Characterization of Robust and Free-Standing 2D-Nanomembranes of

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Characterization of Robust and Free-Standing 2DNanomembranes of UV-Polymerized Diacetylene Lipids Roland Hillmann, Martina Viefhues, Lukas Gött-Zink, Dominic Gilzer, Thomas Hellweg, Armin Gölzhäuser, Tilman Kottke, and Dario Anselmetti Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03403 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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We prepared solid-supported and free-standing 2D-nanomembranes from diacetylene phospholipids and investigated them with AFM, helium ion microscopy and FTIR spectroscopy.

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We prepared solid-supported and free-standing 2D-nanomembranes from diacetylene phospholipids and investigated them with AFM, helium ion microscopy and FTIR spectroscopy. 150x54mm (300 x 300 DPI)


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Characterization of Robust and Free-Standing 2D-Nanomembranes of UV-Polymerized Diacetylene Lipids Roland Hillmann,† Martina Viefhues,† Lukas Goett-Zink,‡ Dominic Gilzer,‡ Thomas Hellweg,‡ Armin Gölzhäuser,¶ Tilman Kottke,‡ and Dario Anselmetti∗,† Experimental Biophysics and Applied Nanoscience, Department of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany, Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany, and Physics of Supramolecular Systems and Surfaces, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany E-mail: [email protected] Phone: +49 (0)521 1065391. Fax: +49 (0)521 1062959

Abstract Free-standing lipid membranes are promising as artificial functional membrane systems for application in separation, filtration and nanopore sensing. To improve the mechanical properties of lipid membranes UV-polymerized lipids have been introduced. ∗

To whom correspondence should be addressed Experimental Biophysics and Applied Nanoscience, Department of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany ‡ Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany ¶ Physics of Supramolecular Systems and Surfaces, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany †

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We investigated free-standing as well as substrate-supported monolayers of 1-palmitoyl2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE) and 1,2-bis(10,12tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and characterized them with respect to their structure, morphology and stability. Using helium ion microscopy (HIM), we were able to visualize the integrity of the lipid 2D-nanomembranes spanning micrometer-sized voids under high vacuum conditions. Atomic force microscopy (AFM) investigations under ambient conditions revealed formation of intact and robust pore-spanning 2D-nanomembranes up to 8 × 2 µm2 in size. Analysis by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) verified a distinct reduction of signal at 2143 cm−1 from diacetylene groups in the 2D-nanomembranes after UV-polymerization. Further high-resolution AFM investigations of unpolymerized lipid monolayers revealed a well ordered two-dimensional network, when deposited on highly oriented pyrolytic graphite (HOPG). These structures were inhibited for polymerized adlayers. Structural models for the molecular arrangement of the adlayers are proposed and discussed.

Introduction Membranes separate distinct volumes from each other, e.g. the inside of a cell from the external medium 1 . When the thickness of the membranes is reduced down to single molecule thickness it is called a 2D-nanomembrane. Free-standing nanomembranes are of paramount importance for many technical applications like separation and filtration 2,3 , an amphiphilic support for polymer films 4,5 or nanopore sensing 6 . The latter has achieved a state of high sensitivity and versatility in the last years 7,8 . Improved techniques enabled the possibility of nucleotide detection with pore-forming proteins embedded within a lipid bilayer membrane 9,10 . Also, fabrication of ultra-thin solid-state nanopores in graphene 11,12 and molybdenum disulfide 13 has become possible, which can be engineered directly by a focused ion beam (FIB) 14 , HIM 15 or electrical breakdown 16 for instance. Although these inorganic materials

