fusion of small unilamellar vesicles onto laterally mixed self-assembled monolayers of...

Journal of Colloid and Interface Science 258 (2003) 298–309www.elsevier.com/locate/jcis

Fusion of small unilamellar vesicles onto laterally mixed self-assembledmonolayers of thiolipopeptides

T. Baumgart,a M. Kreiter,a H. Lauer,a R. Naumann,a,∗ G. Jung,b A. Jonczyk,c

A. Offenhäusser,a and W. Knolla

a Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germanyb University of Tübingen, D-72076 Tübingen, Germany

c Merck KGaA, D-64271 Darmstadt, Germany

Received 6 December 2001; accepted 28 October 2002

Abstract

Monolayers of the thiolipopeptide NH2–Cys–Ala–Ser–Ala–Ala–Ser–Ser–Ala–Pro–Ser–Ser–(Myr)Lys(Myr)–OH (III) were formed ongold surfaces by self-assembly, mixed with a lateral spacer of the same peptide composition, NH2–Cys–Ala–Ser–Ala–Ala–Ser–Ser–Ala–Pro–Ser–Ser–Lys–OH (I). Different mixing ratios were employed ranging from 0.1 to 1, corresponding to 10–100% thiolipopeptide. Theseself-assembled monolayers (SAMs) were then exposed to a suspension of liposomes with the aim of forming lipid bilayers as a function ofthe mixing ratio. A clear optimum with respect to homogeneity and electrical properties of the membranes was obtained in the middle region(0.5) of mixing ratio, as revealed by surface plasmon resonance spectroscopy, impedance spectroscopy, and fluorescence microscopy. Thecombination of these methods was shown to be a powerful tool, although a true lipid bilayer was not obtained. Instead, vesicle adsorptionwas shown to be the predominant process, and FRAP (fluorescence recovery after photobleaching) measurements showed that the films werenot fluid on the micrometer length scale. 2003 Elsevier Science (USA). All rights reserved.

Keywords:Solid-supported lipid bilayers; Solid-supported lipid membranes; Tethered membranes; Thiolipopeptide; Thiopeptide; Surface plasmon resonancespectroscopy; Impedance spectroscopy; Fluorescence microscopy

Abbreviations:SAM = self assembled monolayer; SPS= surface plasmon resonance spectroscopy; IS= impedance spectroscopy;PC= phosphatidylcholine; NBD-PE= N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine;Triethylammonium salt; AFM= atomic force microscopy; FRAP= fluorescence recovery after photo bleaching

1. Introduction

Supported lipid bilayers on gold or other metal films area promising new model of the biological membrane [1,2].The ability of these bilayers for incorporating ion channelsand other complex membrane proteins opens new possibil-ities for investigating signal transduction pathways by bothelectrical and optical methods [3]. In order to accomplishthis goal, strategies have been developed to form supportedlipid bilayers by vesicle fusion onto self assembled mono-layers (SAMs) of pure [4–11] and mixed thiolipids [12–14].Two sorts of hydrophilic spacers have been employed: (i)vertical spacers covalently attached to the lipid thus separat-

* Corresponding author.E-mail address:[email protected] (R. Naumann).

ing the lipid from the solid, and (ii) lateral spacers providingan aqueous layer to allow for the insertion of fluid lipids inbetween, thus providing self-healing properties to the lipidmembrane [15]. Short lateral spacers have been used so farsuch as mercaptoethanol [12,13] or thiodioxyethylenegly-col [14].

In the case of SAMs formed from thiolipopeptides, theyproved to form lipid bilayers incorporating H+-ATPasefrom chloroplasts but only if they were diluted by mercap-toethanol as a lateral spacer [16]. Proton transport across thelipid film by this protein was demonstrated by impedancespectroscopy, although fluorescence methods had revealedthat the films were very inhomogeneous. It was concludedfrom these experiments that lateral spacers the same size asthe vertical spacers could improve the films in terms of ho-mogeneity. Therefore, we mixed the thiolipopeptide, instead

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T. Baumgart et al. / Journal of Colloid and Interface Science 258 (2003) 298–309 299

of mercaptoethanol, with the thiopeptide of the same chem-ical composition as the lipopeptide. The aim was to followthe formation of lipid bilayers by vesicle fusion as a functionof mixing ratio.

Fluorescence microscopy had proved previously to bevery useful to investigate lipid bilayers with respect to ho-mogeneity and fluidity, but mainly on glass [17] and silica[18] substrates that are not amenable to electrical measure-ments. In the earlier investigation these two approaches hadbeen combined, applying fluorescence microscopy togetherwith impedance spectroscopy (IS) to metal substrates [16].Here we present the first systematic study in which we usea combination of these two methods with surface plasmonresonance spectroscopy (SPS) applied to the same sample.Different from our previous studies, we took advantage ofgrating coupled SPS [19], a technique applicable not onlyto a wide variety of film thicknesses but also down to smallsurface areas.

2. Materials and methods

2.1. Materials

Egg PC (phosphatidylcholine) was purchased from Avan-ti Polar Lipids, Inc. Alabaster, USA.N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine, triethylammonium salt (NBD-PE), was fromMolecular Probes, Eugene, OR. KCl, tricine, Na2HPO4,MgSO4 was analytical grade from Riedel de Haen, Seelze,Germany. Hellmanex was from Hellma GmbH, Germany.Trifluoroacetic acid was from Fluka, Buchs, Switzerland.Diisopropylcarbodiimide,N -hydroxybenzotriazole, and di-methylformamide, dichloromethane were from Sigma-Ald-rich Co. Fmoc-Ala, Fmoc-Ser(tBu), Fmoc-Pro, and Fmoc-Lys were from NovaBiochem, Heidelberg.

