorporation of liposomescreating adsorbed layers with structure normal to the electrodeplane (e.g., 3-D structures). The maximum in fluorescenceduring desorption has been observed in our previous work,resulting from a change in the fluorophore organization such asdimer formation.49

The large changes in fluorescence intensity observed duringdesorption can mask smaller details that are important in thisanalysis because the liposomes may incorporate into the layerin particular regions and the influence in structure may remainlocalized. Analyzing for these small features can be accom-plished by removing the featureless background through arolling ball background subtraction (with a 50 pixel (14 μm)radius). This was performed on the fluorescent imagespresented in Figure 8b and shown in Figure 8c. For the layerthat was not exposed to liposomes, the number of features atthe adsorption potential are the same as at the desorptionpotential, although they increase in size. Some subtle changes inthe underlying structure are also noted. These features wereinitially present in the adsorbed layer (shown as outlinedregions in the images) and become easily observable when thelayer is desorbed because of the decrease in quenchingexperienced by the fluorophore. In contrast, most of thefeatures seen in the fluorescent image of the two layers after

interaction with the liposomes come not from the initiallydeposited structure but are a result of interaction with theliposomes. Also present in this analysis are large regions thatalso show increased fluorescence after liposome interaction,which correlate well with the larger changes in fluorescenceobserved in the unprocessed images. In both of the examplesthat were exposed to liposomes and the poration procedure, thenumber of small fluorescent regions also changed with theapplied potential. This is expected because any regions that arefurther from the electrode will increase more rapidly influorescence as a result of the nonlinear quenching withdistance29,50 when the layers are farther away from theelectrode surface. These small features (∼5 pixels in diameter,∼2 μm) are much larger than an individual liposome and socannot be assigned to one specific interaction event.Importantly, a large majority of these features are not presentin the initially adsorbed layer before interaction with liposomes(outlined ROIs), and features of this type are not observed inthe analysis of the layers that were not exposed to liposomesduring the poration procedure. Moreover, a distinct differencein the number of these features is evident when comparing thetwo layers that were exposed to liposomes, indicating that thepotential-controlled liposome interaction is dependent on thestructure of the adsorbed layer, which is not uniform across theelectrode surface.

Poration at −0.4 V/SCE for 15 Minutes. The changesobserved in the adsorbed layer due to the interaction ofliposomes should depend on the time spent at the porationpotential (−0.4 V/SCE), resulting in an increased possibility ofliposome interaction. From the electrochemical measurements,increasing the time from 1 to 15 min resulted in adsorbed layersthat were still intact, with capacitance values that did notchange significantly after the poration process unless liposomeswere present in the subphase. Also, the desorption of theadsorbed layer showed a shift in the potential of the onset ofdesorption that suggested a change in the adsorbed layer afterinteraction with liposomes. The capacitance, fluorescenceintensity, and images of the adsorbed layer during the 15 minporation process in the absence and presence of liposomes areshown in Figure 9. The capacitance changes due to poration arelarger than observed in the electrochemical measurements, butthe trends are similar. In the absence of liposomes, theadsorbed layer capacitance increases when the potential ischanged to −0.4 V/SCE, and a sharp increase in capacitance isalso observed about 7 min after the move to −0.4 V/SCE. Thiscan be explained as a change in the layer, but because nochange in the fluorescence intensity was seen (for the visibleregion), it is more likely due to a change in the wetting of thesides of the electrode held in a hanging meniscus. Thecapacitance decreases during the time at −0.2 V/SCE and whenreturning to 0 V/SCE to a value that is slightly above itsstarting value. In the absence of liposomes in the electrolyte,the adsorbed layer did not experience significant changesduring this extended poration process. The fluorescenceintensity decreases because of photobleaching, with the more-intense ROIs photobleaching more quickly. Small increases areobserved at each change in potential, which is expected becausethe layer will slightly change its organization at these potentials,similar to the behavior in Figure 8. The origin of these smallchanges could be a displacement of the fluorophore to theouter surface of the adsorbed layer, but the change influorescence intensity is much smaller than in the presence ofliposomes.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603356

