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Self-assembly of Fmoc-diphenylalanine inside liquid marbles
Colloids and Surfaces B: Biointerfaces 97 (2012) 43–50
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Self-assembly of Fmoc-diphenylalanine inside liquid marbles Hans-Georg Braun ∗ , André Zamith Cardoso Leibniz Institut of Polymer Research Dresden, Max Bergmann Center of Biomaterials, D-01069 Dresden, Hohe Strasse 6, Germany
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Article history: Received 18 January 2012 Received in revised form 13 March 2012 Accepted 19 March 2012 Available online 27 April 2012 Keywords: Liquid marbles Peptide gels Microreactor Interface bottom-up design Lycopodium clavatum spores Electron diffraction Micro-to-nano hierarchical self-assembly Supramolecular biochemistry
a b s t r a c t Liquid marbles made from Lycopodium clavatum spores are used to encapsulate aqueous solutions of 9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). Acidification of the Fmoc-FF solution at the liquid/air interface of the liquid marble triggers the self-assembly of ribbon-like peptide fibrils into an ultrathin peptide membrane (50–500 nm). The membrane incorporates the lycopodium microparticles and as a result stabilizes the liquid marble against collapse, that could otherwise occur through particle disintegration at the floating interphase. Ultrathin nanostructured peptide membrane formation at the liquid/air interface is also observed within artificial microstructured floating objects. Thus, peptide membranes formed were inspected by SEM and TEM. Electron diffraction data reveal information about the molecular organization inside the oligopeptide membranes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Liquid droplets of millimeter size can easily become wrapped up in a layer of meso- or microsized particles to form a so-called liquid marble [1–3] (see Fig. 1A). The process of liquid marble formation is rather simple, while a droplet is rolled over a layer of loose packed nm- to m-sized powder the particles attach to the droplet surface and engulf it. The powder particles can be either hydrophilic [4] or hydrophobic [5]. The powder particles at the liquid interface assemble into rafts and finally into a dense packed layer which creates a highly structured liquid/air interface around the encapsulated droplet. This microstructured interface of the liquid marble prevents wetting of the entrapped liquid on any other solid or even liquid surface [6,7] as long as the microstructure is not disrupted. In fact, the rough liquid/solid interface of the liquid marble generates a wetting state similar to that described for the Cassie–Baxter configuration of ultrahydrophobic surfaces [8,9]. The particles used for liquid marble stabilization may originate from biological material such as Lycopodium clavatum spores [5] or they may be synthetic particles with functional properties such as: magnetic colloids [10] which can be used to manipulate liquid marbles in a magnetic field; carbon black nanoparticles [11] that can be used to introduce electrical conductivity into the marbles;
∗ Corresponding author. E-mail address: [email protected] (H.-G. Braun). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.03.028
and polymer colloids [12] which respond to variable pH and can be used to generate stimuli responsive liquid marbles. Lycopodium particles are of particular interest because the interior of the spores can be extracted leaving the microstructured capsule with an extreme temperature resistant membrane made of polymer material that has not completely been characterized up to now [13]. The residual microcapsule was used as a drug carrier [14] or, its surface, was used as to physisorb enzymes, therefore adding biofunctionality [15]. Hence, lycopodium spores are not only suitable to stabilize the liquid marble, but also to introduce chemical functionality into liquid marbles, which then represent versatile microreactors [16]. Although evaporation of the liquid entrapped in a liquid marble is significantly reduced, as compared to bare liquid surface [4,17], it still occurs over time and, as a consequence, it can cause collapse the liquid marble during the shrinking process. Nevertheless, it was shown that liquid marbles containing monomer solutions can be stabilized by polymerization [18]. Our approach to stabilize the liquid marble structure focus on pH induced gelation of oligopeptide based hydrogelators [19] entrapped in the liquid marble (Fig. 1). In particular, Fmoc-FF has been extensively studied with respect to pH induced gelation [20,21], which is triggered by pH change from 10.2 to 5.2. Successive protonation of the carboxylic group of Fmoc-FF reduces the negative charge and therefore electrostatic repulsion by the carboxylate anion. In addition the protonated groups become available to molecular aggregation through hydrogen bond formation. The stepwise self-assembly process finally results in a hydrogel network [21]. Gelation in bulk was shown to form an extended
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3-dimensional network of fibrillae with pores from about 10 nm to 1 m, studied by cryo-SEM experiments [22]. 2. Experimental methods 2.1. Materials L. clavatum spores, indicators (cresol red and bromocresol purple) and salts (lithium chloride, potassium carbonate, sodium chloride and potassium chloride) were obtained from Sigma–Aldrich and used without further purification. Sodium hydroxide was obtained from Merk, carbon dioxide from Air Liquid and Fmoc-Diphenylalanine was purchased from Bachem. 2.2. Preparation of L. clavatum liquid marbles with incorporated Fmoc-FF solution In a typical preparation, 10 mmol L−1 solution of Fmoc-FF in millipore water was prepared adapting a procedure described by Ulijn et al. [22] (see Supplementary material). For liquid marble formation (Fig. 1A) a droplet volume of 10 L was deposited on a layer of L. clavatum spores (Fig. 1B) placed in a Petri dish. Intimate contact and rolling the droplet around, completely encapsulates it in a shell of the spores. To control the evaporation conditions from the liquid marbles, they were kept under ambient humidities, which were achieved through vapor pressure control by appropriate salt solutions [23].
Acidification of the encapsulated Fmoc-FF solution to trigger self-assembly was achieved by carbon dioxide exposure from the gas phase. The pH change inside liquid marbles (Fig. 1C) could be detected by color change of appropriate indicators added to the initial solution. The gelation was expected to reach an equilibrium in the first 30 min, indicated by color change and stability of the system. Liquid marbles were handled for wet or dry characterization only after this period. 2.3. Preparation free standing electron transparent Fmoc-FF films A copper grid typically used for as a supporting grid for TEM inspection was placed as free floating object [24] onto an basic Fmoc-FF solution prepared as described before. The copper grid could be used without further surface treatment. Acidification at the liquid/gas interface was achieved by carbon dioxide atmosphere (see Supplementary material). After a short time, typically 5–20 min, ribbon-like aggregates assembled to form an ultrathin film at the meniscus interface inside the meshes. The self supporting peptide film could be used without further treatment for TEM and SEM studies. 2.4. Characterization methods Morphological characterization of all specimen was done in an field emission SEM (Zeiss DSM 962 Gemini) on unsputtered samples. Imaging is done at low electron beam voltage (0.3 KeV E0 1 KeV) in order to avoid surfaces charging.