2.2. Preparation of the thiolipopeptide

The thiopeptide NH2–Cys–Ala–Ser–Ala–Ala–Ser–Ser–Ala–Pro–Ser–Ser–Lys–OH (I), a slight variation of thiopep-tide NH2–Cys–Ala–Ser–Ala–Ala–Ser–Ser–Ala–Pro–Ser–Ser–H (II), described earlier [16], and the thiolipopeptideNH2–Cys–Ala–Ser–Ala–Ala–Ser–Ser–Ala–Pro–Ser–Ser–(Myr)Lys(Myr)–OH (III) were prepared according to the fol-lowing procedure:

The dodecapeptide amide (I) was built up on Rink amideresin (1140 mg) loaded with the C-terminal Fmoc-Cys (Trt)(loading 0.23 mmol/g; Novabiochem) using an Fmoc (9-fluorenyl-methoxycarbonyl) solid-phase peptide synthesisprotocol on the automated multiple peptide synthesizer RSP5032 Tecan (Hombrechtlikon, Switzerland) with SoftwareSyro 1.05 from MultiSynTech (Bochum).

Fmoc-Ala, Fmoc-Ser(tBu), Fmoc-Pro, and Fmoc-Lys(Fmoc, NovaBiochem, Heidelberg) were coupled with diiso-propylcarbodiimide/N -hydroxybenzotriazole in dimethyl-

formamide/dichloromethane (1: 3, v/v) for 70 min using a10-fold excess of each amino acid. Cleavage of the Fmocgroups was done with piperidine/dimethylformamide (1: 4,v/v) for 2 × 10 min. Washes were done with dimethylfor-mamide (8× each) after couplings and Fmoc deprotection;final washes were with methanol(3×). After drying in vacuothe peptidyl resin was divided into two parts.

One part (about 2/3) was used for the cleavage of do-decapeptide amide I from the resin by reagent K (triflu-oroacetic acid/thioanisol/ethanedithiol/phenyl/water, 82.5 :5 : 2 : 5 : 5 : 5) for 3 h. After filtration of the cleavage solu-tion the peptide was precipitated in diethylether at−20◦C.After repeated washings with diethylether the peptide amidewas lyophilized from tert-butyl alcohol/water(4 : 1). Yield:520 mg; HPLC: purity 85% (RP-C18, 214 mn); ES-MS:Mcalc= 1064, Mexp= 1065.6[M + H]+, and[M + 2H]2+ =1066.64. The other part (about 1/3) of the peptidyl resin waslipidated after removal of the two Fmoc protecting groupsfrom the N -terminal lysineNα- and Nε-amino groups.Myristoic acid (18 eq.) was coupled using diisopropylcar-bodiimide (18 eq.) in dimethylformamide/dichloromethane(1 : 1, v/v) and diisopropylethylamine (6 eq.) for 3 h. Af-ter simultaneous cleavage of the side chain protectingand the amide anchor groups with reagent K for 3 hthe resin was filtered off. After concentration of the fil-trate the lipopeptide amide III was precipitated in colddiethylether. Repeated precipitations from trifluoroaceticacid/dichloromethane(1 : 3) with diethylether gave a color-less product (275 mg) which was characterized by ES–MS:Mcalc = 1485, Mexp = 1485.97[M + H]+. Attempts to per-form HPLC for purposes of either analysis or purificationproved unsuccessful, presumably due to the poor solubilityof the product in solvents other than trifluoroacetic acid.

2.3. Preparation of the thiolipopeptide monolayers

Silica substrates covered by gold films (see below forthe preparation) were used either directly following goldevaporation or after treatment with Piranha solution (sul-furic acid, 96%, hydrogen peroxide, 30%, 3: 1 (v/v)) for5 min, thorough rinsing with ultrapure MilliQ water and dry-ing in a stream of nitrogen. (Piranha solution should be usedonly very carefully, avoiding as much as possible contam-ination with organic material.) Immediately thereafter sub-strates were incubated in a solution of the thiopeptide (I) (at aconstant concentration of 0.2 mgml−1) and the thiolipopep-tide (III) (concentration ranging from 0.02 to 0.2 mgml−1)in trifluoroacetic acid (TFA) mixed with 5% water for 65 h,rinsed thoroughly with TFA and ethanol, and dried in astream of nitrogen.

2.4. Vesicle fusion

Liposomes were prepared from egg PC (phosphatidyl-choline) mixed with 1 mol% ofN -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoetha-

300 T. Baumgart et al. / Journal of Colloid and Interface Science 258 (2003) 298–309

Fig. 1. Two alternative methods of exciting a surface plasmon at themetal/dielectric interface with light. In the upper part, the wave vectorsinvolved in grating-coupling are shown. In the lower part, the surfaceplasmon is resonantly excited in the Kretschmann configuration. See thetext for further details.

nolamine, triethylammonium salt (NBD-PE; MolecularProbes, Eugene, OR), by extrusion through 50-nm filters.The extruder was from Milsch Equipment, Laudenbach,Germany, who provided us with the procedures used for thepreparation of liposomes. Substrates with lipid monolayerswere incubated at 30◦C in suspensions of these liposomesin buffer solution (KCl 0.1 mol l−1, tricine 0.01 mol l−1,Na2HPO4 0.01 mol l−1, MgSO4 0.0002 mol l−1, pH= 7.4).