In the presence of liposomes, the adsorbed layer showedmore distinctive changes, similar to the layers exposed to the 1min poration time. On decreasing the potential to −0.4 V/SCE,the capacitance increases dramatically and significant jumps incapacitance are observed during the 15 min at the porationpotential. Similar events were observed for organic dropletadsorption and bursting onto a bare Hg drop11 but cannot beused to explain these changes because the electrode surface iscoated with an organic layer. The capacitance decreases whenthe potential is changed to −0.2 and 0 V/SCE, resulting in anadsorbed layer that has a capacitance of 5 μF/cm2, a largerchange than in the electrochemical measurements. It must beremembered 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 ofliposomes. The initial time spent at 0 V/SCE showed typicalfluorescence bleaching (images in columns A and B in Figure9). Changing to −0.4 V/SCE, the fluorescence intensityincreased uniformly across the image. The fluorescencecontinues to increase when the potential changes to −0.2 and0 V/SCE, again distributed mostly evenly throughout the imagebut with some larger increases seen on the right side of the lastimage. After the liposomes have had sufficient time to interactwith the adsorbed layer, fluorophore may diffuse into theirstructures, resulting in an increase in fluorescence with time. A

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 notinteract with liposomes was very similar to that shown for the 1min poration at −0.4 V/SCE (Figure 8). Similarly, thisindicates that the poration process did not significantly perturbthe adsorbed layer in the absence of liposomes, even though thecapacitance increased slightly in the poration process. Thedesorption of the modified layer in the presence of liposomeswas very different. The capacitance shows the onset ofdesorption at −0.45 V/SCE, with desorption attained by−0.8 V/SCE, as signified by the final capacitance value, similarto that for a water-covered electrode. The fluorescence alsoincreased 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 tothe features that are furthest from the electrode surface becausethese features produce the largest fluorescence signals: smallchanges in the distance from the electrode result in largechanges in the fluorescence. This indicates that features of themodified layer are indeed farther from the electrode surface.Capacitance increases are slight at this potential and do notmirror the large increase in fluorescence because thecapacitance is most sensitive to the dielectric changes on thesurface and less so to changes further from the surface. Afurther increase in fluorescence is also observed up to thedesorption potential. It is important to note that the changes influorescence observed are dependent on the area chosen tostudy, so a direct comparison between the 1 and 15 min resultsis 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 asmaller scale, which can be revealed by treating the images to abackground subtraction routine as described above for the 1min poration case. A comparison of the changes in the smallfeatures observed in the absence of liposome interaction and inthe presence of liposomes is shown in Figure 10. The outlinedfeatures are regions of higher fluorescence that were observedat the start of the poration experiment (e.g., at −15 min). Thelayer that was not exposed to liposomes shows very fewincreases in the features after poration, annealing, anddesorption. In contrast, the layer that was exposed to liposomesthrough the poration procedure displays a significant number ofthese features, far above that observed before the porationprocess. When compared to the 1 min poration studies, thesefeatures are similar, with a density that is between that of thetwo 1 min poration examples. Even though it was expected thatthe time allowed for liposome incorporation should increasethe number of features observed as well as the fluorescenceintensity, the small region chosen for analysis has a largeinfluence on the extent of liposome interaction andincorporation observed, making a comparative analysis difficult.

■ CONCLUSIONSThe interaction of liposomes with a lipid-coated metal surfacewas studied using electrochemical and in situ fluorescencemicroscopy. It was demonstrated that control over theliposome interaction with a physisorbed layer on Au(111)was achieved using the electrical potential for adsorbed layersthat contain few initial defects. The liposome interaction withthe lipid-modified electrode surface depends on the potential-controlled creation of defects in the adsorbed layer. These