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have enhanced mechanical and chemical stability in comparison to biological membranes, synthetic nanopores usually lack the specificity of pore-forming proteins 17 . Synthesis of polydiacetylene (PDA) was first reported from the laboratory of G. Wegner 18 . Polymerization by UV-irradiation establishes a conjugated system 19 with light absorbing properties 20 . PDA then had been utilized for vesicles 21 , Langmuir monolayers 22,23 and self-assembled films 24 . Membranes of UV-polymerizable lipids have two main advantages over conventional lipids with respect to robustness and biomimeticity 25 . The biomimeticity allows functional integration and incorporation of transmembrane proteins like bacteriorhodopsin 25 with very reproducible and appropriate size of the nanopore 26 . Particularly the preparation of phospholipid 2D-nanomembranes can be realized in a straightforward manner by Langmuir-Blodgett (LB) technique. UV-induced polymerization of diacetylene groups in the tail of phospholipids, self-assembled at an air-water interface, gives a possibility to improve the stability of the 2D-nanomembrane 27–29 . Biofunctionalized substrates, such as supported bilayers, also provide a model for artificial and stable biological membranes 30,31 . As lipid bilayers are the key feature of biological cell membranes, self-assembled monolayers (SAMs) of lipids are of great interest for understanding electrical or chemical biosensors 32 or immobilized protein arrays 33 . They provide an interface between biological and technical applications such as a broad variety of physical detection devices of high sensitivity 34 . The mechanism of polymerization of diacetylene groups proceeds by cross-linking due to chain radical polymerization 35 . Raman spectroscopy 36,37 and X-ray studies 38,39 indicate the formation of alternating double and triple bonds in the polymeric backbone (Fig. 1a). The diacetylene group provides a direct and specific marker signal for vibrational spectroscopy that has been exploited to follow the polymerization of polycrystalline material using FTIR spectroscopy 18 and FT-Raman spectroscopy 40 . Other characteristic effects to the signals of acyl chains in FTIR spectra have been revealed for lipid vesicles with diacetylene groups 41,42 . Here, we report on the preparation of robust free-standing artificial 2D-nanomembranes 3


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with sophisticated mechanical properties. This is achieved by using 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1-palmitoyl-2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE), two photopolymerizable diacetylene phospholipids (Fig. 1b). UV-induced polymerization of the lipid monolayers was conducted at the air-water interface of a LB trough, as well as the transfer to various substrates by LB-technique. To investigate the influence on mechanical properties, two different lipids with one (PTPE) and two (DiynePC) diacetylene groups were used. Both, AFM and HIM studies of polymerized lipids revealed distinct differences in the aligning and stability properties of polymeric PTPE and DiynePC. FTIR spectroscopy on the phospholipid monolayers revealed the chemical conversion indicated by a decreasing signal of the diacetylene groups caused by polymerization.

Materials and methods Materials We used 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), 1-palmitoyl2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) from Avanti Polar Lipids (Alabaster, USA). Deionized ultrapure Milli-Q water with an elecR trical resistivity of 18.2 MΩ cm−1 was used for the subphase of the LB trough. Quantifoil

TEM grids were purchased from Plano GmbH (Wetzlar, Germany). Highly oriented pyrolytic graphite (HOPG, grade ZYB, Mosaic Spread 0.8◦ to 1.2◦ ) was purchased from NT-MDT (Moscow, Russia). Germanium crystals were purchased from Korth Kristalle (Altenholz, Germany).

Preparation of lipid film A commercial LB trough (Riegler und Kirstein, Potsdam, Germany) was used for monolayer preparation. Briefly, a lipid solution (1 mg/ml in chloroform) was spread at a 1200 cm2 wide 4


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Figure 1: (a) Tails of three lipid molecules are shown. They are polymerized by UVirradiation at 254 nm. The marked red substituents result from different molecules and are linked through polymerization, the blue substituents show the origin of the new triple bond. Due to the thinning process of the layer, which is accompanied by a lateral expansion, a surface pressure increase in the monolayer at a LB trough can be detected. Polymerization of diacetylene upon light irradiation has been explained by topochemical 1,4polymerization. (b) 1-palmitoyl-2-(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). 18 air-water interface, by carefully dripping the lipid solution using a Hamilton glass pipette. With the Wilhelmy plate method, the surface pressure of monolayers was determined 43 . After evaporation of the chloroform (5 min), the surface pressure was constantly measured as the monolayer was compressed by the trough barrier at 20 cm2 /min. The lipid monolayer films were prepared in symmetrical compression mode using two counter propagating barriers. All experiments were carried out at 16 ◦C subphase temperature.