2.5. Fluorescence microscopy

Fluorescence was observed by an inverted optical micro-scope (Olympus IX-70). The sample was illuminated at awavelength ofλ = 490 nm by means of a high-pressuremercury burner (HBO-100) in combination with an inter-ference filter. Emission was detected atλ = 540 nm by alight-enhancing camera (extended ISIS, Photonic Sciences)and digitized by a frame grabber card (AG-5, Scion, USA).

Fluidity of the samples was tested by illuminating a spotof diameter approximately 12 µm using the field stop withthe whole spectrum of the mercury lamp for 5 min. Alterna-tively, bleaching was performed by illuminating the samplewith a strong laser beam (spot size 4.2 µm) for 40 ms [20].

By these procedures, the fluorescence probes within theilluminated spot were nearly completely bleached. After-wards, the excitation filter was switched back into the illu-minating beam. Samples were regarded as non-fluid if sharpbleaching edges did not change appearance and if recov-ery of the fluorescent intensity could not be observed within10 min.

2.6. Grating coupled surface plasmon spectroscopy (SPS)

The investigation of thin dielectric films on metal surfacesby surface plasmon spectroscopy is now a standard method[19]. However, since instead of the commonly used prismcoupling geometry, optical diffraction gratings were used,the concept of surface plasmon spectroscopy is describedhere with a special emphasis on the peculiarities of gratingcoupling.

A surface plasmon, i.e., an electromagnetic wave at themetal–dielectric interface, can be measured by coupling

plane light waves to this surface mode, therefore allowingoptical detection. Two alternative coupling mechanisms maybe used that are schematically depicted in Fig. 1.

The mechanism of energy exchange of light with thesurface plasmon resonance is best described by expressingboth the incident light and the surface plasmon in terms oftheir wave vectors. The wave vectork of the incident light isdefined as

(1)k = 2π

λ,

whereλ is the wavelength of the incident light. It is pointingin the propagation direction of the light waves.

Coupling of the incident light to the surface plasmonresonance is achieved by matching the in-plane componentof the wavevector of the light(kx) to that of the surfaceplasmon resonance [21].

In the commonly used [19] Kretschmann configuration,the in-plane component of the wavevector of the light inci-dent from a high-index medium matches the in-plane wave-vector of the surface plasmon on the back of a thin metalfilm. The reflected intensity as a function of the angleof incidence shows a characteristic minimum when thecoupling condition is fulfilled. The formation of a dielectricfilm on the metal surface leads to a change of the in-planewave vector of the surface plasmon, which is detected as achange of the angle of minimum reflected intensity.

The use of an optical surface relief grating is an alterna-tive way of establishing coupling to the surface plasmon res-onance. Here, due to the grating geometry, in addition to thespecularly reflected beam, diffracted orders are produced bythe grating structure, which are characterized by an in-planewave vectorkx , which is altered by a multiple integer timesthe reciprocal grating vectorkg ,

(2)kg = 2π

Λ,

whereΛ is the grating pitch. Matching of the in-plane wavevector of a diffracted order to the wave vector of the surfaceplasmon resonance leads to an efficient exchange of energy,which yields again a drastically reduced reflected intensityat a certain angle of incidence.

For investigation of thin films, usually the Kretschmannconfiguration is used because the optical response of a planarmultilayer system can be modeled quantitatively with the aidof Fresnel’s equations [21], which allow a reliable evaluationof the measurements. However, there are certain geometricalconstraints to the prism coupling technique that limit itsapplicability. Thick dielectric films with a high refractiveindex may lead to a surface plasmon resonance with avery largekx , which cannot be excited with this techniquebecause the maximumkx is limited by the refractive indexof the high-index medium. Furthermore, the use of a prismmakes it impossible to fix the position of the laser spot onthe gold surface sufficiently selectively onto areas that aresignificantly smaller than 2 mm in diameter. Neither problemoccurs in grating coupling.


By choosing the appropriate modulation constant of thegrating, kx of the diffracted orders can be tuned to anyvalue. Furthermore, the experimental geometry allows aprecise adjustment of the illuminated spot for all accessibleangles of incidence. The precise mathematical descriptionof the optical response of a grating structure, however isextremely difficult. Until today, only numerical methodsare known to solve this problem [22]. As a way out, it ispossible to assume that the grating coupling experiment witha diffracted order of interest of a certainkx can be describedby a hypothetical prism-coupling experiment leading to thesamekx of the incident beam. In this case, the angle ofincidence in the grating-coupling experiment,θgrat, can betransformed to the angle of incidence in an equivalent prism-coupling,θpris, by means of the equation

(3)kg + n1k0 sin(θgrat) = kx = npk0 sin(θpris).