Figure 9. Poration of the adsorbed octadecanol/3 mol % layer using15 min at −0.4 V/SCE and annealing at −0.2 and 0 V/SCE for 15 mineach, as monitored with capacitance and fluorescence imaging. (a)Capacitance and fluorescence intensity for adsorbed layers undergoingporation without and with DOPC liposomes in the electrolyte. Theaccompanying fluorescent images in part b show the ROIs used in theanalysis. 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 timesindicated in part a. Top row: with liposomes. Bottom row: withoutliposomes. The scale bar is 20 μm.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603357

defects cannot be so extensive as to render the adsorbed layerunstable, and a limited potential range was found where theinteraction is stable and sufficient so as to be observed. Thepotential-dependent incorporation of liposomes is shown usinga combined electrochemical and fluorescence method. In thecases where liposomes are present in the electrolyte, the smallincreases in the adsorbed layer capacitance after the porationprocedure coincide with increases in fluorescence due to aredistribution of fluorophore after liposome interaction. Theincorporation of liposomes into the adsorbed layer was foundto change the desorption potential of the adsorbed layer,desorbing at less negative potentials because of the defectscreated through liposome interaction. Fluorescence imaging ofthe desorption process reveals the presence of small structuresthat may be regions where liposomes are incorporated. These

structures are strongly influenced by the initial quality or natureof the layer deposited, which was found to be neither uniformnor homogeneous. Further work will require the use of a morehomogeneous adsorbed layer so as to interact with liposomesmore uniformly across the interface. In progress are in situAFM studies of the nature of the features observed afterliposome interaction.

■ ASSOCIATED CONTENT

*S Supporting InformationSynthesis of 4,4-difluoro-1,3,5,7-tetramethyl-8-(18-octadeca-nol)-4-bora-3a,4a-diaza-s-indacene (BODIPY-C19-OH).

This material is available free of charge via the Internet athttp://pubs.acs.org/.

Figure 10. Capacitance and fluorescence characterization during a potential desorption step experiment of an adsorbed layer after poration for 15min 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 15 min at −0.4 V/SCE with liposomes in the electrolyte (solid) and without liposomes (dashed). The thin linesrepresent 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 forpotentials during the desorption of the adsorbed layer are also shown for the three experiments. The bottom row shows images from the layer thatwas porated without liposomes in solution, and the top two rows show images for a layer that was porated in the presence of liposomes. The imagesare falsely colored, and the color scale used is shown. The scale bar is 20 μm. (b) Fluorescence images in part a after rolling ball backgroundsubtraction. The images are falsely colored using the same lookup table and a scale that is proportional to the net change in intensity due todesorption.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603358