Polymerization with UV-irradiation Polymerization of PTPE and DiynePC was performed at the air-water interface using an UV-mercury lamp (UVS-14 EL, Upland UVP, Upland, CA, USA) of 4 W (254 nm) at a distance of 11.5 cm above the water level to obtain an UV-intensity of typically 1.0 mW/cm2 . The polymerization only reliably proceeded when the diacetylenes were arranged with appropriate geometry, which can only occur in solids or other highly ordered structures 44 . Subphase temperature of 16 ◦C was chosen advisedly to perform all experiments with lipids in their gel phase (see supporting Fig. S1). An appropriate surface pressure was adjusted in the condensed phase at the LB trough (PTPE: 30.5 mN/m, DiynePC: 28.0 mN/m). Those 5


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parameters were maintained with respect to the maximum surface pressure achievable before reaching the collapse of the monolayer. The area enclosed by the barriers was kept constant during illumination, and we observed that the surface pressure was rising monotonously due to the polymerization process (Fig. 1a). The saturation of the increase in surface pressure was typically reached after 45 min, although the final surface pressure depended both on the used lipid and the compression parameters. After illumination with UV-light, typical final surface pressures were 43 mN/m for PTPE and 48 mN/m for DiynePC, respectively.

Deposition of monolayer R We used HOPG, germanium and Quantifoil as substrate materials for investigation of

the lipids. HOPG is a layered, planar material. To prepare a clean sample of HOPG for usage with the LB trough, adhesive tape was used to remove several surface layers of material just before film preparation. No further cleaning or functionalization was necessary. Monolayers of lipids were deposited onto hydrophobic samples (HOPG, germanium) from the air-water interface by Langmuir-Schaefer (LS) technique. There, the hydrophobic sample was approached and withdrawn parallel, thus horizontally orientated, to the interface (LS technique) with a velocity of 50 µm/s. The sample was slightly tilted by 5◦ , to drain the water R while withdrawing the sample (see supporting Fig. S2). Quantifoil is a TEM grid laminated

with a thin carbon layer and contains small aperture arrays with various diameters. Coating R of Quantifoil with the phospholipids was performed by a horizontal drawing-up method,

were a solid support is pulled up trough a monolayer from the water phase 29 . Thus, the TEM grid was submerged in the subphase, before spreading and polymerizing the phospholipids. The horizontally orientated sample is then raised trough the air-water interface, slightly tilted by 5◦ .

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AFM Characterization AFM measurements of lipids on different surfaces were performed at ambient conditions (20 ◦C) using a Nanoscope IIIa Multimode (Digital Instruments, Santa Barbara, CA, USA) with Tap300Al-G cantilevers (BudgetSensors, Sofia, Bulgaria) in tapping mode of operation. The images were routinely treated by offset and plane correction algorithms in the software Nanoscope 5.30. Afterwards, figures were visualized by the public domain software package Gwyddion 2.44.

HIM Characterization The samples were investigated using a scanning helium ion microscope (HIM, Zeiss Orion Plus). Helium ion microscopy does not only provide superb resolution but also offers high materials contrast and surface sensitivity, and it can also provide sharp images from electrically insulating samples without a conductive coating 45 .

FTIR Spectroscopy Lipid monolayers were investigated by FTIR spectroscopy using a germanium crystal with the dimensions 72 × 10 × 2 mm3 as internal reflection element for attenuated total reflection (ATR) spectroscopy 46,47 with 17 active reflections. The monolayer was deposited at a surface pressure of 30.5 mN/m if not stated otherwise. Drying at room temperature for at least three hours was done in complete darkness to avoid polymerization reactions, the substrate was then mounted in a Gateway ATR cell (Specac, Orpington, UK). Intensity spectra of the lipids were recorded by 1024 scans with an IFS 66v spectrometer (Bruker Optik, Ettlingen, Germany) at a spectral resolution of 2 cm−1 . To record a reference intensity without monolayer, the crystal was removed from the ATR setup, cleaned by an argon plasma (Zepto, Diener Electronic, Ebhausen, Germany) at 0.4 mbar and 100 W for 5 min, and remounted subsequently. DiynePC was irradiated before deposition as described above. PTPE was

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irradiated after deposition with 254 nm light for 120 min at a distance of 7 cm. The temperature was kept constant at 20 ◦C during illumination. To avoid stray light on the detector, a germanium filter restricted the recording range to below 6000 cm−1 .