Fig. 1. (A) Lycopodium clavatum liquid marble on a solid surface. (B) An SEM image of Lycopodium clavatum spores, constituted by a shell, named the sporopollenin exine capsule. This capsule have a cellular morphology with hexagonal or pentagonal shaped indentations of 5 m diameter. The indentations are interconnected by submicrometer sized holes inside the indentation walls. (C) pH induced Fmoc-FF gelation system in Lycopodium clavatum liquid marble.
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Additionally the penetration depth of electrons is only a few ten nanometers under these conditions causing contrast in thin films to increase. Observations of macroscopic liquid marbles were recorded by a stereo microscope (Zeiss Stemi 2000-C) connected to a Nikon D100 camera and the SEM. TEM (Zeiss Libra 120) was used in diffraction mode to characterize free standing ultrathin peptide films. The camera length for quantification of lattice spacings was calibrated with TlCl standard (PLANO) and diffraction patterns were recorded with a 2K × 2K slow scan CCD camera (TRS sharp:eye, Troendle, Germany). Selected area electron diffraction patterns (SAED) were analyzed with the public domain software ImageJ [25].
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3.1. Morphological observations of the Fmoc-FF system in the liquid marble
the particle layer, which separates the liquid inside the marble from the underlying substrate, gets disrupted, capillary bridges between engulfed liquid and the underlying surface can form and the surface will immediately become wetted. The particle coated interface disrupts and particles spread over the surface. Otherwise, if the interior of the liquid marble has become stabilized by polymerization, the liquid marble is prevented from disintegration, but as the liquid phase inside the marble will transform into a solid phase or at least a bulk gel phase, an essential feature of liquid marbles will get lost. Fig. 2A reveals the typical shape of a liquid marble in which an Fmoc-FF solution has initially been encapsulated and selfassembled by exposure to carbon dioxide atmosphere, prior to drying. The marble is neither disintegrated nor has it kept its original spherical shape. Instead SEM microscopy of the specimen reveals a shape like the shell of an deflated object (Fig. 2A). This deflated-like liquid marble appears gradually during the drying step. Observed from the interior, it is possible to identify an interconnected membrane which attaches to the L. clavatum spores (Fig. 2B) and which we assume to consist of self-assembled FmocFF. This behavior differs remarkable from that to be expected, if a bulk gel phase would have formed during acidification of the Fmoc-FF solution [21,22,26]. Such a volume gel phase would have stabilized the spherical shape of the marble even after drying [20]. The following observations strongly support a mechanical stabilization of the Fmoc-FF treated marbles:
In general, liquid marbles disintegrate during the evaporation process of the engulfed liquid phase due to mechanical stresses within the particle layer (shrinking process). If during this process
1. Liquid marbles as-described were capable to float on water surface for 5 days without deflation, collapse or disintegration of L. clavatum particles.
3. Results and discussion First, SEM morphological studies of self-assembled Fmoc-FF membranes at the liquid/air interface are discussed and a model for membrane formation is proposed. Second, TEM diffraction experiments were done to characterize structural features inside the free standing membranes on a molecular scale. ATR-IR spectra of the Fmoc-FF layers prepared by the interfacial assembly were almost similar to that observed for bulk gelation. A representative spectrum is part of the Supplementary materials.
Fig. 2. (A) Deflated-like Fmoc-FF liquid marble formed after the final drying step; (B) inside view of the liquid marble reveals a Fmoc-FF membrane interconnectivity between the individual lycopodium spores, formed at the liquid/air interface.
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Fig. 3. (A) Bare Lycopodium clavatum spore; (B) thick peptide membrane coating a spore; (C) thin peptide membrane coating a spore (approximately 50 nm). (D) Closer view of the peptide membrane at one indentation of the Lycopodium clavatum spore in (C).
2. Even after drying, the deflated marble shows a remarkable mechanical stability and deflated marbles could be handled by tweezers. It was found that the thickness of the membrane strictly depends on the time allowed for gelation. The evaporation rate, present during gelation could be reduced by appropriate humidity control. High humidity favors the formation of thicker membranes than low humidity conditions. Another relevant phenomena is the heterogeneity in membrane thickness through the liquid marble interface. It is clear, from the deflated-like shape and from SEM inspection of the membrane fragments, that the thickness on the superior part of the liquid marble was larger than the thickness on the interior part, which turn out to be difficult to quantify in this multiscale system. Nevertheless, with this L. clavatum (Fig. 3A) gelation system, the formation of an interwoven network of ribbons was expected to constitute the observed peptide membrane (Fig. 3B–D). Over several observations under different experimental conditions, it became clear that the different times chosen for carbon dioxide acidification of the aqueous phase, originated membranes of different thickness. In short carbon dioxide exposure (under 30 min) a thin membrane the individual ribbons could be recognized (Fig. 3C and D), while in longer (over 30 min) a thicker membrane of the overlapping ribbons did not allow identification of individual ribbons in the peptide (Fig. 3B). The irregular folding of the deflated liquid marbles appears most probably due to capillary forces between the evaporating liquid and the solid interface during the water evaporation. Even thought, the peptide membrane
bends and follows the liquid/air interface morphology structured by the L. clavatum particles. The membrane thickness differences in the same liquid marble might happen due to the same physical phenomena driving droplet evaporation [27]: such as heat transfer by convection (Marangoni effects and normal convection); modified wettability due to molecular scale interactions between the liquid and the particles; and the the different vapor mass diffusion around the drop. Another possibility might be related to the higher access of the carbon dioxide though the superior part of the liquid marble, in contrast with the lower part. Another remarkable observation for the self-assembly inside the lycopodium based liquid marbles is the peptide membrane formation not only between the lycopodium particles, but also across the open chambers, arranged in a honeycomb like morphology (Fig. 3). The membrane forming ribbons always span over the cavity of the spores. They never appeared coated at the interior of the cavity. In a closer look at this indentations, the ribbon assemblies are seen to attach to each-other coating the individual indentations (Fig. 3D). With all the observations in mind, a model for the pH induced gelation at the gas/liquid/solid interface of L. clavatum encapsulated liquid marble is proposed and schemed in Fig. 4. Initially, microparticles arranged in a highly condensed packing are floating at the liquid interface of the alkaline Fmoc-FF solution. The movement and interaction of the floating microparticles does not only depend on the surface tension of the aqueous solution, but also to size, shape and electronic charge interactions [28,29] of the floating particles. Upon exposure of the liquid marbles to carbon dioxide, carbon dioxide dissolution and finally dissociation
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Fig. 4. Schematic view to illustrate the model proposed for Fmoc-FF self-assembly at interface, before (A) and after (B) the pH induced assembly of the Fmoc-FF molecules. This interface is an approximately 80 m cut-through sectional view of the liquid marble. Lycopodium particles float at the liquid/air interface between the alkaline Fmoc-FF drop and the gas phase. This surface is non-planar and strongly determined by capillary forces between the liquid and the floating particles. Finally peptide membranes are formed which interconnect the microparticles.