■ AUTHOR INFORMATIONCorresponding Author*E-mail: bizzotto@chem.ubc.ca.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe spectroelectrochemical cell was created by Brian Ditchburn(UBC, chemistry, glassblowing), for which we are indebted.A.M. acknowledges support from the Agnes and Gilbert HooleyScholarship in Chemistry (UBC). This research was funded byNSERC (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 bilayersmodified by Au nanoparticles. Biosens. Bioelectron. 2010, 25, 2253−2258.(3) Merz, C.; Knoll, W.; Textor, M.; Reimhult, E. Formation ofsupported bacterial lipid membrane mimics. Biointerphases 2008, 3,FA41−FA50.(4) Ye Fang, B. W.; Hong, Y.; Lahiri, J. Applications ofbiomembranes in drug discovery. MRS Bull. 2006, 31, 541−545.(5) McConnell, H.; Watts, T.; Weis, R.; Brian, A. Supported planarmembranes 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 phospholipidmolecules at a Au(111) electrode surface. J. Am. Chem. Soc. 2004, 126,12276−12277.(7) Reviakine, I.; Brisson, A. Formation of supported phospholipidbilayers from unilamellar vesicles investigated by atomic forcemicroscopy. Langmuir 2000, 16, 1806−1815.(8) Schonherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S.G. Vesicle adsorption and lipid bilayer formation on glass studied byatomic force microscopy. Langmuir 2004, 20, 11600−11606.(9) Ivosevic, N.; Tomaic, J.; Zutic, V. Organic droplets at anelectrified interface: critical potentials of wetting measured bypolarography. Langmuir 1994, 10, 2415−2418.(10) Ivosevic, N.; Zutic, V. Spreading and detachment of organicdroplets at an electrified interface. Langmuir 1998, 14, 231−234.(11) Denardis, N. I.; Zutic, V.; Svetliccic, V.; Frkanec, R. Adhesionsignals of phospholipid vesicles at an electrified interface. J. Membr.Biol. 2012, 245, 573−582.(12) Hellberg, D.; Scholz, F.; Schauer, F.; Weitschies, W. Burstingand spreading of liposomes on the surface of a static mercury dropelectrode. Electrochem. Commun. 2002, 4, 305−309.(13) Hellberg, D.; Scholz, F.; Schubert, F.; Lovric, M.; Omanovic, D.;Hernandez, V. A.; Thede, R. Kinetics of liposome adhesion on amercury electrode. J. Phys. Chem. B 2005, 109, 14715−14726.(14) Agmo Hernandez, V.; Scholz, F. Kinetics of the adhesion ofDMPC liposomes on a mercury electrode. effect of lamellarity, phasecomposition, size and curvature of liposomes, and presence of the poreforming peptide mastoparan. Langmuir 2006, 22, 10723−10731.(15) Nelson, A.; Auffret, N.; Borlakoglu, J. Interaction of hydro-phobic organic compounds with mercury adsorbed dioleoylphospha-tidylcholine monolayers. Biochim. Biophys. Acta 1990, 1021, 205−216.(16) Nelson, A. Electrochemical studies of thallium(I) transportacross gramicidin modified electrode-adsorbed phospholipid mono-layers. 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 toubiquinone-10 in a self-assembled phospholipid monolayer. Biophys.J. 1996, 70, 2716−2726.

(18) Nelson, A.; Bizzotto, D. Chronoamperometric study of Tl(I)reduction at gramicidin-modified phospholipid-coated mercuryelectrodes. Langmuir 1999, 15, 7031−7039.(19) Bizzotto, D.; Nelson, A. Continuing electrochemical studies ofphospholipid monolayers of dioleoyl phosphatidylcholine at themercury-electrolyte interface. Langmuir 1998, 14, 6269−6273.(20) Rueda, M.; Navarro, I.; Ramirez, G.; Prieto, F.; Nelson, A.Impedance measurements with phospholipid-coated mercury electro-des. 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 throughgramicidin channels in self-assembled gramicidinmodi fied dioleoyl-phosphatidylcholine monolayers on mercury electrodes. Langmuir1999, 15, 3672−3678.(22) Becucci, L.; Moncelli, M. R.; Herrero, R.; Guidelli, R. Dipolepotentials 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-assembledphosphatidylcholine and octadecanethiol monolayers and octadecane-thiol/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 onmercury. Solid State Ionics 2002, 150, 13−26.(25) Becucci, L.; Moncelli, M. R.; Guidelli, R. Ion carriers andchannels in metal-supported lipid bilayers as probes of transmembraneand dipole potentials. Langmuir 2003, 19, 3386−3392.(26) Stoodley, R.; Bizzotto, D. Epi-fluorescence microscopiccharacterization of potential-induced changes in a DOPC monolayeron a Hg drop. Analyst 2003, 128, 552−561.(27) Agak, J. O.; Stoodley, R.; Retter, U.; Bizzotto, D. On theimpedance of a lipid-modified Hg-electrolyte interface. J. Electroanal.Chem. 2004, 562, 135−144.(28) Shepherd, J.; Yang, Y.; Bizzotto, D. Visualization of potentialinduced formation of water insoluble surfactant aggregates by epi-fiuorescence 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. Electrochemicaland spectroelectrochemical characterization of lipid organization in anelectricfield. J. Electroanal. Chem. 2004, 574, 167−184.(30) Zawisza, I.; Lipkowski, J. Layer by Layer characterization of 1-octadecanol films on a Au(111) electrode surface. in situ polarizationmodulation infrared reflection absorption spectroscopy and electro-chemical studies. Langmuir 2004, 20, 4579−4589.(31) Shepherd, J. L.; Bizzotto, D. Characterization of mixed alcoholmonolayers adsorbed onto a Au(111) electrode using electro-fluorescence microscopy. Langmuir 2006, 22, 4869−4876.(32) Weaver, J. C.; Chizmadzhev, Y. A. Theory of electroporation: areview. Bioelectrochem. Bioenerg. 1996, 41, 135−160.(33) Bizzotto, D.; Pettinger, B. Fluorescence imaging studies of theelectrochemical adsorption/desorption of octadecanol. Langmuir1999, 15, 8309−8314.(34) Mayer, L.; Hope, M.; Cullis, P. Vesicles of variable sizesproduced by a rapid extrusion procedure. Biochim. Biophys. Acta 1986,858, 161−168.(35) Marathe, D.; Mishra, K. P. Radiation-induced changes inpermeability in unilamellar phospholipid liposomes. Radiat. Res. 2002,157, 685−692.(36) Casanova-Moreno, J.; Bizzotto, D. Electrochemistry and in situfluorescence microscopy of octadecanol layers doped with a BODIPY-labeled phospholipid: Investigating an adsorbed heterogeneous layer. J.Electroanal. Chem. 2010, 649, 126−135.(37) Zawisza, I.; Bin, X.; Lipkowski, J. Spectroelectrochemical studiesof 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 polarizationmodulation infrared reflection absorption spectroscopy study of the