Results and discussion Pore spanning 2D-nanomembranes We prepared free-standing 2D-nanomembranes of DPhPC monolayers, a phospholipid without any polymerizable groups, by LB-technique. Inspected in the HIM, they were found to be very unstable and disrupted instantly. Therefore, UV polymerizable phospholipids, DiynePC and PTPE, were investigated next. Successful formation of robust pore-spanning monolayers up to 8 × 2 µm2 could be verified for UV-polymerized PTPE and DiynePC by HIM and AFM imaging. In contrast to the DPhPC monolayers, we found that the membranes remained intact and functional for several hours both under laboratory and high vacuum conditions. Figs 2a and 2c show polymerized 2D-nanomembranes of PTPE and DiynePC taken by HIM where apertures of various sizes were suspended (Fig. 2a). Marked by a red circle, an 8 × 2 µm2 aperture was partially covered (Fig. 2a). In rare cases, the polymerized monolayers could be locally damaged during the scan with higher energy per unit area, allowing to investigate the behavior of forming defects. Although punctures were occurring, most parts of the membrane remained functional. For polymerized PTPE 2D-nanomembranes we observed defects to be exclusively circular and smooth in shape. In contrast, HIM-images of pore suspending 2D-nanomembranes of polymerized DiynePC (Fig. 2c) revealed somewhat different properties of beam resistance and stability. Whereas microapertures up to 8 × 2 µm2 could be covered likewise, the geometry of defects was more irregular indicating a high-grade crosslinked and stiffer membrane. Membrane-covered and uncovered voids were also imaged with AFM. Successful for8


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Figure 2: (a) HIM-image of PTPE (UV-polymerization time: 45 min). One pore (blue circle) is not covered, defects (red circle) are circular, ion dose: 5.44 × 1012 cm−2 (b) AFM scan of polymeric pore-spanning PTPE. The membrane bends down to 65 nm, along the line profile (supporting Fig. S3). (c) HIM-image shows intact pore-spanning 2D-nanomembranes of polymeric DiynePC on the TEM grid, ion dose: 7.25 × 1013 cm−2 . In contrast to PTPE membranes the ruptures (e.g. red circle) have an elongated shape (UV-polymerization time: 45 min). (d) AFM image of polymeric pore-spanning DiynePC. No sagging of the membrane is visible along the line profile. mation of pore-spanning membranes can be seen in Figs 2b and 2d, where several intact 2D-nanomembranes of polymerized PTPE and DiynePC were covering the microapertures. Although there was a moderate interaction of the AFM-tip with the sample under ambient conditions, 2D-nanomembranes were kept intact during scanning. The line scan profile

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for PTPE taken from Fig. 2b revealed the downward bending of the membrane during AFM scanning (see supporting Fig. S3) as a consequence of the applied forces by the AFM cantilever and gravitational forces. In contrast, the line scan profile for the polymerized DiynePC 2D-nanomembrane in Fig. 2d exhibited a constant height level with oscillations of the membrane due to the tapping image mode and resulting resonance. These observations can be explained with a stiffer and more rigid DiynePC nanomembrane, which complies with the higher density of polymerizable diacetylene groups available. Although we used two different lipid head groups in our experiments, we expect no influence on the mechanical properties of our membranes. Molecular models showed that the possible increase in profile width of a lipid layer caused solely by head group conformational changes is small compared to the effects of chain tilt 48 . Regardless of the used head group, polymerization of diacetylene groups led to tilted configuration of the hydrocarbon chains in membrane systems 49 .

Analysis of lipid adlayer Analysis of substrate-supported monolayers was conducted to provide further insights into the nanomembrane stability. Investigations of many different self-assembling molecules on substrates have been reported in the past decades 50–52 . Those AFM and scanning tunneling microscopy (STM) studies revealed that molecules like alkanes, alcohols and fatty acids organize in lamellae with the extended alkyl chains oriented parallel to a lattice axis within the basal plane of graphite 53,54 , although amphiphilic molecules are orientated vertically at the air-water interface of the Langmuir trough in general. In this work, we prepared unpolymerized PTPE and DiynePC monolayers on HOPG using the LB-technique and investigated them with AFM. We found the monolayer of monomeric DiynePC and PTPE to be well organized showing a two-dimensional network of molecularly ordered domains (Figs 3a and 3d). The domains exhibited different orientation angles of 60◦ or 120◦ between them, displaying the threefold symmetry of the HOPG lat10


