Electrically induced lipid migration in non-lamellar phase

Journal of Colloid and Interface Science 386 (2012) 421–427

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Electrically induced lipid migration in non-lamellar phase Kaori Sugihara a,⇑, Janick Stucki a, Lucio Isa b, János Vörös a, Tomaso Zambelli a a b

Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zurich, Gloriastrasse 35, CH-8092 Zurich, Switzerland Laboratory for Surface Science and Technology, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland

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Article history: Received 20 January 2012 Accepted 12 April 2012

Keywords: 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) Electrophoresis Electrical manipulation

a b s t r a c t Inverted hexagonal blocks of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipid adsorbed on a polyethyleneimine (PEI)-coated surface in deionized water transformed its shape upon the application of an electric field, forming lipid objects in a variety of shapes (e.g. lines with a width of 10–50 lm). The phenomenon was driven by the electrophoresis, because the zwitterionic lipid, DOPE turned out to be highly negatively charged in deionized water. The interaction between DOPE and the PEI surface stabilized the system, assuring a lifetime over several weeks for the formed structures after the electric field was switched off. The free-drawing of microscopic objects (lines, crosses, and jelly fish) was also achieved by controlling the direction of the lipid movement with the field direction. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Lipid-electric field interactions play a key role in electrophysiology [1] and cell motility [2] and also provide possibilities to manipulate lipids with a good controllability in macro- and nano-scales. Typically, tens-of-mV membrane potential (i.e. a potential difference between inside and outside of cells) exists across severalnm-thick cell membranes. Therefore, the electric field in the lipid membranes can be as large as E  100,000 V/cm. Surprisingly, lipid bilayers survive such a high field and function as an insulator [1]. On the contrary, when electric fields are applied parallel to bilayers, much smaller fields (e.g. 10–100 V/cm) influence the lipid arrangements. Stelzle et al. [3] and Groves and Boxer [4] formed supported lipid bilayers (SLBs) made of a mixture of electrically neutral (zwitterionic) and electrically charged lipids and achieved the lipid-composition gradient by applying an electric field. The reason for the required weaker field is attributed to the high lateral fluidity of lipid molecules in the bilayers. Apart from the fundamental studies, these systems have been used for membrane protein [5], lipopolymer [6] and vesicle [7] separation and purification by exchanging the charged lipids with charged-lipid-tagged proteins, polymers, and vesicles. Adding cholesterol decreases the lipid diffusion, hindering the re-mixing of the electrically separated molecules [8]. Such electrical manipulations have been performed with phospholipids in lamellar phase such as SLBs as described above or adsorbed giant vesicles [9]. However, the electrical properties of lipids in non-lamellar phase (e.g. hexagonal and cubic phase) are much less known. In addition, in SLB-based ⇑ Corresponding author. Fax: +41 44 632 11 93. E-mail address: [email protected] (K. Sugihara). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.04.031

systems, lipid bilayers are confined in micro-fabricated flow cells, barriers or surface contrasts, since otherwise the lipids would self-spread over the surface [10]. Consequently, the electrical manipulation focuses on the spatial-density control of charged molecules within the confined SLBs without inducing any movement going over the border of the pattern. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is a zwitterionic conical-shaped lipid. It has garnered attention due to its inverted hexagonal phase (HII) at full hydration and room temperature (Fig. 1B) [17,18]. In aqueous solution, it forms HII blocks, characterized by electron microscopy [11,12], including our previous work [13]. It is also known to consist 80% of the inner membrane of Escherichia coli [14], where it has a critical function in membrane curvature and vesicle fusion [15,16]. In this work, we report that DOPE lipid blocks, adsorbed as patches on a polyelectrolyte-coated (polyethyleneimine, PEI) surface, migrate upon the application of an electric field E by changing their shape. The macroscopic shape change we observed is distinct from the conventional electrophoretic lipid polarization within a confined bilayer. The direction of the movement correlates with the direction of the applied field. After the electric field was turned off, the electrically formed objects are stable for weeks. Taking the advantage of the phenomenon, we created lipid objects with a shape of lines, crosses, etc. It can be considered as a free-drawing in a sense that it requires no pre-patterning of the shapes of the objects on the surface. Such a lipid drawing was possible due to (1) the interaction between the lipid and the polyelectrolyte that promotes the lipid adsorption but avoids the complete self-spreading all over the surface and (2) the feature of DOPE that constructs macroscopic objects that change their shape flexibly. DOPE is a zwitterionic lipid, which generally has little net electrical charge.