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603359

electric field driven transformations of a phospholipid bilayersupported at a gold electrode surface. Biophys. J. 2003, 85, 4055−4075.(39) Buess-Herman, C. Non-faradaic phase transition in adsorbedorganic layers part I. General theory. J. Electroanal. Chem. 1985, 186,27−40.(40) Buess-Herman, C. Non-faradaic phase transition in adsorbedorganic layers part II. Experiemental aspects. J. Electroanal. Chem.1985, 186, 41−50.(41) Moncelli, M.; Guidelli, R. Test of Gouy-Chapman theory onphosphatidylcholine-coated mercury from differential capacity meas-urements. J. Electroanal. Chem. 1992, 326, 331−338.(42) Becucci, L.; Moncelli, M. R.; Guidelli, R.; Kelley, D. Surfacecharge density measurements on mercury electrodes covered byphospholipid monolayers. J. Electroanal. Chem. 1998, 413, 187−193.(43) Stauffer, V.; Stoodley, R.; Agak, J. O.; Bizzotto, D. Adsorption ofDOPC onto Hg from the G S interface and from a liposomalsuspension. J. Electroanal. Chem. 2001, 516, 73−82.(44) Becucci, L.; Moncelli, M. R.; Guidelli, R. Thallous ionmovements through gramicidin channels incorporated in lipidmonolayers supported by mercury. Biophys. J. 2002, 82, 852−864.(45) Bizzotto, D.; Wong, E.; Yang, Y. Adsorption of octadecanol/1-pyrenenonanol mixed monolayers of insoluble surfactant ontoAu(111): an electrochemical study. J. Electroanal. Chem. 2000, 480,233−240.(46) Tleugabulova, D.; Zhang, Z.; Brennan, J. D. Characterization ofbodipy 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 andbiology. J. Am. Chem. Soc. 2002, 124, 196−204.(48) Musgrove, A.; Kell, A.; Bizzotto, D. Fluorescence imaging of theoxidative desorption of a BODIPY-alkyl-thiol monolayer coated Aubead. Langmuir 2008, 24, 7881−7888.(49) Shepherd, J. L.; Bizzotto, D. On the apparent fluorescencerecovery due to electrosorption. J. Phys. Chem. B 2003, 107, 8524−8531.(50) Chance, R. R.; Miller, A. H.; Prock, A.; Silbey, R. Fluorescenceand energy transfer near interfaces: the complete and quantitativedescription of the Eu+3/mirror systems. J. Chem. Phys. 1975, 63, 1589−1595.

Langmuir Article

dx.doi.org/10.1021/la400042c | Langmuir 2013, 29, 3347−33603360