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Figure 3: (a) Unpolymerized PTPE layer coated onto HOPG. A triangular pattern of lipid domains can be observed, showing well-ordered lamellae. (b) Polymerized PTPE layer on HOPG. (c) Close-up view of polymerized PTPE, where self-assembling structures can be discerned. (d) AFM image of an unpolymerized DiynePC layer, where molecular steps are visible. The step height along the white line profile amounts to 0.9 nm. (e) On polymerized DiynePC layer on HOPG an irregular patchy surface structure is visible. (f) Close-up view of polymerized DiynePC. The step height along the line profile is 1.4 nm high. No organized structures can be observed. tice (see Figs 3a and d as well as supporting Figs. S4 and S5). The monolayer thickness was determined to be 0.9 nm for unpolymerized DiynePC (Fig. 3d), which is much smaller than the DiynePC chain length. Thus, a monolayer structure with vertically oriented hydrocarbon chains can be excluded. However, the film thickness of 0.9 nm is consistent with the DiynePC chains lying parallel to the HOPG basal plane 55 . The distance of periodicity between neighboring lamellae was measured to be 6.2 ± 0.6 nm for DiynePC (line profile 3d) and 6.0 ± 0.6 nm for PTPE layers (line profile 3a), respectively, as expected from the molecular geometry of the aligned SAM (see supporting Fig. S5). 11


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In contrast, we assume that the polymeric 2D-nanomembrane transferred to Quantifoil still exhibited the arrangement of the Langmuir film at the air-water interface of the trough. There, the polymerized lipids are vertically orientated with a 36◦ 49 tilt in the lipids hydrocarbon tails, assuming that the lipids are rigid rods and their long axis is initially orientated perpendicular to the air-water interface. A polymerized monolayer of DiynePC transferred to HOPG is showing no alignments and its thickness was estimated with 2 nm (supporting Fig. S6). In Figs 3b, c, e and f AFM images of the polymerized PTPE and DiynePC are presented. A PTPE monolayer, polymerized before LS transfer on HOPG (Fig. 3b), showed a complete surface coverage with only a few defects. The typical step-structure of HOPG remains faintly visible. A close-up scan of 0.5 µm2 in size (Fig. 3c) revealed ordered structures, similar to unpolymerized adlayers. In contrast, polymerized DiynePC on HOPG (Fig. 3e) showed a patchy and defective layer. In the close-up scan (Fig. 3f) the nanomembrane showed no regular alignment. We suppose, that those aligned structures were caused by unpolymerized lipids, which could freely migrate during LS transfer, whereby the horizontal arranged sample was withdrawn, and thus moved in the voids of the polymerized DiynePC layer. We assume that the different appearance of the polymerized adlayer was caused by the higher density of polydiacetylenes in DiynePC compared to those of PTPE. To confirm that the polymerization indeed impacts the diacetylene groups, spectroscopic experiments with FTIR were performed. The experiments revealed that the polymerization process strongly depends on the diacetylene density of the lipid films. Based on the molecular structure, we expect filamentous 1D-polymerization for PTPE, whereas DiynePC lipids can polymerize in a 2D-mesh-like structure. Therefore the resulting monolayer of polymerized PTPE remained more fluid-like and could align to the atomic structure of HOPG. In contrast, the polymerization degree for DiynePC is higher exhibiting a stiff and solid-like behavior with no molecular alignment of the polymerized monolayer to HOPG.

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Chemical analysis of monolayers before and after irradiation The structure of the lipid monolayers was characterized by FTIR spectroscopy using germanium as an ATR substrate (Fig. 4). The lipid monolayer was transfered to the substrate by Langmuir-Schaefer technique and dried afterwards at room temperature in the dark. DiynePC showed the characteristic signals of lipids with bands originating from C-H stretches at 2960 to 2850 cm−1 , from C=O stretches at around 1730 cm−1 , and from C-H bends at around 1460 and 1378 cm−1 , as described in literature 41,42,56 . The head group contributes with signals at 1485 and 1262 cm−1 . The direct comparison to spectra of a similar lipid without diacetylene groups, DPPC, shows that the bands of DiynePC are broadened with a slight upshift in frequency of C-H stretch and a downshift of C=O (Fig. 4b). These bands are sensitive probes for changes in the packing, flexibility and chemical structure of the lipids by the diacetylene moieties. Here, one contribution to the broadening is an additional band at 2937 cm−1 caused by the chemical variation 42 . The UV-irradiated DiynePC showed the same overall band pattern as without treatment. Both C=O and C-H stretches respond to the irradiation by a small upshift in frequency. To analyze the chemical conversion taking place by irradiation, we aimed to resolve the signal of the diacetylene group. Previous FTIR spectroscopic analyses of bulk diacetylenic acids as model compounds revealed a signal at around 2139 cm−1 that was assigned to the asymmetric stretch by quantum chemical calculations 57 . Indeed, we resolved a very small signal of the monolayer at 2143 cm−1 with a shoulder at 2150 cm−1 that rises with increasing surface pressure during deposition from 10 to 30.5 mN/m and then remains constant for a further increase to 45 mN/m (Fig. 4c). When these three spectra were scaled to the amount of lipid using the C=O band integral, all three signal strengths were equal in intensity, which further supports a differentiation of the signal from the noise level. Irradiation of the DiynePC strongly reduced the signal of the diacetylene group and thereby provides first evidence, to our knowledge, from FTIR spectroscopy for a chemical conversion of diacetylene in the monolayer. However, this band should not be confused with that from the symmetric 13