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The reason for such a dramatic reaction of DOPE against electric fields will be discussed. 2. Materials and methods 2.1. Polyelectrolytes Polyethyleneimine (PEI, MW = 25,000 g/mol, branched, purity P 99%, #408727) was purchased from Sigma-Aldrich Chemie GmbH (Switzerland). It was dissolved at a concentration of 1 mg/mL in deionized water filtered through MilliQ Gradient A10 filters (Millipore AG, Switzerland). For coating, glass slides were incubated in the solution for 15 min and rinsed with deionized water. 2.2. DOPE lipid solutions 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, purity > 99%, #850725), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Liss Rhod PE, purity > 99%, #810150), and 1-Oleoyl-2-[12-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]lauroyl]-sn-Glycero-3-phosphocholine (NBD-PC, purity > 99%, #810133) were purchased from Avanti Polar

Lipids and stored in chloroform (purity > 99%). The lipid solution was prepared by taking DOPE + 2% Liss Rhod PE (or NBD-PC) into a flask, drying for 30 min under a nitrogen stream, and adding deionized water, followed by a sonication at the highest power using Elma Transsonic Digital S (T710DH, IS@Work Pte Ltd., Singapore) at a concentration of 0.1 mg/mL. The lipid solution was further diluted to control the lipid patch density on the surface if necessary. The PEIcoated glass slides were incubated in the lipid solution for around 30 min to deposit the lipids onto the surface. During the adsorption, the samples were imaged with a fluorescent microscope from time to time to stop the lipid adsorption before the complete lipid coating of the whole surface. 2.3. Flow cells The straight flow cell consists of a PDMS block with a channel, a plastic homemade clamping system and a glass slide cleaned by an oxygen-plasma cleaner (PDC-32G, Harrick, USA) just before the experiments. The channel was 0.5 or 1 cm long, 2 mm wide and 1 mm high, fabricated by cutting out a part of PDMS with a razor blade. The flow cell for the free-drawing (Fig. 7) was also made similarly but with a cross-shaped channel of 2 mm width, 1 mm height with four electrode reservoirs.