into hydrogenecarbonate ions reduces the pH at liquid/air interface inside the liquid marble and consequently creates a pH gradient inside. Acidification triggers the self-assembly process into ribbon like peptide aggregates at the liquid/air interface resulting in a membrane formation. As this membrane formation occurs only at the liquid/gas interface, the resulting membrane/particle morphology is to discuss as a replication of this interface. Membrane formation across and not inside the cavities of the L. clavatum spores clearly indicates that the cavities withstand the penetration of liquid at the interface both into these spore cavities but also into the gas filled gaps between floating spores. This is a consequence of the Laplace pressure which suppresses the penetration of liquid into the lycopodium cavities. The interconnection of the cavities by holes as it is evident from Fig. 3A, guarantees a carbon dioxide transport to the liquid/air interface of the single cavities. The gas transport in between the cavities, through their pores, allows a local pH change at the liquid/air interface of an individual cavity and therefore triggers the membrane formation across the cavity. If the carbon dioxide source is detached, the pH of the solution slightly increases again, which can be followed by an indicator (e.g. with bromocresol red, the final pH after detachment is above pH 6.8) encapsulated in the liquid marble. A particular difference of this system resides in the avoidance of high local gradients in the alkaline Fmoc-FF solution, which in the bulk may suppress inhomogeneous Fmoc-FF fiber growth.
3.2. Morphological observations of the Fmoc-FF system in floating grids In order to study the membrane formation with respect to a single floating L. clavatum spore (Fig. 5 (I)) or at the bare liquid/air interface (Fig. 5 (II)) an experimental setup as schemed in Fig. 5 was used. A copper grid with appropriate mesh size (hexagonal meshes) is placed as microstructured floating object at the liquid/air interface of a Fmoc-FF solution. In some of the grids cavities single lycopodium spores were placed (Fig. 5 (I)), while others were free of particles and therefore create a bare liquid/air interface (Fig. 5 (II)). Through this in-grid system, both single microparticle to membrane connectivity (Fig. 5, Case I.A and B) and thin membranes can be formed with high thickness control (Fig. 5, Case II.C) for microand nano-structure characterization [24]. In the Case I, the patterning of the membrane formation at the grid cavities allows observation of the connectivity from both sides of the microparticle–membrane connection. The bottom view (Fig. 5A), confirms that the membrane formation follows the liquid/air interface and connects to the spore exine capsule. This results in free standing peptide membranes at each of the bottom indentations of the L. clavatum particle. In the top view (Fig. 5B), the conditions are different, since no contact with the liquid phase exists, the membrane is not formed and does not completely coats the microparticles. This situation, permits diffusion of the gas
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Fig. 5. Schematics of in-grid gelation system: Case I: single lycopodium microstructure (green) integrated into the peptide membrane (red); Case II: free standing peptide membrane (red). Morphological (SEM) observations of spore-membrane at a single grid mesh, from the top side (A) and the bottom side (B). (C) SEM image of the free standing peptide membrane reveals distinguishable single ribbons of thickness approximately 50 nm. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
through the pores in the spore exine capsule, which allows the diffusion of the carbon dioxide at the liquid/air interface of the microparticle indentations. As observed initially in Fig. 2B, not all indentations are filled in all lycopodium spores. These phenomena might be related to the time carbon dioxide has to diffuse through the inter-indentation cavities, essential for this liquid/air gelation at the lycopodium spore (in water) indentations. A representative film segment which has formed as free standing Fmoc-FF membrane inside a single mesh of the grid and which was suitable for TEM diffraction experiments is shown in Fig. 5C. Although the low density density of peptide ribbons causes defects as the single hole shown the ultrathin membrane is remarkable stable with respect to electron radiation damage in the SEM. The samples reveal a single peptide ribbon 2D-layer perpendicular to the electron beam, with an approximate thickness of 50 nm, when the exposure time to carbon dioxide is set to 5 up to 10 min. Longer than this times revel a increasingly thick layer (observed via SEM). On one hand, multi-ribbon layers, for the purpose described in the next section (in the selected area for diffraction), might introduce additional features to the following described diffraction experiments, on another hand this might be exploited for biosensor applications, kinetics of diffusion through hydrogelcells membranes or pharmacokinetics and pharmacodynamics in vitro studies of hydrogel membranes with a pharmacological compound of interest, done at a relative low cost and over a large area. 3.3. Electron diffraction experiments A typical SAED pattern obtained from an Fmoc-FF assembly of extended ribbons within an ultrathin membrane is shown in Fig. 6 (left). The sample was prepared as-described, a free standing thin membrane grown within the holes of a floating TEM grid. The pattern is a superposition of two zone axis diffraction patterns, one
appearing as a set of well defined sharp reflections, which are located on an orthogonal set of equidistant layer lines. The axis aligned with equidistant reflections of a periodic spacing of 23 A˚ is assigned the reciprocal axis a∗ , while the orthogonal axis aligned refections with a real lattice spacing of 4.8 A˚ is assigned as the reciprocal b∗ axis. The second pattern in this figure generates a set of reflections, which show the same lattice spacings dh k l as those already measured for the single crystal diffraction pattern. The cor responding reciprocal lattice vectors a∗ and b∗ are indicated in Fig. 6 (left). The broadening of the reflections with an arc-segment (red dashed circles) indicates a texture of several crossing highly oriented ribbons, and additionally have a similar zone axis as both axis with the extended single crystal ribbon, as well as with respect to each other. The common zone axis is indicated as the [0 0 w] axis (Fig. 6A). A second typical SAED pattern taken from an area with a large assembly of ribbons crossing each other is shown in Fig. 7. It is worth noticing that all rings observed can be indexed using the dh k l spacings calculated from the single crystalline diffraction pattern that has been described previously (Fig. 6). This clearly indicates that although the ribbons are randomly oriented, they are randomly oriented around one common zone axis, similar to the one observed for a single ribbon [0 0 w] (Fig. 6 (left)). Curiously, the ribbons are not twisted at least within the sample area contributing to the SAED. The lack of twisting may result from the self-assembly of ribbon structures at the liquid/air interface. Concerning a discussion of the observed lattice spacings of 23 A˚ and 4.8 A˚ on the perpendicular axis within a molecular model, we refer to the data and structure proposal recently published by Ulijn et.al. [20]. Although the group also could not determine a complete set of crystallographic data on an elementary cell, they proposed a molecular model based on X-ray powder diffraction data and on the interpretation of periodic striations observed in high resolution TEM image analyzed by FFT analysis. Ulijn et. al.