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Figure 4: FTIR absorption spectra of lipid monolayers on a germanium substrate. (a) DiynePC was investigated in the dark and after irradiation with UV light. The polymerization induced only minor changes in the overall band pattern. The signal of residual atmospheric CO2 at 2349 cm−1 was removed for clarity. (b) The spectral regions of C-H stretch and C=O stretch were selected for direct comparison of DiynePC without and with polymerization. Spectra were scaled to the integral of the C=O band. Upshifts in frequency and a decrease in absorbance were observed after polymerization. Both vibrations are sensitive to changes in the structure and packing of the lipids. A spectrum of DPPC was added for comparison and scaled by 0.5 for clarity. (c) DiynePC was deposited at different surface pressures. An increase in signal at 2143 cm−1 was found with an increase in pressure from 10.0 to 30.5 and 45 mN/m. This band is assigned to the asymmetric stretch of the diacetylene group. The signal strongly decreased after UV irradiation, which provides evidence for the conversion of the diacetylene. Four and three independent experiments were averaged without and with irradiation, respectively. (d) PTPE was investigated in the dark and after irradiation with UV light. Spectra were scaled to the integral of the C=O band. Changes in frequency by irradiation were observed that were smaller than for DiynePC. stretch of diacetylene, which has been observed previously by FT-Raman spectroscopy on model compounds in the bulk at around 2257 and 2220 cm−1 , respectively 40,57 . Signals of the product were not found in our analyses, which can be attributed to the small signal strength and the expected broadening by a distribution of polymeric aromatic groups formed

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according to the reaction model (Fig. 1a). An overview of the band assignments for the DiynePC monolayer is given in Table 1. Table 1: Selected FTIR bands of the DiynePC monolayer and their assignment.a DiynePC monolayer

DiynePC vesicleb

DiynePC powderb

DiynePC theoryc

lipids generald

assignment

2957

2956

2957

2953

2956

CH3 asym. stretch

2937

-

2939/2934

-

CH2 asym. stretch close to diacetylene

2921

2926

2917

2920

CH2 asym. stretch

2896

2899

2895

-

CH2 sym. stretch close to diacetylene

-

2872

2870

CH3 sym. stretch

2851

2854

2850

2873

2850

CH2 sym. stretch

2143

-

-

2162

-

diacetylene asym. stretch

1734

-

1818

1730

-

a d

2922

−1

b

42

c

Band positions are given in cm . Lee . Roman and Baranska Tamm and Tatulian and references therein 56

C=O stretch 57

The monolayer of PTPE was analyzed by FTIR spectroscopy before and after irradiation with UV light on the substrate (Fig. 4d). Both C-H and C=O stretches were slightly upshifted in frequency as compared to DiynePC. The band of the C=O stretch shows a strongly asymmetric distribution which is attributed to the two chemically different tails of the lipid. UV irradiation led to very minor changes in the band positions with a downshift of the C=O band by only 1 cm−1 . Besides, the relative absorption of the C-H stretches with respect to the C=O stretches was reduced by illumination. These deviations might be attributed to changes in orientation relative to the substrate plane and to a chemical conversion, which awaits further quantitative analysis. In summary, the chemical changes in the DiynePC monolayer were more easily detected than in PTPE because of the two reactive groups present. Direct changes by irradiation were identified by resolving the diacetylene signal and its reduction in the monolayer. Additionally, the light-induced modifications in packing and structure of diacetylene-containing lipids were indirectly monitored via the C=O 15


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and C-H signals.