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2.4. Confocal laser scanning microscope (CLSM) and fluorescent recovery after photobleaching (FRAP) We used a CLSM 510 microscope (Zeiss, Germany) equipped with an argon (488 nm) and a DPSS laser (561 nm) using a 40  (LD, NA 0.7) objective. The intensity profiles were analyzed with ImageJ (Image processing and analysis in Java, National Institute of Health; http://rsb.info.nih.gov). 2.5. CCD camera used for movies Movies, from which snapshots were taken for figures, were recorded by a CCD camera (C9100-13, Hamamatsu, Japan), combined with a fluorescent microscope (Zeiss, Germany). 2.6. Electrical manipulation The voltage was applied maximum at V = 25 V with an EC-TwinPlus voltage generator (Dr. K. Witmer Elektronik AG, Switzerland). The current was measured with a multimeter purchased from Voltcraft, Germany. In our two-electrode setup, the circuit includes two charge transfer resistance 2Rct at Pt/solution interface and the solution resistance in the channel Rsolution. The current is typically I = 0.4 lA at V = 25 V with the straight flow cell (R = 60 MX = 2Rct + Rsolution). It is slightly lower than the solution resistance estimated from the dimension of the flow cell and the resistivity of deionized water (q = 18.2 MX cm), Rsolution = 450 MX, presumably because unavoidable ion contaminations from the atmosphere (CO23 ), PDMS or glass lowered q. Nevertheless, it implies that Rct is negligible at such a high voltage, which is reasonable since the hydrolysis occurs already at a few V, lowering Rct significantly. Therefore, we describe the electric field E simply as E = (V/ distance between electrodes), neglecting the voltage drop at Rct. 2.7. Zeta potential measurements Zeta potential measurements were carried out using a NanoZetasizer (Malvern Instruments, UK). Approximately 1 mL of solution was loaded into a disposable cell (DTS1060C, Malvern Instruments, UK), and zeta potential measurements were acquired automatically (automatic attenuation and voltage selection mode). Ten measurements were performed in a sequence to verify the reproducibility, and the reported values of zeta potentials are the average over these 10 measurements, while the error bars are the measurements’ standard deviations. Each measurement consisted of a minimum of 10 runs until the instrument convergence criteria were fulfilled. 2.8. Atomic force microscopy (AFM) The samples were prepared in the same way as the ones for the optical studies with the flow cells. The field was applied for 1– 5 min. To be able to access the lipid object with AFM, the PDMS block (the channel part of the flow cell) was gently removed after the sample fabrication under water. We used the Nanowizard I BioAFM (JPK Instruments, Germany) and the Mikromasch CSC38/ noAl cantilevers in tapping mode. The imaging was always combined with optical microscope (with CLSM for Fig. 5, and with a fluorescent microscope for Fig. S3) to compare the optical and AFM images. 3. Results and discussions Fig. 1A shows a schematic of the experimental setup. The design of the flow cell was inspired by the one used in the electrical

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manipulation of lipid bilayers [4]. Deionized water (pH = 5.5) was used for all the experiments to minimize the ion screening effect [3]. First, a glass slide was coated with PEI to promote the lipid adhesion, since DOPE did not adsorb on glass surfaces very well without PEI. PEI is a positively charged polyelectrolyte at pH = 5.5. Second, the HII lipid-block solution was made with DOPE with 2% fluorophore-tagged lipid Liss Rhod PE and adsorbed on the PEI-coated surface for 30 min and rinsed with deionized water. Fig. 1C at 11 s shows a representative lipid patch adsorbed on the surface. Such patches typically have a size between 10 and 100 lm with constant intensity and some higher-intensity small dots inside the patch. 3D CLSM imaging (Fig. S1) confirmed that those dots are higher (tens of lm) than the rest of the patch which has a height below the optical limit (see also the AFM characterization later). Only negligible movement of the patches was detected in the absence of potential. Lipids started migrating along the field when a voltage V = 25 V (E = 25 V/cm) was applied parallel to the surface (Fig. 1C at 90 s). The lipids moved opposite to the field direction, indicating that the major driving force of the phenomenon is the interaction between the field and negatively charged molecules. Next, the direction of the field was reversed (Fig. 1C at 290 s). Lipids moved in the opposite direction from the field as previously. At 362 s, when the inverse field was turned off, the lipid patch remained elongated along the direction of the field. Interestingly, most bright dots that existed in the patch before the field application disappeared. The chronological change in the intensity profile along the white line in Fig. 1C is plotted in Fig. 1E and F. The bright dots within the structure are visible as spikes in the profiles. While E was applied, the right edge of the lipid patch moved in the opposite direction from E, while the left edge kept its position (Fig. 1E). In addition, the curve at 170 s has about the half number of spikes compared to the curve at 12 s. When the field was reversed, both the left and the right edge of the patch moved left (Fig. 1F). Remarkably, the spikes completely disappeared in the intensity profile at 362 s. In the Movie S1, one can observe that each dot disappears one by one abruptly but not gradually, indicating that this phenomenon is not due to the photobleaching. We assume that the lipids in the bright dots act as a reservoir, from which lipids are taken and used to elongate the patch (Fig. 1D). Previously in our group, we have found that the HII DOPE blocks interact with the PEI surface differently in physiological buffers, assembling lipid nanotubes upon the application of a solution flow [13]. In the assembly, the block also acts as a reservoir to form the tubes. The lipid migration tends to slow down after a voltage application of more than 30 min, presumably due to the lack of further lipids. Apart from the growth of the lipid structure, a slight intensity increase at the left edge of the patch was observed during the E application within the structure (the dotted arrow in Fig. 1E). We assume this is due to the integration of the bright dots (lipid reservoirs) into the patch, increasing the intensity locally. Nevertheless, the final intensity of the main patch area (around 200 (a.u.) at t = 362 s in Fig. 1F) is roughly the same as that of the original one (around 200 (a.u.) at t = 12 s in Fig. 1E), indicating that the main patch area did not change its thickness significantly. The shape of the migrated lipids depended on the initial shape of the lipid patches. Fig. 2A shows the lipid migration from another lipid patch. When E = 0, no lipid movement was noticed, as previously. When the field was turned on, this time the lipids drew several lines with a few lm width (Fig. 2A in 89–116 s), which is ten times smaller than the one in Fig. 1C. We observed that the rougher the edge of the patch, the larger the number of the fingers. To study the velocity of the lipid movement, we chose three fingers from Fig. 2A, and the migrated distances (xi, xii, xiii) were plotted as a function of time in Fig. 2B. The three points showed similar x vs t curves. All the points showed a major change in x around 10 s after

