Surface Patterning DOI: 10.1002/anie.201204836 Immobilization of Liposomes and Vesicles on Patterned Surfaces by a Peptide Coiled-Coil Binding Motif** Jens Voskuhl, Christian Wendeln, Frank Versluis, Eva-Corinna Fritz, Oliver Roling, Harshal Zope, Christian Schulz, Stefan Rinnen, Heinrich F. Arlinghaus, Bart Jan Ravoo,* and Alexander Kros* Coiled-coil motifs are abundant in proteins where they exhibit an array of functions like gene regulation, [1] cell signaling, [2] transport of small molecules, [3] and membrane fusion. [4] Native SNARE proteins control fusion processes between and within cells (for example, exocytosis). The common feature of all these coiled-coils is that at least two a- helical peptide strands bind, thereby acting as molecular Velcro. The specific molecular recognition between helices has enabled scientists to develop self-assembled, highly structured materials based on the coiled-coil motif. [5–7] Herein we describe a completely new function for the coiled-coil peptide binding units, namely their application in materials science and surface modification. We report that an a-helical coiled-coil pair exclusively forms parallel hetero- dimers, denoted “peptide E” (EIAALEK) 3 and “peptide K” (KIAALKE) 3 and acts as selective recognition unit through which liposomes and cyclodextrin (CD) vesicles can be selectively immobilized in surface patterns obtained using microcontact printing. The immobilization of vesicles and liposomes by recog- nition units, such as complementary DNA strands, [8] electro- static interactions [9] and protein–ligand pairs, [10] has attracted increasing attention in recent years. By using these recog- nition units, it is possible to attach liposomes and vesicles to a variety of substrates, to prepare microarrays of lipo- somes, [11, 12] to construct sensing platforms, [13–15] and to inves- tigate reactions in immobilized liposomes, [16–19] including single-molecule reactions. [20] In this study we used microcontact printing to produce well-defined patterns of peptide E (1) by the formation of a covalent bond (i.e. a triazole unit) between an azide self- assembled monolayer (SAM) and the alkyne-terminated peptide E in the presence of Cu I . Surface patterning by microcontact chemistry has been widely studied by several groups during the last years, and it was successfully applied for the preparation of various functional surfaces, including carbohydrate, [21, 22] DNA, [23] and peptide microarrays. [24] Pep- tide E is able to bind to the complementary peptide K by forming a coiled-coil binding motif, which has previously been used to induce liposomal fusion processes in buffered aqueous media. [25–27] The main benefits of this complementary peptide binding motif include its simplicity, selectivity, pH and temperature stability, and low cost. Figure 1 describes the process of liposome and vesicles immobilization on patterned surfaces, and Scheme 1 shows the molecular structures of the key components. After functionalization of a glass or silicon slide with an azide SAM (see the Supporting Information), patterns of the alkyne-terminated peptide E (1) were prepared by inducing the Cu I -catalyzed azide–alkyne cycloaddition (CuAAC) with a structured polydimethylsiloxane (PDMS) stamp. The inter- space was passivated with the alkyne–tetraethylene glycol derivative 2. After incubation with either liposomes deco- rated with peptide K 4 or CD vesicles [28] functionalized by host–guest complexation with peptide K 5, patterns of liposomes/CD vesicles were obtained. The noncovalent, reversible nature of liposome immobilization was checked by washing the liposome surface with ethanol, or with an excess of a buffered b-CD solution for the immobilized CD vesicles. The immobilization of peptide E (1) results in a significant increase in the wettability of the surface, which is consistent with the hydrophilic nature of peptide E. This behavior was observed by water condensation on the printed surface, which shows clear dot patterns of water at the hydrophilic islands (Supporting Information, Figure S1). After CuAAC using flat PDMS stamps, the static water contact angle decreased from around 838 for the azide SAM to around 508 for the surface immobilized peptide E (Supporting Information, Figure S1). Furthermore, the presence of carbonyl carbon atoms and amide groups was verified by X-ray photoelectron spectros- copy (XPS). After printing by using a flat PDMS stamp, an additional band (288.5 eV) in the C 1s region belonging to the [*] Dr. J. Voskuhl, M. Sc. F. Versluis, M. Sc. H. Zope, Dr. A. Kros Soft Matter Chemistry, Leiden Institute of Chemistry P.O. Box 9502, 2300 RA Leiden (The Netherlands) E-mail: a.kros@chem.leidenuniv.nl Dr. C. Wendeln, M. Sc. E.-C. Fritz, M. Sc. O. Roling, Dr. C. Schulz, Prof. Dr. B. J. Ravoo Organic Chemistry Institute and CeNTech, Westfälische Wilhelms- Universität Münster, Corrensstrasse 40, 48149 Münster (Germany) E-mail: b.j.ravoo@uni-muenster.de Dipl.-Phys. S. Rinnen, Prof. Dr. H. F. Arlinghaus Physikalisches Institut, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 10, 48149 Münster (Germany) [**] A.K. acknowledges the support of the European Research Council by an ERC starting grant. B.J.R. acknowledges DFG for financial support (grant Ra 1732/1). Silicon wafers were kindly donated by Siltronic AG. Patrick Seelheim and Prof. Dr. H. J. Galla are acknowledged for access to and discussion of QCM-D measure- ments. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201204836. . Angewandte Communications 12616  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012, 51, 12616 –12620