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Fig. 6. Selected area electron diffraction (SAED) pattern of Fmoc-FF membrane ribbons (left) and schematic arrangement of nanostructural entities (right). The scheme (A) indicates the assigned axis to the diffractogram on the left. The scheme (B and C) illustrates various packing arrangements for a dense packed assembly of helical motifs, either as perfect 3D crystal (B) or as assembly with translational and/or rotational disorder of stacked helices (C).The scheme (D) figures an alternative, so called packed column model [30].
assumed a helical arrangement of individual Fmoc-FF entities due to – interactions of the aromatic groups and further stabilization by hydrogen bonds along the stacking or helical axis. The helical assemblies simplified as tubes are dense packed and the periodicity between the cylinders should be represented by the 23 A˚ spacing. Ulijn et.al. assigned the spacing between the cylinders to 26 A˚ as concluded from a strong reflection in the WAXS powder diagram although this is in conflict with the spacing of 35 A˚ which he proposed from the FFT of band periodic features, observed in the TEM images. Concerning different assembly conditions and less accuracy evaluation of lattice parameters, from a electron beam sensitive specimen by TEM, as compared to X-ray diffraction data. We assume the observed difference in lattice spacing acceptable.
Based on the packing of cylindrical units within the ribbons as it is indicated in Fig. 6B and C, only the packing shown in Fig. 6B would be reasonable resulting in a single crystalline zone axis diffraction pattern. Any translational or rotational disorder (Fig. 6C) of the cylinders can be excluded due to the lack of streaks, which should appear along the (h 1 0) lattice planes. The 4.8 A˚ spacing observed for the b-axis, which is close to the 4.3 A˚ spacing, previously discussed, should originate from the periodic spacing of aromates along the cylinder axis. Nevertheless it should be mentioned that aside the helical Fmoc-FF model, a planar compact columns stacking model as schemed in Fig. 6D could offer an alternative packing arrangement as-described in literature [30], for many similar gelation systems of short sequenced peptides. A recent X-ray diffraction
Fig. 7. Indexed electron diffraction pattern (left) of a ultrathin, few layered ribbon structures, with a main ribbon direction as indicated the meridional (h 0 0) reflections and the longitudinal (0 k 0) reflections. The fit of the main crystallographic axis previously described with the circular pattern, suggests a general flat ribbon assembly randomly oriented in a plane normal to the diffraction beam [0 0 w].
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result on shear oriented Fmoc-FF gels [31], indicates a cross -type structure (in line with Fig. 6D). 4. Conclusions Liquid marbles were stabilized by pH triggered membrane formation, of self-assembling peptide units, in particular of Fmoc-FF. The interfacial self-assembly process controlled by acidification with gaseous carbon dioxide, results in interwoven meshes of peptide ribbons. The crystallographic data obtained from electron diffraction experiments, extend the existing information based on X-ray powder diffraction of the Fmoc-FF system. The mechanical stabilization will open potential application fields for liquid marbles as chemical microreactors, as encapsulating systems for removal of unwanted materials, as drug carriers or as sensing devices when filled with appropriate analytes. Mechanical stabilization of marbles will be a necessary condition to actively move these on surfaces, for example, by surface acoustic wave based movement. A complete crystallographic evaluation of lattice parameters by TEM diffraction experiments is a future goal. Acknowledgements A.Z.C. gratefully acknowledges the financial support from an Erasmus Mundus scholarship. We thank Dr. P. Formanek for technical assistance with TEM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2012.03.028. References [1] P. Aussillous, D. Quere, Properties of liquid marbles, Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 462 (2067) (2006) 973–999. [2] G. McHale, N. Newton, Liquid marbles: principles and applications, Soft Matter 7 (5) (2011) 5473–5481. [3] E. Bormashenko, Liquid marbles: properties and applications, Curr. Opin. Colloid Interface Sci. 16 (2011) 266–271. [4] M. Dandan, H.Y. Erbil, Evaporation rate of graphite liquid marbles: comparison with water droplets, Langmuir 25 (14) (2009) 8362–8367. [5] P. Aussillous, D. Quere, Liquid marbles, Nature 411 (6840) (2001) 924–927. [6] E. Bormashenko, Y. Bormashenko, A. Musin, Z. Barkay, On the mechanism of floating and sliding of liquid marbles, Chemphyschem 10 (4) (2009) 654–656. [7] B.P. Binks, A.N. Boa, M.A. Kibble, G. Mackenzie, A. Rocher, Sporopollenin capsules at fluid interfaces: particle-stabilised emulsions and liquid marbles, Soft Matter 7 (8) (2011) 4017–4024. [8] A.B.D. Cassie, S. Baxter, Wettability of porous surfaces, Trans. Faraday Soc. 40 (1944) 0546–0550.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Self-assembly of Fmoc-diphenylalanine inside liquid marbles Hans-Georg Braun ∗ , André Zamith Cardoso Leibniz Institut of Polymer Research Dresden, Max Bergmann Center of Biomaterials, D-01069 Dresden, Hohe Strasse 6, Germany
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Article history: Received 18 January 2012 Received in revised form 13 March 2012 Accepted 19 March 2012 Available online 27 April 2012 Keywords: Liquid marbles Peptide gels Microreactor Interface bottom-up design Lycopodium clavatum spores Electron diffraction Micro-to-nano hierarchical self-assembly Supramolecular biochemistry