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the voltage was applied (the field was applied at t = 65 s, as indicated as an arrow in Fig. 2B). Once they started moving, x increased linearly with a velocity of v = 0.46 ± 0.03 lm/s (E = 25 V/cm) (note that the minus sign ‘‘–’’ is used to indicate that the direction of the lipid migration is opposite to that of E). To estimate the effective mobility of the lipids l, we studied the voltage dependence of v. Electric fields E = 0, 5, 10, 15, 20, 25 V/cm were applied for 2 min in series on each sample. 2 min is long enough to obtain the velocity v for each E, since the velocity becomes stable after 20 s. v was plotted against E for three individual experiments in Fig. 2C. The averaged data points were fitted linearly (black line) with l = v/E = (1.7 ± 0.2)  10 2 lm cm/Vs and the threshold field of Ethreshold = 2.9 ± 2.4 V/cm. In addition, we studied the inherent fluidity of lipids with fluorescence recovery after photobleaching (FRAP) (Fig. 3). Patch areas, from which fingers protrude, were chosen for the place for photobleaching, since most of the time finger areas were too small. The bleached circle fully recovered in 45 min. The diffusion coefficient was obtained as D = (1.1 ± 0.5)  10 9 cm2/s by fitting the data with the commonly used equation [17]. The value is one order of magnitude

20 µm Fig. 3. The internal fluidity of the lipid objects. Fluorescence recovery after photobleaching (FRAP) conducted on a lipid patch. The diffusion coefficient was obtained as D = (1.1 ± 0.5)  10 9 cm2/s, where the error comes from three individual experiments.

