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Hierarchical assembly of diphenylalanine into dendritic nanoarchitectures
Colloids and Surfaces B: Biointerfaces 79 (2010) 440–445
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Hierarchical assembly of diphenylalanine into dendritic nanoarchitectures Tae Hee Han a , Jun Kyun Oh a , Gyoung-Ja Lee b , Su-Il Pyun a , Sang Ouk Kim a,∗ a b
Department of Materials Science and Engineering, KI for the Nanocentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea Nuclear Nano Materials Development Laboratory, Korea Atomic Energy Research Institute, Daejeon 305-353, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 October 2009 Received in revised form 1 May 2010 Accepted 3 May 2010 Available online 9 May 2010 Keywords: Hierarchical self-assembly Peptides Dendrites Fractal dimension
a b s t r a c t Highly ordered, multi-dimensional dendritic nanoarchitectures were created via self-assembly of diphenylalanine from an acidic buffer solution. The self-similarity of dendritic structures was characterized by examining their fractal dimensions with the box-counting method. The fractal dimension was determined to be 1.7, which demonstrates the fractal dimension of structures generated by diffusion limited aggregation on a two-dimensional substrate surface. By confining the dendritic assembly of diphenylalanine within PDMS microchannels, the self-similar dendritic growth could be hierarchically directed to create linearly assembled nanoarchitectures. Our approach offers a novel pathway for creating and directing hierarchical nanoarchitecture from biomolecular assembly. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hierarchical nanostructures are of growing interest due to their potential applications to photonics [1], energy devices [2], biosensors [3], separation media [4], catalysts [5], lithographic masks [6] and so on. The multilevel ordering of hierarchical structures enables the simultaneous incorporation of diverse functionalities. It is well known that the complex multi-functionalities of living systems are largely owing to their hierarchical structures [7,8]. Biomolecular self-assembly is considered a highly efficient route to hierarchical structures [9]. A wide spectrum of biomolecules such as peptides [10], proteins [11], DNA [12], and lipids [13] spontaneously assemble into hierarchical structures. Unlike synthetic self-assembly, which is usually dominated by undirectional weak intermolecular interactions [14–17], biomolecular self-assembly takes place via a subtle interplay of numerous highly specific interactions, such as hydrogen bonding, charged termini or sidechain interactions, and hydrophobic interactions, etc. [18,19]. As a consequence, the surrounding environment may significantly influence the outcome of biomolecular self-assembly. Recently, it has been reported that an aromatic peptide of diphenylalanine, which was introduced as the -amyloid fractal motif associated with Alzheimer’s disease, assembles into a variety of nanostructures such as nanotubes, nanowires, and nanoribbons according to its growth conditions [20–26]. Nonetheless, the organization of one-dimensional nanoscale objects into multi-dimensional struc-
∗ Corresponding author. Tel.: +82 42 350 3339; fax: +82 42 350 3310. E-mail address: [email protected] (S.O. Kim). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.05.003
tures and the theoretical analysis on this assembly behaviour have been rarely explored [19]. In this work, we present a hierarchical assembly method for generating multi-dimensional structures of diphenylalanine, which proves highly useful for advanced nanofabrication applications [27]. The aromatic peptide of diphenylalanine was found to spontaneously assemble into a highly ordered dendritic morphology during solidification from an acidic buffer solution. The assembly consisted of self-similar, multi-scale orderings, whose characteristic dimensions ranged from nanoscale to microscale. The dendritic growth was hierarchically directed into a well-ordered, linear array of nanoarchitectures by applying a physical confinement in a capillary [28].
2. Experimental 2.1. Materials The lyophilized form of the diphenylalanine (NH2 -PhePhe-COOH) peptide was purchased from Bachem (Bubendorf, Switzerland). KCl, citric acid, KH2 PO4 , glycine, and KCl were obtained from Sigma–Aldrich. All reagents were used as received without any further purification. Water used in all reactions was deionized by using Milipore MilliQ® . An aqueous buffer solution of pH 1 was prepared with 50 mM KCl. The pH 4, 7, 10, and 13 buffer solutions were prepared with 50 mM citric acid, KH2 PO4 , glycine, and KCl, respectively.