a b s t r a c t Liquid marbles made from Lycopodium clavatum spores are used to encapsulate aqueous solutions of 9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). Acidification of the Fmoc-FF solution at the liquid/air interface of the liquid marble triggers the self-assembly of ribbon-like peptide fibrils into an ultrathin peptide membrane (50–500 nm). The membrane incorporates the lycopodium microparticles and as a result stabilizes the liquid marble against collapse, that could otherwise occur through particle disintegration at the floating interphase. Ultrathin nanostructured peptide membrane formation at the liquid/air interface is also observed within artificial microstructured floating objects. Thus, peptide membranes formed were inspected by SEM and TEM. Electron diffraction data reveal information about the molecular organization inside the oligopeptide membranes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Liquid droplets of millimeter size can easily become wrapped up in a layer of meso- or microsized particles to form a so-called liquid marble [1–3] (see Fig. 1A). The process of liquid marble formation is rather simple, while a droplet is rolled over a layer of loose packed nm- to m-sized powder the particles attach to the droplet surface and engulf it. The powder particles can be either hydrophilic [4] or hydrophobic [5]. The powder particles at the liquid interface assemble into rafts and finally into a dense packed layer which creates a highly structured liquid/air interface around the encapsulated droplet. This microstructured interface of the liquid marble prevents wetting of the entrapped liquid on any other solid or even liquid surface [6,7] as long as the microstructure is not disrupted. In fact, the rough liquid/solid interface of the liquid marble generates a wetting state similar to that described for the Cassie–Baxter configuration of ultrahydrophobic surfaces [8,9]. The particles used for liquid marble stabilization may originate from biological material such as Lycopodium clavatum spores [5] or they may be synthetic particles with functional properties such as: magnetic colloids [10] which can be used to manipulate liquid marbles in a magnetic field; carbon black nanoparticles [11] that can be used to introduce electrical conductivity into the marbles;
∗ Corresponding author. E-mail address: [email protected] (H.-G. Braun). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.03.028
and polymer colloids [12] which respond to variable pH and can be used to generate stimuli responsive liquid marbles. Lycopodium particles are of particular interest because the interior of the spores can be extracted leaving the microstructured capsule with an extreme temperature resistant membrane made of polymer material that has not completely been characterized up to now [13]. The residual microcapsule was used as a drug carrier [14] or, its surface, was used as to physisorb enzymes, therefore adding biofunctionality [15]. Hence, lycopodium spores are not only suitable to stabilize the liquid marble, but also to introduce chemical functionality into liquid marbles, which then represent versatile microreactors [16]. Although evaporation of the liquid entrapped in a liquid marble is significantly reduced, as compared to bare liquid surface [4,17], it still occurs over time and, as a consequence, it can cause collapse the liquid marble during the shrinking process. Nevertheless, it was shown that liquid marbles containing monomer solutions can be stabilized by polymerization [18]. Our approach to stabilize the liquid marble structure focus on pH induced gelation of oligopeptide based hydrogelators [19] entrapped in the liquid marble (Fig. 1). In particular, Fmoc-FF has been extensively studied with respect to pH induced gelation [20,21], which is triggered by pH change from 10.2 to 5.2. Successive protonation of the carboxylic group of Fmoc-FF reduces the negative charge and therefore electrostatic repulsion by the carboxylate anion. In addition the protonated groups become available to molecular aggregation through hydrogen bond formation. The stepwise self-assembly process finally results in a hydrogel network [21]. Gelation in bulk was shown to form an extended
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3-dimensional network of fibrillae with pores from about 10 nm to 1 m, studied by cryo-SEM experiments [22]. 2. Experimental methods 2.1. Materials L. clavatum spores, indicators (cresol red and bromocresol purple) and salts (lithium chloride, potassium carbonate, sodium chloride and potassium chloride) were obtained from Sigma–Aldrich and used without further purification. Sodium hydroxide was obtained from Merk, carbon dioxide from Air Liquid and Fmoc-Diphenylalanine was purchased from Bachem. 2.2. Preparation of L. clavatum liquid marbles with incorporated Fmoc-FF solution In a typical preparation, 10 mmol L−1 solution of Fmoc-FF in millipore water was prepared adapting a procedure described by Ulijn et al. [22] (see Supplementary material). For liquid marble formation (Fig. 1A) a droplet volume of 10 L was deposited on a layer of L. clavatum spores (Fig. 1B) placed in a Petri dish. Intimate contact and rolling the droplet around, completely encapsulates it in a shell of the spores. To control the evaporation conditions from the liquid marbles, they were kept under ambient humidities, which were achieved through vapor pressure control by appropriate salt solutions [23].
Acidification of the encapsulated Fmoc-FF solution to trigger self-assembly was achieved by carbon dioxide exposure from the gas phase. The pH change inside liquid marbles (Fig. 1C) could be detected by color change of appropriate indicators added to the initial solution. The gelation was expected to reach an equilibrium in the first 30 min, indicated by color change and stability of the system. Liquid marbles were handled for wet or dry characterization only after this period. 2.3. Preparation free standing electron transparent Fmoc-FF films A copper grid typically used for as a supporting grid for TEM inspection was placed as free floating object [24] onto an basic Fmoc-FF solution prepared as described before. The copper grid could be used without further surface treatment. Acidification at the liquid/gas interface was achieved by carbon dioxide atmosphere (see Supplementary material). After a short time, typically 5–20 min, ribbon-like aggregates assembled to form an ultrathin film at the meniscus interface inside the meshes. The self supporting peptide film could be used without further treatment for TEM and SEM studies. 2.4. Characterization methods Morphological characterization of all specimen was done in an field emission SEM (Zeiss DSM 962 Gemini) on unsputtered samples. Imaging is done at low electron beam voltage (0.3 KeV E0 1 KeV) in order to avoid surfaces charging.