lower than that of lipid bilayers on glass surfaces [17] but is comparable to the one for the lipid bilayers on a PEM film reported previously (0.21–0.72  10 9 cm2 s 1), where the one or two orders of magnitudes smaller value of D compared to that on a glass slide has been linked either to the strong coupling between the lipid molecules and the oppositely charged PEM or to the result of insufficient connectivity of the lipid bilayer [18–20]. Note that the lipid structures tend to change their appearance when they are heated locally by the laser during the photo-bleaching (see the bright dots that appeared during the recovery in Fig. 3). Since it was unavoidable, diffusion coefficient was estimated by neglecting those areas. In the following, we discuss the mechanism of the lipid migration. First, we consider the electrophoretic effect. Generally, even vesicles made of zwitterionic phospholipids have a non-zero zeta potential f – 0 in physiological buffer (pH = 7.4) (e.g. f  8 mV for POPC vesicles) [21]. Therefore, the lipid migration may be attributed to the interaction between the field and the charged lipid molecules. The zeta potential of pure DOPE and DOPE + 2% Liss Rhod PE was measured, and the results are shown in Table 1. It shows that DOPE has an unexpectedly large negative charge. The head group of DOPE consists of amine (+) and phosphate ( ). The large f implies the imperfect ionization of the amine group in deionized water (pH = 5.5) since ammonium is a weak base (pKa = 9.3), while phosphates ionize completely since it is a strong acid (pKa1 for H+ + H2PO = 2.1). The sign of the charge ( ) is consistent with the direction of the lipid migration. Since the 2%added fluorophore-tagged lipid (Liss Rhod PE) is negatively

Table 1 Zeta potential of different lipids in deionized water (pH = 5.5, T = 25 °C). Lipids DOPE DOPE + 2% Liss Rhod PE DOPE + 2% NBD PC DOPE + 2% DOTAP

f (mV) 26 ± 15 55 ± 8 36 ± 13 45 ± 19

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from the field (Fig. 4C and D), suggesting that the electrophoresis is the dominant effect. The speed of the movement was v = 0.025 ± 0.001 lm/s (at E = 25 V/cm), which is one order of magnitude smaller than the one on PEI. It may be largely because the electro-osmotic flow hindered the lipid movement, although the change in the surface may have also affected the speed. The electrically formed lipid structures often present a fluorescent intensity gradient within the object as mentioned previously. Next, we studied the origin of such an intensity difference at the local scale. A representative AFM image and an optical image (the inset) of the same lipid object are shown in Fig. 5A. Fig. 5B and C correspond to the cross-sections of (B) the optical image and (C) the AFM image at the white lines indicated in Fig. 5A. Unfortunately, the AFM-derived absolute height of the lipid object (10.5 ± 1.3 nm on average among several objects from a few samples where the field was applied for 1–5 min) is not perfectly reliable because the cantilever also detects the electrostatic forces in water [24]. Nevertheless, the small standard deviation suggests that the thickness of the object is relatively uniform independent of the initial patch size and the duration of the field application in agreement with the optical study (see the discussion for Fig. 1). To estimate the real height of the object, we imaged a lipid patch with an AFM at different salt concentrations in solution (Fig. S3). The result suggested that the height of the lipid object is underestimated three fold when AFM imaging is conducted in deionized water (see the detail in SI). However, this calibration is still not optimal, since the salt concentration change may have affected the lipid structure. Therefore, here, we focus on the topology of the object. In the optical image, the intensity of the edge of the structure is lower than the inner part. On the other hand, the AFM image shows a smooth inner part with a fuzzy edge (Fig. 5A). The imaged edge structure resembles to the reported edge structure of SLBs [25]. The cross-section shows a continuous height for the inner part, while the height fluctuates at the edge (Fig. 5C). Importantly, it suggests that the lower intensity in the optical image