UV-Polymerization kinetics Polymerization of DiynePC and PTPE was monitored by UV-VIS spectroscopy in a bulk setup. A Shift in absorption to higher wavelength was evident resulting from the formation of conjugated systems with large π-systems. With progression of polymerization, peaks of absorption of visible light arose (supporting Fig. S7).

Figure 5: (a) Isotherm of PTPE, measured by extrapolation of the condensed phase. The inset shows the rising surface pressure during UV-polymerization. (b) Isotherm of DiynePC. The progressing polymerization (inset) started at high speed (PTPE), and finally reached a saturation level, when most of the monomeric molecules were cross-linked, whereas the polymerization for DiynePC was retarded in the first 10 min of the illumination. Polymerization of the Langmuir monolayers resulted in conformational change 19 of the hydrocarbon chains of the lipids. AFM studies 58 revealed a thinner film thickness after polymerization which supported the accepted model of polymerization mechanics of polydiacetylenes. As a result, the area per molecule in the polymerized Langmuir monolayer is of a higher value, or, for a fixed surface area, the pressure increases 49 . Consequently, the surface pressure is suitable to monitor the polymerization. The polymerization kinetics that were measured by monitoring the surface pressure, ex16


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hibited different characteristics for DiynePC and PTPE (insets in Fig. 5). The rate of polymerization for PTPE was at its maximum right in the beginning as expected from an approximately first order kinetics, whereas the polymerization for DiynePC was retarded in the first 10 min of the illumination exhibiting a more sigmoidal dependence 59 . It is known and accepted, that the polymerization starts by UV-induced formation with a free radical as initiator 60 . Chain propagation by adding monomer units and termination by recombination with another radical are therefore in direct competition. As a consequence, the frequency of termination events depends on the amount of free radicals per unit area 60 . Since DiynePC has a high density of diacetylene groups per unit area, chain termination during polymerization can occur very likely in the beginning. This also explains the observed retarded polymerization of DiynePC in the first 10 min of illumination.

Conclusion The present study describes the preparation and characterization of free-standing and supported UV-polymerized lipid 2D-nanomembranes. Due to covalent bonds in the polymeric membrane, advanced mechanical properties lead to pore-spanning membranes in the order of micrometers not achievable with ordinary lipid membranes. Free-standing and porespanning 2D-nanomembranes of DiynePC and PTPE were prepared with LB-technique, investigated with AFM and HIM and clearly revealed robust polymerized 2D-nanomembranes for DiynePC and PTPE. In contrast, unpolymerized DPhPC could not form stable 2Dnanomembranes. Further investigations were conducted with DiynePC and PTPE transferred onto HOPG. Adlayers of DiynePC exhibited a rigid structure after polymerization showing no alignment to the underlying crystalline structure of the HOPG. In contrast, polymerized PTPE organized into lamellae, indicating a flexible structure. We found that the mechanical properties of polymerized 2D-nanomembranes were attributed to the amount of diacetylene groups per molecule. This was confirmed by ATR-FTIR studies at the monolayers, which revealed a distinct reduction in the number of the diacetylene groups by the 17


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polymerization process.

Associated content DSC measurements (Fig. S1), conducted transfer-techniques (Fig. S2), AFM line profiles of freestanding membranes (Fig. S3), AFM images of lipid alignment to HOPG (Fig. S4), structural models for the molecular arrangement (Fig. S5), AFM line profile of polymerized DiynePC monolayer (Fig. S6) and UV-VIS spectra of PTPE and DiynePC (Fig. S7).

Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft (DFG) under grant AN370/7-1 and by a Heisenberg fellowship to TK (KO3580/4-1). We thank Marlén-Viviane Eickmann for the preliminary studies on monomeric DiynePC on HOPG by AFM, Christoph Pelargus for his assistance with the HIM measurements, Niklas Biere for valuable discussions and suggestions on the AFM and Carina Dargel for her assistance with the DSC measurements. We also thank Dr. Katja Tönsing for her support.

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Characterization of Robust and Free-Standing 2D-Nanomembranes of

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