Fig. 1. (A) Lycopodium clavatum liquid marble on a solid surface. (B) An SEM image of Lycopodium clavatum spores, constituted by a shell, named the sporopollenin exine capsule. This capsule have a cellular morphology with hexagonal or pentagonal shaped indentations of 5 m diameter. The indentations are interconnected by submicrometer sized holes inside the indentation walls. (C) pH induced Fmoc-FF gelation system in Lycopodium clavatum liquid marble.
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Additionally the penetration depth of electrons is only a few ten nanometers under these conditions causing contrast in thin films to increase. Observations of macroscopic liquid marbles were recorded by a stereo microscope (Zeiss Stemi 2000-C) connected to a Nikon D100 camera and the SEM. TEM (Zeiss Libra 120) was used in diffraction mode to characterize free standing ultrathin peptide films. The camera length for quantification of lattice spacings was calibrated with TlCl standard (PLANO) and diffraction patterns were recorded with a 2K × 2K slow scan CCD camera (TRS sharp:eye, Troendle, Germany). Selected area electron diffraction patterns (SAED) were analyzed with the public domain software ImageJ [25].
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3.1. Morphological observations of the Fmoc-FF system in the liquid marble
the particle layer, which separates the liquid inside the marble from the underlying substrate, gets disrupted, capillary bridges between engulfed liquid and the underlying surface can form and the surface will immediately become wetted. The particle coated interface disrupts and particles spread over the surface. Otherwise, if the interior of the liquid marble has become stabilized by polymerization, the liquid marble is prevented from disintegration, but as the liquid phase inside the marble will transform into a solid phase or at least a bulk gel phase, an essential feature of liquid marbles will get lost. Fig. 2A reveals the typical shape of a liquid marble in which an Fmoc-FF solution has initially been encapsulated and selfassembled by exposure to carbon dioxide atmosphere, prior to drying. The marble is neither disintegrated nor has it kept its original spherical shape. Instead SEM microscopy of the specimen reveals a shape like the shell of an deflated object (Fig. 2A). This deflated-like liquid marble appears gradually during the drying step. Observed from the interior, it is possible to identify an interconnected membrane which attaches to the L. clavatum spores (Fig. 2B) and which we assume to consist of self-assembled FmocFF. This behavior differs remarkable from that to be expected, if a bulk gel phase would have formed during acidification of the Fmoc-FF solution [21,22,26]. Such a volume gel phase would have stabilized the spherical shape of the marble even after drying [20]. The following observations strongly support a mechanical stabilization of the Fmoc-FF treated marbles:
In general, liquid marbles disintegrate during the evaporation process of the engulfed liquid phase due to mechanical stresses within the particle layer (shrinking process). If during this process
1. Liquid marbles as-described were capable to float on water surface for 5 days without deflation, collapse or disintegration of L. clavatum particles.
3. Results and discussion First, SEM morphological studies of self-assembled Fmoc-FF membranes at the liquid/air interface are discussed and a model for membrane formation is proposed. Second, TEM diffraction experiments were done to characterize structural features inside the free standing membranes on a molecular scale. ATR-IR spectra of the Fmoc-FF layers prepared by the interfacial assembly were almost similar to that observed for bulk gelation. A representative spectrum is part of the Supplementary materials.
Fig. 2. (A) Deflated-like Fmoc-FF liquid marble formed after the final drying step; (B) inside view of the liquid marble reveals a Fmoc-FF membrane interconnectivity between the individual lycopodium spores, formed at the liquid/air interface.
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Fig. 3. (A) Bare Lycopodium clavatum spore; (B) thick peptide membrane coating a spore; (C) thin peptide membrane coating a spore (approximately 50 nm). (D) Closer view of the peptide membrane at one indentation of the Lycopodium clavatum spore in (C).
2. Even after drying, the deflated marble shows a remarkable mechanical stability and deflated marbles could be handled by tweezers. It was found that the thickness of the membrane strictly depends on the time allowed for gelation. The evaporation rate, present during gelation could be reduced by appropriate humidity control. High humidity favors the formation of thicker membranes than low humidity conditions. Another relevant phenomena is the heterogeneity in membrane thickness through the liquid marble interface. It is clear, from the deflated-like shape and from SEM inspection of the membrane fragments, that the thickness on the superior part of the liquid marble was larger than the thickness on the interior part, which turn out to be difficult to quantify in this multiscale system. Nevertheless, with this L. clavatum (Fig. 3A) gelation system, the formation of an interwoven network of ribbons was expected to constitute the observed peptide membrane (Fig. 3B–D). Over several observations under different experimental conditions, it became clear that the different times chosen for carbon dioxide acidification of the aqueous phase, originated membranes of different thickness. In short carbon dioxide exposure (under 30 min) a thin membrane the individual ribbons could be recognized (Fig. 3C and D), while in longer (over 30 min) a thicker membrane of the overlapping ribbons did not allow identification of individual ribbons in the peptide (Fig. 3B). The irregular folding of the deflated liquid marbles appears most probably due to capillary forces between the evaporating liquid and the solid interface during the water evaporation. Even thought, the peptide membrane
bends and follows the liquid/air interface morphology structured by the L. clavatum particles. The membrane thickness differences in the same liquid marble might happen due to the same physical phenomena driving droplet evaporation [27]: such as heat transfer by convection (Marangoni effects and normal convection); modified wettability due to molecular scale interactions between the liquid and the particles; and the the different vapor mass diffusion around the drop. Another possibility might be related to the higher access of the carbon dioxide though the superior part of the liquid marble, in contrast with the lower part. Another remarkable observation for the self-assembly inside the lycopodium based liquid marbles is the peptide membrane formation not only between the lycopodium particles, but also across the open chambers, arranged in a honeycomb like morphology (Fig. 3). The membrane forming ribbons always span over the cavity of the spores. They never appeared coated at the interior of the cavity. In a closer look at this indentations, the ribbon assemblies are seen to attach to each-other coating the individual indentations (Fig. 3D). With all the observations in mind, a model for the pH induced gelation at the gas/liquid/solid interface of L. clavatum encapsulated liquid marble is proposed and schemed in Fig. 4. Initially, microparticles arranged in a highly condensed packing are floating at the liquid interface of the alkaline Fmoc-FF solution. The movement and interaction of the floating microparticles does not only depend on the surface tension of the aqueous solution, but also to size, shape and electronic charge interactions [28,29] of the floating particles. Upon exposure of the liquid marbles to carbon dioxide, carbon dioxide dissolution and finally dissociation
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Fig. 4. Schematic view to illustrate the model proposed for Fmoc-FF self-assembly at interface, before (A) and after (B) the pH induced assembly of the Fmoc-FF molecules. This interface is an approximately 80 m cut-through sectional view of the liquid marble. Lycopodium particles float at the liquid/air interface between the alkaline Fmoc-FF drop and the gas phase. This surface is non-planar and strongly determined by capillary forces between the liquid and the floating particles. Finally peptide membranes are formed which interconnect the microparticles.