Here, n1 and np are the refractive indices of the sub-phase and the hypothetical prism, respectively, andk0 is thewavevector of light in vacuo. As it was pointed out [23], thisevaluation procedure is valid as long as thekx of the surfaceplasmon is not influenced by the existence of a grating orby the finite thickness of the metal film in the Kretschmanngeometry. In general, this assumption is not justified. Nev-ertheless, this evaluation procedure can be used for the de-termination of the optical thickness of thin films, as we haveshown recently [24] by rigorous grating theory. If the thick-ness and refractive index of the hypothetical metal film areadjusted to give the right position, width, and depth of thesurface plasmon minimum, the thickness of an additionaldielectric film as determined by evaluating the equivalentprism geometry is in agreement with rigorous grating the-ory. Therefore, the evaluation method as described abovewas used for the data analysis in this work.

2.7. The preparation of the gold substrates for theself-assembly of the thiopeptides

Surface-relief gratings with a constant of roughlyΛ =490 nm were prepared on fused silica slides (2×12×4 mm),thus allowing coupling to the surface plasmon resonancein first diffracted order close to normal incidence. Forthe preparation of the grating, standard photolithographictechniques [25] were used.

A photoresist layer (Shipley microposit) deposited onthe glass surface by spin coating was illuminated by twointerfering beams of a He–Cd laser (λHe–Cd = 442 nm,Optilas). The resulting sinusoidal intensity distribution leadsto destruction of the resist in the illuminated areas, thusleaving only the not-illuminated portion of the resist afterdeveloping.

This structure was transferred into the fused silica sub-strate by reactive ion beam etching (Roth & Rau). Subse-quently, the surface was covered by electrothermal evapora-tion by a chromium layer of thickness 2 nm, followed by agold layer of thickness 150 nm. The chromium is to enhance

the adhesion between gold and glass. The thickness of thegold film is chosen to give the same optical response as aninfinitely thick material.

2.8. Impedance spectroscopy

Impedance spectra were recorded using a three-electrodearrangement (with platinum counter and Ag/AgCl, sat.KCl reference electrodes) and a Solartron 1260 FrequencyResponse Analyzer in conjunction with an EG&G 273Potentiostat utilizing the Zplot and Zview software fromSolartron (Version 2.1). An area of 0.283 cm2 was utilizedas the working electrode by pressing the sealing lip of theTeflon cell body (see Fig. 2), onto the gold coated substrate.Parameters used were amplitude 15 mV; frequency range50 kHz to 0.003 Hz. Spectra were recorded at a bias potentialof ±0 mV vs Ag/AgCl, sat. KCl, the open circuit potentialwas observed to be at about+0.3 V. No faradaic processeswere detected by cyclic voltammetry in the range+0.2 V to−0.7 V.

2.9. Set-up for the simultaneous grating coupled SPS,impedance spectroscopy and fluorescence microscopy

The set-up used for the simultaneous grating-coupledSPS and impedance spectroscopy is depicted schematicallyin Fig. 2.

The optics is designed to record the reflectivity from thegrating as a function of the angleθ of the incident lightbeam relative to the surface normal. The incident light (λ =632.8 nm) is TM-polarized. Measurements were performedin the “classical mount” (the reciprocal grating vector islying in the plane of incidence).

The set-up includes a measuring cell (Fig. 2B) designedto be easily disassembled in order to perform fluorescencemicroscopy on the same substrate together with the SPS andIS experiment.

The measuring cell is characterized by a Teflon bodycovered by quartz plates on both sides. One is designed asthe pathway for the laser beam. The other one constitutesthe substrate for the grating covered with the gold film asdescribed above. Both are sealed by O-rings, on the side ofthe substrate by an additional sealing lip. The two quartzplates are pressed to the Teflon body by two metal barsfixed by four screws, making it possible at the same timeto adjust the grating in the correct position with respectto the laser beam. When the screws are taken away thewhole assembly can be easily removed and disassembledunder water or buffer solution in order not to disturb thelipid bilayer. The substrate can then be transferred to thefluorescence apparatus.

The Teflon body also contains outlet and inlet ports forsyringes to fill the cell with buffer solution or provide asuspension of liposomes, as well as an opening each for thecounter and reference electrodes.


(A) (B)

Fig. 2. Set-up and measuring cell for (prism (A) and alternatively grating (B) coupled) surface plasmon resonance spectroscopy to be used simultaneouslywith impedance spectroscopy. For the set-up: C= light chopper, A= aperture, P= polarizer, M= mirror, G = goniometer, D= detector, L= lens, FC=flow cell; for the grating coupled SPS measuring cell: T= Teflon body, S= quartz substrate with grating, gold film and thiopeptide supported lipid film, C=quartz cover slip, Pt= platinum contact to the gold film operating as working electrode, Ref= opening for reference and/or counter electrode, SR= sealingrings, SL= sealing lip, CD= clamping device, HB= heating block.

2.10. Atomic force microscopy (AFM)

For the AFM imaging, particularly flat gold (111) sam-ples were prepared as described earlier [26]. Mica wascleaved and dried at 650◦C for 1 min under a nitrogenatmosphere in a tubular oven (Heraeus). After drying, themica was immediately put into an evaporation system (Balz-ers) and a 60-nm-thick gold film was subsequently evapo-rated at a pressure of 4× 10−6 mbar. The mica/gold samplewas then annealed at 650◦C for 30 s under a nitrogen at-mosphere. This procedure yields mica/gold samples with asurface structure exhibiting flat terraces.

Monolayers of thiolipopeptide and thiopeptide were pre-pared immediately following evaporation as describedabove.