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charged, we initially thought that it was the origin of the net negative charge. However, the effect on f due to the addition of 2% Liss Rhod PE was not clearly detected, suggesting that the addition of Liss Rhod PE is not necessary for the lipid migration. Indeed, even if Liss Rhod PE was replaced by zwitterionic fluorophore-tagged lipids (NBD-PC), the blocks have a negative charge (Table 1) and showed the lipid migration at a speed of v = 0.50 ± 0.15 lm/s at V = 25 V, which is roughly the same as the one with Liss Rhod PE (v = 0.46 ± 0.03 lm/s, Fig. S2). Next, we consider the effect of electro-osmosis. Electro-osmosis is a solution flow close to the surface due to the interaction between the field and the counter ions accumulated on the charged surface. Generally, solution flows are known to push and move SLBs on glass [22], therefore the electro-osmotic flow may also function similarly. This effect was reported as negligible for lipid bilayers [4], but it becomes significant when vesicles are attached to the bilayers [7]. In our system, the major counter ions accumulated above PEI (+), and DOPE lipids ( ) are anions (e.g. OH ) and cations (e.g. H+), respectively (Fig. 4A). Since the anions and the cations produce flows in the opposite direction, the distribution of the total electro-osmotic flow must be complex. To study only the effect of the electro-osmosis, we attempted to suppress the electrophoresis by reducing the charge of the lipid blocks. However, f did not change significantly even if 2% positively charged phospholipids (DOTAP) were added to DOPE (see Table 1), since the majority of the lipids DOPE are negatively charged. It is reasonable, since in case of vesicles made of neutral lipids (DPPC), more than 10% addition of cationic lipids (DDAB) is required to change f by tens of mV [23]. A further addition of cationic lipids is unreasonable, because it influences the phase of the lipid structure, changing the phenomena fundamentally. As a second try, we attempted to rather alter the surface charge from positive into negative. If the surface is negatively charged, the electro-osmosis has the same direction as the field because cations accumulate on the surface (Fig. 4B). In such a case, the direction of the electro-osmotic and the electrophoretic lipid movements are the opposite. Thus, we can determine which effect (electrophoresis vs electro-osmosis) is dominant by monitoring the direction of the lipid movement. We used a glass slide as the negatively charged surface. DOPE blocks do not adsorb on glass surfaces very well as mentioned previously, because of the electrostatic repulsion, since both DOPE and glass surfaces are negatively charged. However, sometimes we observed an irregular adsorption of a DOPE block on glass. We observed such a lipid block while applying a field. The lipid block moved in the opposite direction

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Fig. 5. AFM imaging of the created lipid object. (A) An AFM image and a fluorescent optical image (inset) of an identical electrically formed lipid object. The crosssection of (B) the optical image and (C) the AFM image at the white lines are shown.

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introduced by sonication was used to increase the surface area (the outer most lipid bilayer that covers the block). Now, the question is what will happen when these lipid blocks in HII were ‘‘re-adsorbed’’ onto the surface. There is a high chance that the inner structure remains HII, since they were originally surface-bound HII structures before the sonication. A similar structure in air has been reported with Cardiolipin which is another molecule known for inverted hexagonal phase [29]. Nevertheless, further studies on the internal architecture are difficult, since the surface-bound microscopic lipid patches do not give enough signals in NMR or X ray diffraction, while cryoTEM is for samples in solution but not for surface-bound molecules. Surface sensors such as optical waveguide lightmode spectroscopy [30] (OWLS), quartz crystal microbalance with dissipation monitoring [31,32] (QCM-D), surface plasmon resonance [33] (SPR) can tell adsorbed mass; however, it is impossible to derive structural information from the mass because lipids adsorb as patches but not homogeneously on the surface. Finally, we show examples of objects that one can draw with this phenomenon. The ‘‘canvass’’ is a few mm2 PEI-coated glass surface without any pre-microfabrication of patterns, barriers or channels, thus this is free-drawing. Fig. 6 shows a multiple patterning of jelly fish. Individual lipid patches react to the electric field in a similar way; therefore, the method allows shaping of objects simultaneously (e.g. three jelly fish in Fig. 6). The lifetime of such objects was more than 3 weeks (Fig. S4). Furthermore, we demonstrate a 2D lipid drawing by manipulating the direction of the field in both x and y. A cross-section shaped channel was fabricated in a PDMS block (Fig. 7A). Four reservoirs for electrodes were made at the end of each channel. When a field is applied along x, two electrodes were inserted in the two corresponding reservoirs. In case of y direction, the position of the two electrodes was rearranged accordingly. The images in the first

500 µm Fig. 6. The multiple patterning of jelly fish. Many patches react in a similar way under the voltage application, resulting in multiple patterning.