T.H. Han et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 440–445
2.2. Methods 2.2.1. Diphenylalanine assembly into peptide nanostructures A predetermined amount of diphenylalanine was completely dissolved in the buffer solution to form a transparent solution (peptide concentration: 0.5–3 mg/mL). To prepare dendrites, an aliquot (20 L) of transparent peptide solution (pH 1) was dropped onto a cleaned silicon wafer and allowed to dry at room temperature. After complete drying, the dendrites remained on the substrate surface. In other buffer solutions, the diphenylalanine was also completely dissolved at 2 mg/mL. After leaving the vial at room temperature for several hours, nanostructures were formed in the vial in contrary with pH 1 condition. An aliquot (20 L) of peptide solution containing peptide nanostructures was dropped onto a cleaned silicon wafer and allowed to dry at room temperature. 2.2.2. Characterization The dendritic morphology was analyzed using a field emission scanning electron microscope (Hitachi S-4800, Japan). A thin layer of osmium was coated on the sample surface to enhance image contrast and sample stability. The dendritic assembly behaviour of diphenylalanine was observed under an optical microscope (Leitz Laborlux 12 Pols, Germany). The turbidity of the diphenylalanine solution was measured with a UV-Vis 1201 spectrometer (Shimadzu, Japan). The measuring sample was prepared by dissolving diphenylalanine in a pH controlled aqueous solution (concentration: 1–20 mg/mL) and kept for 5 h at an ambient condition prior to measurements. The fractal dimension of the dendritic morphology was determined by the box-counting method using Image J software version 1.40 (National Institutes of Health, USA). The SEM images of dendrites were converted into 8-bit binary format images, which were covered by square box arrays. The number of boxes occupied by the underlying dendritic morphology
441
(N) and the side length of boxes (s) were plotted in logarithmic scale to determine the fractal dimension of the dendrites (D). 2.2.3. Directed dendritic assembly in microchannel Directed dendritic assembly was achieved by physically confining the dendritic growth in a polydimethylsiloxane (PDMS) microchannel. The micromold for confining the peptide solution was prepared as follows: A PDMS prepolymer (Sylgard 184, Dow Corning) was mixed with a curing agent and poured in a pattern master having parallel grid relief structure and cured at 75 ◦ C. 3. Results and discussion The dendritic assembly of diphenylalanine was achieved through a simple process of solution casting from an acidic buffer solution. Diphenylalanine was completely dissolved in a buffer solution with the pH value of 1. A drop of the prepared solution was dispensed onto a clean silicon substrate and left to dry under ambient conditions. After complete drying, a highly ordered hierarchical morphology was obtained on the substrate surface. Fig. 1a shows the scanning electron microscopy (SEM) image of a self-assembled dendrite that resembles the ice crystal structures in snowflakes. The dendrite extends over several hundreds of micrometers. Fig. 1b is a magnified image of a dendrite edge, which reveals the rod-shaped nanoscale primary frameworks. The frameworks have an average diameter of approximately 840 nm. The growth of each linear framework was nucleated from the previously generated frameworks and preceded laterally at an angle of 45◦ or 90◦ with respect to previous frameworks. In the early stage of growth, the frameworks had a 4-fold symmetry. As growth continued, the dendrites formed highly branched structures with a specific branching angle of 45◦ . The nanoscale frameworks grew in both the out-of-substrate
Fig. 1. SEM images of dendrites self-assembled from an acidic buffer solution of diphenylalanine. (a) SEM image showing the morphology of an entire dendritic structure, (b) SEM image of a magnified view of a dendrite edge. (c and d) Magnified SEM images of the central region of a dendrite.
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Fig. 2. The fractal dimension analysis of dendrites using the box-counting method. (a and b) The black and white binary images of dendrites converted from SEM images. (c and d) The number of boxes needed to cover the dendritic patterns (N(s)) in figures (a) and (b), plotted as a function of the side length of boxes (s). The slopes in the logarithmic scale demonstrate the fractal dimensions (D) of 1.71 and 1.73 for (a) and (b), respectively.