AFM images were recorded on a multimode Nano-scope III (Digital Instruments) using the tapping mode.Images were taken with a 14-µm piezoelectric scanner andsilicon tips (NanoSensors) having a spring constant of 30–52 N/m and a resonant frequency between 309 and 368 kHz,depending on the tip.

In the tapping mode the tip is forced to oscillate inthe z direction above the sample and touches the surfaceperiodically. In this way, the friction forces are eliminatedand the interactions between tip and sample decrease.Therefore even very soft surfaces such as our samples can beanalyzed without damage. It is, however, difficult to obtainmolecular resolution in this mode, particularly in aqueoussolutions.

2.11. Contact angle measurements

Contact angle measurements were done in the advancingangle mode in demineralized water utilizing a G1 instrumentfrom Krüss GmbH, Hamburg, Germany.

Table 1Thickness measured by prism-coupled SPS of self-assembled monolayersof pure and mixed thiopeptide I and thiolipopeptide III in buffer solution,compared with the length of the molecules estimated by tentative modeling(ChemDraw)

dSAM dSAM(nm peptide part) (nm lipid part)

Thiopeptide (I) 3.0± 0.2Thiolipopeptide (III) 0.8± 0.1 0.4± 0.10.5 mixture thiopeptide (I)and thiolipopeptide (III)

1.7± 0.2 0.8± 0.2

Estimated length of thethiolipopeptide

3.6 2.2

SPS spectra were simulated taking into account the four-layer modelmentioned above and the lengths of the peptide and lipid part distributedat a ratio of 2: 1, according to the estimated dimensions.

3. Results and discussion

3.1. Contact angle and layer thickness of the SAMs

The thicknesses of the pure and mixed thiolipopeptideSAMs were determined separately using the prism-coupledSPS [19] as described earlier [7]. SPS spectra were simu-lated using a four-layer model including the prism, gold,peptide, and lipid layers. The parameters used for the pep-tide and lipid layers were the refractive indicesn = 1.41and n = 1.52, respectively, determined independently ear-lier [7]. As illustrated in Table 1, the thickness of the SAMsmeasured by SPS is smallest for the largest molecule, thethiolipopeptide (III). This indicates a poor coverage and/ora large tilt angle of the self-assembled molecules. A largetilt angle, would mean that molecules are arranged almostparallel to the surface rather than perpendicular. This wouldagree with the contact angle (Table 2), which is too smallcompared to that of a truly hydrophobic surface, pointing tothe more hydrophilic parts of the molecules being exposedto the outside. The thickness of the thiopeptide (I) mono-layer, on the other hand, is relatively larger, considering the


Table 2Parameters of the lipid films formed by vesicle fusion with self-assembled monolayers (SAMs) of thiolipopeptide/thiopeptide mixtures as a functionof mixingratio

Mixing ratio: Θ (◦) dll (nm) dll (nm) Fluorescence R (M�cm2) C (µF cm−2) R (M�cm2) C (µF cm−2)thiolipo-peptide/ (SAM on (SAM on gold treated image before vesicle before vesicle after vesicle after vesicle

thiopeptide untreated gold) with Piranha) fusion fusion fusion fusion

1 69 3.3±0.2 1.5± 0.2 grainy 0.2–1.1 27± 3 1–2.2 23± 40.8 66 3.2±0.2 3.0± 0.2 grainy 0.3–1.5 23± 4 1–1.4 19± 30.5 63 11±1.0 2.4± 0.2 homogeneous 0.2–0.7 21± 3 4–17 14± 20.43 63 n.d. n.d. homogeneous 0.2–0.3 22± 4 2–12 13± 20.33 49 7.1±0.8 3.3± 0.2 homogeneous 0.2 30± 3 1–2 26± 40.1 46 0.5±0.2 1.1± 0.2 inhomogeneous 0.2 27± 3 0.4 26± 3

Θ = contact angle of the SAM;dll = increase in layer thickness (measured by SPS and evaluated usingn = 1.52 for the lipid) due to vesicle fusion withSAMs on plain gold substrates or on gold substrates hydrophilized by treatment with Piranha solution;R andC, resistance and capacitance of the SAM onuntreated gold before and after vesicle fusion obtained from the fits to the equivalent circuit 1 given in Fig. 5b. Values are given as mean and standard deviationout of three measurements, or a range of values in cases of larger variability.

length of the molecule estimated to be 3.6 nm (in the ex-tended state because of the helix breaker proline). However,the estimated length, close to the thickness found by SPS,indicates a better arrangement more or less perpendicular tothe surface. The contact angle, on the other hand, does notcorrespond to that of a hydrophilic surface, which is no con-tradiction to the presumed high packing density because hy-drophilicity only comes from the OH-group of the serinesand from the NH2- of the lysine, respectively. The thicknessof the 0.5 mixing ratio lies in between the two extremes,pointing to a higher packing density and/or a lower tilt an-gle than for the lipopeptide alone. Low thicknesses measuredwhenever the thiolipopeptide was involved could be due tothe fact that lateral dimension of the molecule is determinedby the peptide spacer rather than the alkane chains. Hencehydrophobic interaction can occur but only of the alkanechains of single molecules with the gold surface rather thanbetween alkane chains of different molecules. This wouldresult in molecules being arranged parallel to the surfacerather than perpendicular. Another reason for the low thick-ness could be impurities reducing the concentration of theactive material in the SAM-forming solution. No indicationof such impurities could be detected but only by ES–MS, theonly method applicable to test the purity of lipopeptide III,since HPLC could not be used due to solubility problems.