does not originate from thinner areas but indicates the presence of defects in the structure. DOPE lipids prefer to be in HII phase in aqueous solution at room temperature. The structure has been extensively characterized by differential scanning calorimetry (DSC) [26], X ray diffraction [26,27], and nuclear magnetic resonance (NMR) [28]. In such studies, dried DOPE lipids (typically several mg) are simply mechanically mixed with water or hydrated by vapor phase equilibration corresponding to the desired hydration rate (0–100%), and stabilized in the chamber before the measurement. Especially, when the hydration rate is low, the lipids are in a bulk crystal, which is adsorbed on the chamber wall. Therefore, in that sense, many of those works have actually studied the surface-bound bulk HII structures. In our study, high power sonication detaches the hydrated lipids from the glass wall, making the microscopic blocks. Since the cryoTEM imaged typical patterns for HII inside the blocks [13], the original lipid architecture on the glass wall before the sonication must be surface-bound HII structures. The energy

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(i) 100 µm Fig. 7. The free-drawing of lipid objects by controlling the field directions. (A) A schematic of a cross-shaped flow cell. Images of lipids upon voltage applications at (B) position B, (C) position C, and (D) position D, indicated with white boxes in (A). The voltage (V = 25 V) was applied in ±x or ±y direction for 2 min. The direction of the electrical field is indicated by red arrows while the yellow arrows indicate the direction of finger growth. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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column in Fig. 7B–D show the adsorbed lipid patches at three positions in the flow cell indicated with white boxes in Fig. 7A. After the field was applied in y direction (the images in the second column in Fig. 7A–D), the lipid patches in the position B did not show a major change, suggesting the effect of the field at the place is not significant. In the position C, the patch deformed in the opposite direction from the field. In position D, which is the edge of the cross-section, (i) some lipids moved in y direction, while (ii) some moved diagonally. It is interesting to note that the presence of the lipids enabled the visualization of the field lines at an area where the field effect is not trivial. Next, a voltage was applied in the -x direction (the third column images in Fig. 7A–D). In the position B, the lipids moved this time, showing there is an effect of the field. In the position C, lipids also detected the field, resulting in a structure with fingers both in x and -y direction. In the position D, lipids drew a curvy pattern. It suggests that one can pattern also curves by using the right design of flow cells. Similarly, we applied a field in the direction of y (the fourth column images in Fig. 7A–D) and x (the fifth column images in Fig. 7A–D). After the field applications, the lipids in each position showed (B) a structure elongated in x direction, (C) that with fingers in four directions (x, x, y, y), and (D) that with curvy fingers, reflecting the history of the field effect at corresponding positions. In conclusion, we reported the electrically induced migration of DOPE lipid molecules on a polyelectrolyte-functionalized surface. DOPE prefers a non-lamellar phase (inverted hexagonal phase), which differentiates the current work from bilayer studies. Although DOPE is a zwitterionic lipid, it turned out to be highly negatively charged in deionized water. We found that the electrophoretic interaction between the charge of the lipids and the electric field is the dominant cause for the phenomenon. The observed macroscopic movement of surface-bound lipids is unique compared to the electrophoretic polarization of charged lipids within confined SLBs. Such a lipid immobilization was possible because (i) DOPE blocks adsorb on PEI surfaces as patches without selfspreading all over the surfaces, (ii) the lipid blocks transform their macroscopic architecture upon electric field applications incorporating the lipid reservoir, and (iii) the interaction between the PEI-coated surface and the lipids preserve the formed objects for a few weeks. Changing the direction of the field could be used to control the direction of the lipid movement. We used this phenomenon to demonstrate the free-drawing of microscopic objects in several shapes (lines, crosses, etc.). The simultaneous patterning of several objects was also performed, since similar lipid patches react to the electric field in a similar way. Acknowledgments This work was supported by EU Seventh Research Framework Program (FP7, ASMENA Project). We are grateful to Stephen

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