into hydrogenecarbonate ions reduces the pH at liquid/air interface inside the liquid marble and consequently creates a pH gradient inside. Acidification triggers the self-assembly process into ribbon like peptide aggregates at the liquid/air interface resulting in a membrane formation. As this membrane formation occurs only at the liquid/gas interface, the resulting membrane/particle morphology is to discuss as a replication of this interface. Membrane formation across and not inside the cavities of the L. clavatum spores clearly indicates that the cavities withstand the penetration of liquid at the interface both into these spore cavities but also into the gas filled gaps between floating spores. This is a consequence of the Laplace pressure which suppresses the penetration of liquid into the lycopodium cavities. The interconnection of the cavities by holes as it is evident from Fig. 3A, guarantees a carbon dioxide transport to the liquid/air interface of the single cavities. The gas transport in between the cavities, through their pores, allows a local pH change at the liquid/air interface of an individual cavity and therefore triggers the membrane formation across the cavity. If the carbon dioxide source is detached, the pH of the solution slightly increases again, which can be followed by an indicator (e.g. with bromocresol red, the final pH after detachment is above pH 6.8) encapsulated in the liquid marble. A particular difference of this system resides in the avoidance of high local gradients in the alkaline Fmoc-FF solution, which in the bulk may suppress inhomogeneous Fmoc-FF fiber growth.
3.2. Morphological observations of the Fmoc-FF system in floating grids In order to study the membrane formation with respect to a single floating L. clavatum spore (Fig. 5 (I)) or at the bare liquid/air interface (Fig. 5 (II)) an experimental setup as schemed in Fig. 5 was used. A copper grid with appropriate mesh size (hexagonal meshes) is placed as microstructured floating object at the liquid/air interface of a Fmoc-FF solution. In some of the grids cavities single lycopodium spores were placed (Fig. 5 (I)), while others were free of particles and therefore create a bare liquid/air interface (Fig. 5 (II)). Through this in-grid system, both single microparticle to membrane connectivity (Fig. 5, Case I.A and B) and thin membranes can be formed with high thickness control (Fig. 5, Case II.C) for microand nano-structure characterization [24]. In the Case I, the patterning of the membrane formation at the grid cavities allows observation of the connectivity from both sides of the microparticle–membrane connection. The bottom view (Fig. 5A), confirms that the membrane formation follows the liquid/air interface and connects to the spore exine capsule. This results in free standing peptide membranes at each of the bottom indentations of the L. clavatum particle. In the top view (Fig. 5B), the conditions are different, since no contact with the liquid phase exists, the membrane is not formed and does not completely coats the microparticles. This situation, permits diffusion of the gas
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Fig. 5. Schematics of in-grid gelation system: Case I: single lycopodium microstructure (green) integrated into the peptide membrane (red); Case II: free standing peptide membrane (red). Morphological (SEM) observations of spore-membrane at a single grid mesh, from the top side (A) and the bottom side (B). (C) SEM image of the free standing peptide membrane reveals distinguishable single ribbons of thickness approximately 50 nm. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
through the pores in the spore exine capsule, which allows the diffusion of the carbon dioxide at the liquid/air interface of the microparticle indentations. As observed initially in Fig. 2B, not all indentations are filled in all lycopodium spores. These phenomena might be related to the time carbon dioxide has to diffuse through the inter-indentation cavities, essential for this liquid/air gelation at the lycopodium spore (in water) indentations. A representative film segment which has formed as free standing Fmoc-FF membrane inside a single mesh of the grid and which was suitable for TEM diffraction experiments is shown in Fig. 5C. Although the low density density of peptide ribbons causes defects as the single hole shown the ultrathin membrane is remarkable stable with respect to electron radiation damage in the SEM. The samples reveal a single peptide ribbon 2D-layer perpendicular to the electron beam, with an approximate thickness of 50 nm, when the exposure time to carbon dioxide is set to 5 up to 10 min. Longer than this times revel a increasingly thick layer (observed via SEM). On one hand, multi-ribbon layers, for the purpose described in the next section (in the selected area for diffraction), might introduce additional features to the following described diffraction experiments, on another hand this might be exploited for biosensor applications, kinetics of diffusion through hydrogelcells membranes or pharmacokinetics and pharmacodynamics in vitro studies of hydrogel membranes with a pharmacological compound of interest, done at a relative low cost and over a large area. 3.3. Electron diffraction experiments A typical SAED pattern obtained from an Fmoc-FF assembly of extended ribbons within an ultrathin membrane is shown in Fig. 6 (left). The sample was prepared as-described, a free standing thin membrane grown within the holes of a floating TEM grid. The pattern is a superposition of two zone axis diffraction patterns, one
appearing as a set of well defined sharp reflections, which are located on an orthogonal set of equidistant layer lines. The axis aligned with equidistant reflections of a periodic spacing of 23 A˚ is assigned the reciprocal axis a∗ , while the orthogonal axis aligned refections with a real lattice spacing of 4.8 A˚ is assigned as the reciprocal b∗ axis. The second pattern in this figure generates a set of reflections, which show the same lattice spacings dh k l as those already measured for the single crystal diffraction pattern. The cor responding reciprocal lattice vectors a∗ and b∗ are indicated in Fig. 6 (left). The broadening of the reflections with an arc-segment (red dashed circles) indicates a texture of several crossing highly oriented ribbons, and additionally have a similar zone axis as both axis with the extended single crystal ribbon, as well as with respect to each other. The common zone axis is indicated as the [0 0 w] axis (Fig. 6A). A second typical SAED pattern taken from an area with a large assembly of ribbons crossing each other is shown in Fig. 7. It is worth noticing that all rings observed can be indexed using the dh k l spacings calculated from the single crystalline diffraction pattern that has been described previously (Fig. 6). This clearly indicates that although the ribbons are randomly oriented, they are randomly oriented around one common zone axis, similar to the one observed for a single ribbon [0 0 w] (Fig. 6 (left)). Curiously, the ribbons are not twisted at least within the sample area contributing to the SAED. The lack of twisting may result from the self-assembly of ribbon structures at the liquid/air interface. Concerning a discussion of the observed lattice spacings of 23 A˚ and 4.8 A˚ on the perpendicular axis within a molecular model, we refer to the data and structure proposal recently published by Ulijn et.al. [20]. Although the group also could not determine a complete set of crystallographic data on an elementary cell, they proposed a molecular model based on X-ray powder diffraction data and on the interpretation of periodic striations observed in high resolution TEM image analyzed by FFT analysis. Ulijn et. al.