3.2. AFM images of the SAMs

AFM images were recorded in the tapping mode fromthe pure thiopeptide (I) and thiolipopeptide (III) SAM, andSAMs with the mixtures with the ratios 0.5 and 0.1. Theimages of the two pure compounds (I) and (III) show asmooth surface, i.e., the roughness is not different from thatof the gold film, indicating homogeneous monolayers. Theimage of the pure thiolipopeptide (III) is given in Fig. 3Aas an example. The roughness increases for the 0.1 mixture(Fig. 3B) and becomes significant for the 0.5 mixture(Fig. 3C), indicating different lengths of the molecules. Thiswould mean that the lipid tails extend into space above thelevel of the thiopeptide monolayer where they appear to

be distributed at random; i.e., there is no indication of aphase separation here. In summary of the last two sections,the lateral spacers in the mixed SAMs are considered aspacking molecules [14], as concluded from the SPS andAFM measurements.

3.3. Tracking vesicle fusion by SPS

Starting with the pure and mixed SAMs, the fusionprocess was then followed by grating coupled SPS. Spec-tra were recorded before and after vesicle fusion when thethiolipopeptide SAMs were exposed to the suspension of li-posomes. The thicknesses of the lipid layers thus obtainedwere used to calibrate the kinetic traces as described [7]. Ki-netic traces were thus directly given in terms of the increasein layer thickness as indicated in Fig. 4 at different mixingratios. (Mixing ratios are given from 0 to 1 corresponding to100 to 0% of the lateral spacer.) The layer thicknesses ob-tained varied as a function of mixing ratio. Values obtainedin the middle region of 0.5= 50% are high compared tothe increase in layer thickness expected for a second leafletof a lipid bilayer (2.5 nm). They decrease on both sides ofthe mixing ratio to almost zero on the side of 100% of thethiopeptide as the lateral spacer and to about 2–3 nm on theside of 100% thiolipopeptide (Fig. 4A and Table 2). Thatmeans that there is no correlation with the contact angles ofthe SAMs, also displayed in Table 2, decreasing graduallyfrom mixing ratio 1 to 0. Contact angles did not vary appre-ciably, particularly since the thiolipopeptide and thiopeptideSAMs were not really hydrophobic or hydrophilic, respec-tively, as mentioned above, in contrast to other mixed sys-tems [14,27]. For example, in the case of cholesterol deriv-atives mixed with shorter packing molecules, both havingethyleneoxyl chains close to the surface, the contact anglevaries monotonously from a perfectly hydrophobic to a hy-drophilic surface [14]. Fusion of liposomes then brings aboutincreases in layer thickness, also changing monotonically inthe direction opposite to that of a lipid monolayer, towardthat of a lipid bilayer. The maximum in layer thickness inthe middle region of mixing ratio obtained with the mixed


Fig. 3. AFM images of thiolipopeptide (III) (A), mixtures of the thiolipopeptide (III) and the thiopeptide (I) with a mixing ratio of 0.1 (B), and a mixing ratio0.5 (C). All images were obtained in tapping mode.

(A) (B)

Fig. 4. SPS measurements showing the increase in optical thickness as a function of time. The kinetic traces show the fusion process of liposomes followed at30◦C, on thiolipopeptide SAMs prepared on untreated gold films (A) as a function of mixing ratio as indicated on the graph, and alternatively, on the mixedSAM (mixing ratio 0.5) prepared on a gold film hydrophilized by treatment with Piranha solution (B).

thiolipopeptide SAMs, on the other hand, has some simi-larity to a vesicle fusion experiment on SAMs mixed fromalkanethiols and hydroxy-functionalized alkanethiols [28].Layer thicknesses obtained after vesicle fusion, in this case,were too high compared to those of a lipid bilayer indicat-ing the additional adsorption of vesicles, particularly in theregion between mixing ratios 30 and 80%. The same phe-

nomenon was observed in our case. Hence mixing of SAMsappears to enhance the tendency toward adsorption of vesi-cles. The reason could be the pronounced roughness as seenin the AFM images, Fig. 3C.

Interestingly, when the gold film was treated with Piranhasolution prior to self-assembly, the increase in layer thick-ness corresponds much better to the expected thickness of


the second leaflet of a lipid bilayer (2.5 nm), particularly atthe 50% mixing ratio, as shown in Fig. 4B and Table 2. Thetreatment with Piranha solution is expected to form surfacefunctionalities such as OH- and COOH-groups, thus lendingsome hydrophilicity to the otherwise hydrophobic surface ofpolycrystalline gold. The preferential formation of bilayersinstead of multilayers on such surfaces had been shown pre-viously [28]. Unfortunately, this treatment also increases theinhomogeneity in terms of the impedance spectra of the re-sulting lipid film presumably due to an increased roughnessof the gold. Therefore, the phenomenon was not pursued fur-ther. The parameters measured, collected in Table 2, with thesole exception of the thickness of the lipid layer, refer to theuntreated gold (contact angles are measured before and allother parameters after fusion of the vesicles).