and the in-substrate planes. Fig. 1c and d shows the resulting framework structures with a typical branching angle of 90◦ , which were frequently observed in the central part of a dendrite. The average diameter of a primary framework was approximately 530 nm, and the distance between two neighboring branches of a framework was approximately 3 m. Unlike the dendritic morphology of other soft materials [27,29,30], the dendrite of diphenylalanine exhibited a highly ordered regular morphology. Fig. 2 shows a dimensional analysis of a dendritic morphology. This analysis provides a useful basis for investigating the selfsimilarity of hierarchical structures [31,32]. To date, a few methods such as the box-counting method [29], the divider method [27], and the perimeter-area method [33,34], have been developed to determine fractal dimensions from an image analysis. In this work, the box-counting method has been used. A dendritic morphology was covered by square box arrays. When more than half of a square box area is occupied by underlying dendritic morphology, the box is considered occupied [35]. The number of boxes required to completely cover a dendritic morphology (N(s)) was counted as a function of the side length of the square box (s). The relation between s and N(s) provides the fractal dimension of a dendritic morphology (D) according to the following relationship: D = lim − s→o
log N(s) log(s)
(1)
We applied this straightforward method to determine D of the dendritic morphologies using a computer-aided image processing. Fig. 2a and b are black and white binary images of a dendrite transformed from the digitized SEM images of Fig. 1a and b. For each s, N(s) was counted using an automatic image analysis and plotted in Fig. 2c and d. Linear-regression was used to determine D, which was 1.71 and 1.73 for Fig. 2a and b, respectively. The dimensional consistency at different length scales verifies the self-similarity of the dendritic structures [29]. Moreover, the determined D value was
close to the ideal value of 5/3 for the structure generated by ‘diffusion limited aggregation’ (DLA) in two-dimensions [36–38] (see Supplementary information, Fig. S2). Fig. 3 shows a series of optical micrographs taken in succession at the various stages of dendrite growth. A drop of transparent diphenylalanine solution was dispensed onto a glass substrate. As water evaporated from the acidic peptide solution, convective flow occurred at the edge of the droplet, and this flow was visible as the movement of air bubbles (Fig. 3b). Water evaporation increased the local concentration of the diphenylalanine, particularly at the edge of a droplet, and eventually caused the nucleation of a dendrite at the position indicated by a white arrow in Fig. 3c. (The critical concentration for assembly was found to be approximately 7 mg/mL from the turbidity test [39], as shown in Fig. 4.) The growth of dendrites proceeded very rapidly after this nucleation event. Initially, the dendrites grew laterally over the substrate plane and eventually they grew out of the substrate plane (see Supplementary information, Fig. 3d and e). Our experimental observations of the dendritic assembly process along with the fractal dimension D value determined by the box-counting method suggest that the dendritic growth of diphenylalanine should occur via DLA on two-dimensional substrate surfaces [40–42]. In general, DLA occurs in a dilute solution or suspension, in which particles diffuse by Brownian motion until they contact or adhere to another particle. The dendritic patterns are formed only when the pattern formation is highly anisotropic. In the early stages of assembly, peptide molecules associate to form oligomeric aggregates. The oligomeric aggregates rapidly grew in a specific direction to form a rod-shaped nanoscale framework. This anisotropic assembly repeats at various length scales and leads to growth of a multi-dimensional dendritic architecture. Dendritic assembly of diphenylalanine was observed only at strong acidic conditions with the pH value around 1. Otherwise, diphenylalanine assembly generated completely different mor-
T.H. Han et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 440–445
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Fig. 3. Mechanism of hierarchical dendritic growth. (a) An aromatic diphenylalanine molecule. (b and c) Nucleation of dendrites at the edge of peptide solution. (d) Growth of dendrites spreading over the substrate surface. (e) Multi-dimensional growth of dendrite architecture. The sizes of all images are 1000 m × 750 m.
phologies, as shown in Fig. 5. In the pH range of 4–7 nanotubular structures were observed (Fig. 5a and b). In a basic condition of a pH value around 10 irregularly aggregated short peptide nanowires were obtained (Fig. 5c and d). The generation of similar patterns by the pH-dependent hierarchical self-assembly of a cross-linkable coiled-coil peptide has been recently reported [27]. In general, the assembly of peptide molecules results from the subtle interplay of noncovalent interactions, such as ionic interaction, van der Waals interaction, hydrophobic interaction and hydrogen bonding [19,26]. Tamamis et al. [19] also revealed through both experiments and theoretical study that the self-assembly of diphenylalanine and triphenylalanine (FFF) into ordered discrete nanotubes and planar nanostructures, respectively, occurred through diverse molecular interactions. Diphenylalanine used in this work undergoes the tran-
Fig. 4. Variation of peptide solution turbidity as a function of peptide concentration, measured at the wavelength of 600 nm. The critical concentration for diphenylalanine assembly was found to be 7 mg/mL in acidic buffer solution (pH 1).