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Fig. 6. Selected area electron diffraction (SAED) pattern of Fmoc-FF membrane ribbons (left) and schematic arrangement of nanostructural entities (right). The scheme (A) indicates the assigned axis to the diffractogram on the left. The scheme (B and C) illustrates various packing arrangements for a dense packed assembly of helical motifs, either as perfect 3D crystal (B) or as assembly with translational and/or rotational disorder of stacked helices (C).The scheme (D) figures an alternative, so called packed column model [30].
assumed a helical arrangement of individual Fmoc-FF entities due to – interactions of the aromatic groups and further stabilization by hydrogen bonds along the stacking or helical axis. The helical assemblies simplified as tubes are dense packed and the periodicity between the cylinders should be represented by the 23 A˚ spacing. Ulijn et.al. assigned the spacing between the cylinders to 26 A˚ as concluded from a strong reflection in the WAXS powder diagram although this is in conflict with the spacing of 35 A˚ which he proposed from the FFT of band periodic features, observed in the TEM images. Concerning different assembly conditions and less accuracy evaluation of lattice parameters, from a electron beam sensitive specimen by TEM, as compared to X-ray diffraction data. We assume the observed difference in lattice spacing acceptable.
Based on the packing of cylindrical units within the ribbons as it is indicated in Fig. 6B and C, only the packing shown in Fig. 6B would be reasonable resulting in a single crystalline zone axis diffraction pattern. Any translational or rotational disorder (Fig. 6C) of the cylinders can be excluded due to the lack of streaks, which should appear along the (h 1 0) lattice planes. The 4.8 A˚ spacing observed for the b-axis, which is close to the 4.3 A˚ spacing, previously discussed, should originate from the periodic spacing of aromates along the cylinder axis. Nevertheless it should be mentioned that aside the helical Fmoc-FF model, a planar compact columns stacking model as schemed in Fig. 6D could offer an alternative packing arrangement as-described in literature [30], for many similar gelation systems of short sequenced peptides. A recent X-ray diffraction
Fig. 7. Indexed electron diffraction pattern (left) of a ultrathin, few layered ribbon structures, with a main ribbon direction as indicated the meridional (h 0 0) reflections and the longitudinal (0 k 0) reflections. The fit of the main crystallographic axis previously described with the circular pattern, suggests a general flat ribbon assembly randomly oriented in a plane normal to the diffraction beam [0 0 w].
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result on shear oriented Fmoc-FF gels [31], indicates a cross -type structure (in line with Fig. 6D). 4. Conclusions Liquid marbles were stabilized by pH triggered membrane formation, of self-assembling peptide units, in particular of Fmoc-FF. The interfacial self-assembly process controlled by acidification with gaseous carbon dioxide, results in interwoven meshes of peptide ribbons. The crystallographic data obtained from electron diffraction experiments, extend the existing information based on X-ray powder diffraction of the Fmoc-FF system. The mechanical stabilization will open potential application fields for liquid marbles as chemical microreactors, as encapsulating systems for removal of unwanted materials, as drug carriers or as sensing devices when filled with appropriate analytes. Mechanical stabilization of marbles will be a necessary condition to actively move these on surfaces, for example, by surface acoustic wave based movement. A complete crystallographic evaluation of lattice parameters by TEM diffraction experiments is a future goal. Acknowledgements A.Z.C. gratefully acknowledges the financial support from an Erasmus Mundus scholarship. We thank Dr. P. Formanek for technical assistance with TEM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2012.03.028. References [1] P. Aussillous, D. Quere, Properties of liquid marbles, Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 462 (2067) (2006) 973–999. [2] G. McHale, N. Newton, Liquid marbles: principles and applications, Soft Matter 7 (5) (2011) 5473–5481. [3] E. Bormashenko, Liquid marbles: properties and applications, Curr. Opin. Colloid Interface Sci. 16 (2011) 266–271. [4] M. Dandan, H.Y. Erbil, Evaporation rate of graphite liquid marbles: comparison with water droplets, Langmuir 25 (14) (2009) 8362–8367. [5] P. Aussillous, D. Quere, Liquid marbles, Nature 411 (6840) (2001) 924–927. [6] E. Bormashenko, Y. Bormashenko, A. Musin, Z. Barkay, On the mechanism of floating and sliding of liquid marbles, Chemphyschem 10 (4) (2009) 654–656. [7] B.P. Binks, A.N. Boa, M.A. Kibble, G. Mackenzie, A. Rocher, Sporopollenin capsules at fluid interfaces: particle-stabilised emulsions and liquid marbles, Soft Matter 7 (8) (2011) 4017–4024. [8] A.B.D. Cassie, S. Baxter, Wettability of porous surfaces, Trans. Faraday Soc. 40 (1944) 0546–0550.
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