3.4. Impedance spectroscopy

Impedance spectra of the SAMs were recorded before andafter vesicle fusion. Examples of the 0.5 mixture and of thepure thiolipopeptide are given in Fig. 5. They show a sin-gle time constant different from the spectra obtained frommercaptoethanol-mixed SAMs [16]. Hence instead of usinga series arrangement of two RC circuits [20,29] or a capaci-tance in series with an RC [13], data were fitted to circuit 1 inFig. 5b, also used by other groups [12]. A certain distributionof time constants can be deduced from the suppressed arcs ofthe frequency-normalized admittance plots compared to thehalf-circles obtained from the fits. A constant phase elementwas not taken into account, however, because of the limitedinformation gained. Parameters obtained as a result from thefitting procedure for the particular samples given in Fig. 5aare listed in the figure caption. Average values of the resis-tance and capacitance out of three measurements obtainedbefore and after vesicle fusion for the different mixing ra-tios are given in Table 2. Changes are more pronounced onvesicle fusion of the mixed SAMs than those observed forthe pure monolayer. The capacitance goes through a min-imum and the resistance reaches a maximum in the mid-dle region. The capacitance of the 50% mixture, however, isstill much too high compared to that found by other groups(0.5 µF cm−2) for a supported lipid bilayer [13], whereas theresistance is relatively high, at least in many instances; seethe range of data in this case, given in Table 2. A possibleexplanation of this discrepancy would be that fusion of vesi-cles does occur to form lipid bilayers on small areas of theSAM whereas defect areas are covered by tightly packedvesicle aggregates rather than pinholes of plain gold filledwith electrolyte. Such layers of adsorbed vesicles could wellrepresent a high resistance, considering that liposomes arepossibly hemifused with the mixed monolayer. This wouldagree with the thickness data mentioned above as well asthe fluorescence measurements discussed below. This con-cept was further tested by simulating the impedance spectra,e.g., the one shown in Fig. 5a1, by the equivalent circuit 2 inFig. 5b. In this circuit, two RC circuits are arranged in paral-

lel, one for a lipid bilayer and one for a defect layer of vesicleaggregates. The capacitance and the resistance of the lipidbilayer,Cll andRll , are fixed to 1 µF cm−2 and 5 M�cm2,respectively. According to this simulation (simulated curvesnot shown), the impedance spectra are compatible with thepartial formation of a lipid bilayer, particularly in the middleregion of mixing ratios where the capacitance was found todecrease upon vesicle fusion.

3.5. Fluorescence measurements

Fluorescence measurements of mixed lipid bilayers havebeen mostly carried out on glass and other oxidic surfaces,e.g., [18]. Such measurements are usually more difficult toobtain on metal surfaces because fluorescence of a moleculeattached to a metal is quenched due to energy transfer in thedirection perpendicular to the surface [30,31]. Energy trans-fer critically depends on the distance the molecule is locatedapart from the surface. This phenomenon can be used tostudy the architecture of surface-bound molecules providedthe fluorophor is located at a distance not far exceeding acritical transfer distance (Foerster radius) [32,33]. This crit-ical transfer distance was determined on gold films to beapproximately 10 nm, by varying the fluorophor–substratespacing by evaporated SiOx layers and measuring the result-ing fluorescence intensities [34]. A quantitative analysis ofmembrane–substrate spacings, however, has not been carriedout up to now.

Fluorescence microscopy images of the SAMs after fu-sion with NBD-PE-labeled egg PC are shown in Fig. 6.(Their appearance did not depend on whether or not the goldsurface had been treated with Piranha solution.) Uniform im-ages are obtained in the middle region, showing a consider-able improvement compared to films obtained on the basisof mercaptoethanol-mixed SAMs [16]. They become onlyslightly more heterogeneous for the higher proportions ofboth thiolipopeptide and thiopeptide.

Attempts to detect fluorescence recovery after photobleaching (FRAP) [20,35] of these NBD-PE-labeled lipidbilayers formed by vesicle fusion indicated that there was nofluidity, i.e., no detectable mobility in the lipid matrix overdistances of micrometers.

These findings are well in agreement with the notionmentioned above of small areas covered by lipid bilayers, ifany, whereas vesicle adsorption is the predominant process.The homogeneous coverages on the 50, 43, and 33% sur-faces, however, could well indicate vesicle aggregates hemi-fused with the mixed lipopeptide monolayer. They wouldprevent both the layers from being fluid and the electrolytefrom penetrating the film.

Therefore, in order to further test this hypothesis, at-tempts were made to transfer egg PC monolayers labeledwith NBD-PE by the Langmuir–Blodgett (LB) technique.For example, a single lipid monolayer was transferred ontothe SAM made from the 50% mixture of thiopeptide (I) andthiolipopeptide (III), by an upstroke movement, so as to fill


(a1)

(a2)

(b1) (b2)

Fig. 5. (a) Impedance spectroscopy of gold films coated with mixed (mixing ratio 0.5, a1) and pure thiolipopeptide monolayers (a2) before and after fusion withegg PC vesicles 50 nm, in contact with buffer solution: (1) Re(Z) vs frequency/Hz and phase angle vs frequency/Hz plots; (2) complex admittance dividedby frequency plots. (A) thiopeptide I-mixed thiolipopeptide III layers (mixing ratio 0.5), (B) pure thiolipopeptide III layers. Curves (�–�–) and (�–�–)monolayers, curves (�–�–) and (�–�–) monolayers following fusion with egg PC vesicles. Solid lines represent data obtained for the bilayer fitted accordingto the equivalent circuit, given in (b1).RS = solution resistance, Rll = resistance of the lipid layer,Cll = capacitance of the lipid layer. Parameter valuesobtained from the fits are listed in Table 3. (b) Equivalent circuits (b1) for fitting of the impedance spectra shown in (a). (b2) For simulation of the impedancespectra shown in (a),RS = solution resistance,Rll = resistance of the lipid layer,Cll = capacitance of the lipid layer,Rdefect= resistance of the defects,Cdefect= capacitance of the defects.

Table 3Parameter values obtained from the fits (Fig. 5a)

Mixing ratio Before vesicle fusion After vesicle fusion

Rll (M�cm2) Cll (µF cm−2) Rll (M�cm2) Cll (µF cm−2)

0.5 0.2 21 17 141 0.2 24 1.0 23


Fig. 6. Fluorescence images of the lipid films obtained after vesicle fusion on thiopeptide I–mixed thiolipopeptide III SAMs at different mixing ratios.

(A) (B)

Fig. 7. Fluorescence images of lipid films obtained by Langmuir–Blodgett and subsequently by Langmuir–Schäfer transfer of egg PC monolayers labeledwithNBD-PE on a thiopeptide I SAM on a gold film evaporated on a hydrophilic glass substrate. (A) The borderline between glass and gold showing the extent offluorescence quenching on gold compared to the glass surface; (B) fluorescence-labeled lipid film on gold covered with a thiopeptide SAM.

the spaces left between the thiolipids. Then a second lipidmonolayer was transferred by Langmuir–Schäfer (LS) trans-fer so as to form a lipid bilayer with the lower leaflet par-tially covalently attached to the gold surface. No fluores-cence was observed in this case. In the case of the SAMmade from the 100% thiopeptide (I), the same procedurewas applied so as to eventually form a lipid bilayer on top of

the thiopeptide SAM, this time both leaflets held by forcesother than covalent binding. Fluorescence could be observedin this case (Fig. 7), although it is quenched to a consid-erable extent as demonstrated in Fig. 7A, showing the bor-derline between the bare quartz substrate and the gold filmcovered by the bilayer on top of the 100% thiopeptide SAM.Fluorescence intensity is much higher for the lipid bilayer


(A) (B)

Fig. 8. Fluorescence images of a spreading experiment of egg PC (doped with NBD-PE) as a lipid reservoir placed on the borderline between glass (A) andgold film evaporated on the hydrophilic glass substrate and coated with a thiopeptide I SAM (B).

on quartz glass (the glass surface had been subjected to thesame treatment regarding LB/LS transfer together with thegold film; hence a lipid bilayer was formed there too.) How-ever, a uniform fluorescence can be clearly seen on top ofthe thiopeptide SAM, Fig. 7B. Recovery of fluorescence in-tensity upon bleaching could be observed, for the same film,within a short period of time (not shown). However, imagesof the recovery process were not recorded and diffusion co-efficients were not measured, due to the low intensity of thesignal. Quenching is, however, compatible with the thick-ness of a lipid bilayer (5 nm) on top of the thiopeptide mono-

layer, the thickness of which was determined by SPS to be3 nm (Table 1).

Finally, experiments were carried out into the spreadingof a lipid bilayer extending from a lipid reservoir, whichhad been deposited in dry crystalline form on the substratewhen it is immersed in buffer solution. The 100% thiopep-tide (I) film was used since the contact angle is the best oneavailable in terms of a hydrophilic surface. Figure 8 showsagain the gold film (Fig. 8B) compared to the surroundingquartz surface (Fig. 8A). Spreading can be clearly seen onthe quartz surface being hydrophilic due to the cleaning with


Hellmanex prior to evaporation of the gold film. No spread-ing occurred, however, on the thiopeptide-covered gold film.

It is shown by these experiments that a fluid lipid bilayercan be formed, however, by LB transfer only and on the purethiopeptide monolayer rather than the mixed SAMs.

As to vesicle fusion on hydrophilic surfaces, vesiclefusion was discussed as a three-step process [15,35,36]:(1) adsorption, (2) rupture, (3) spreading. The tendencytoward adsorption seems to be low on the pure thiopeptideSAM as deduced from the kinetic traces shown in Fig. 4.

Hydrophobic surfaces [37], on the other hand, are consid-ered not to adsorb lipid vesicles unless a defect in the vesi-cle wall opens up presenting the hydrophobic interior to thesurface and leading to the splitting of the lipid bilayer in azipper-like fashion. This mechanism does not apply to thepure thiolipopeptide (III) SAM because it is not sufficientlyhydrophobic as deduced from the contact angle. It shouldapply to the mixed SAMs considering the interaction of hy-drophobic forces to bilayer adhesion and fusion [38] and thelipid tails extending above the level of the lateral spacer, dis-cussed above. This accounts for the clear optimum, both re-garding fluorescence images as well as impedance spectra,obtained in the middle region of mixing ratios.

4. Conclusion

Lipid films formed by vesicle fusion with mixed thi-olipeptide SAMs having identical lateral and vertical spac-ers are shown to be more homogeneous than those basedon the respective mercaptoethanol-mixed SAMs. The addi-tional adsorption of vesicles, however, cannot be avoided,possibly due to the selection of molecules used for thepresent investigation.

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fusion of small unilamellar vesicles onto laterally mixed self-assembled monolayers of...

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