sition of ionic forms at three pH values including the pK of the carboxylic group (2.2), the pK of amine group (9.3) and the isoelectric point of free phenylalanine (5.7). At a very low pH below the pK of the carboxylic group, the carboxylic acid groups lose their negative charge, which eliminates the electrostatic repulsion between their C-terminal ends. Additionally, the increase in ionic strength of the medium by acidification with HCl could generate the intercalation of Cl− ions between positively charged N-terminals to form the FF+ · · ·Cl− · · ·FF+ salt bridges [43,44] (see Supplementary information, Fig. S1). We investigated the evolution of the dendritic morphology under physical confinement in a soft mold. The strong selfsimilarity of the dendritic growth of diphenylalanine allows for the production of hierarchically directed nanoarchitecture. The procedure for creating the physically confined dendritic structures is schematically illustrated in Fig. 6a. An acidic peptide solution was dispensed onto a silicon substrate. A PDMS soft mold engraved with microchannels was placed over the solution and left until drying was complete. We note that the acidic peptide solution (pH 1) oxidized the surface of the microchannels of the PDMS mold such that the peptide solution completely wet the walls of the microchannels (see Supplementary information, Fig. S3). Upon drying, the peptide solution was concentrated along the sidewalls of the PDMS channel. The nucleation and growth of dendrites occurred in this confined solution. Fig. 6b shows a large field view of a linear array of dendritic structures created using the PDMS mold with the channel width of 25 m. Fig. 6c is a magnified image of Fig. 6b, and demonstrates that lateral confinement of the solution at the sub-micrometer scale (600 nm) resulted in a highly regular nanoarchitecture of “zig-zag” shaped linear arrays. Fig. 6d and e are SEM images of the dendrites formed in a PDMS channel with the channel width of 50 m. The dendritic structure laterally confined in a width of 5.5 m, showed linearly organized primary peptide frameworks connected to one another at right angles. These highly regular, linear arrayed morphologies clearly demonstrate that physically confined den-
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Fig. 5. Various morphologies of diphenylalanine assemblies according to pH conditions. Nanotubes were formed at the pH values of 4 (a) and 7 (b). Fibril like aggregation was observed at the pH value of 10 (c) and (d).
Fig. 6. Directed assembly of diphenylalanine. (a) Schematically illustrated procedure for hierarchically directed dendrite assembly in capillary confinement. (b–e) SEM images of linearly ordered dendritic morphologies. (c and e) Magnified images of (b) and (d), respectively.
T.H. Han et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 440–445
dritic growth may generate the hierarchically directed assembly of nanoscale frameworks. 4. Conclusions We have fostered the highly ordered dendritic assembly of diphenylalanine during its solidification from an acidic buffer solution. The hierarchical peptide structures were found to show self-similar fractal dimensions by dimensional analysis using the box-counting method. The dendritic assembly could be hierarchically directed into linearly arrayed nanoarchitectures by confining diphenylalanine solution within PDMS microchannels, and the assembled morphology could also be tuned. The large surface area and the tunability of the assembled morphology make the dendritic growth of diphenylalanine potentially useful for advanced nanofabrication. Furthermore, the fractal dimension analysis of the box-counting method used in this work would be useful for the analysis of various biomolecular assembled morphologies. Acknowledgements We thank Dr. S.-J. Lee for helpful discussions. This work was supported by the National Research Laboratory Program (R0A2008-000-20057-0) and the Fundamental R&D Program for Core Technology of Materials funded by the Korean government. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2010.05.003. References [1] [2] [3] [4]
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Hierarchical assembly of diphenylalanine into dendritic nanoarchitectures Tae Hee Han a , Jun Kyun Oh a , Gyoung-Ja Lee b , Su-Il Pyun a , Sang Ouk Kim a,∗ a b
Department of Materials Science and Engineering, KI for the Nanocentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea Nuclear Nano Materials Development Laboratory, Korea Atomic Energy Research Institute, Daejeon 305-353, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 October 2009 Received in revised form 1 May 2010 Accepted 3 May 2010 Available online 9 May 2010 Keywords: Hierarchical self-assembly Peptides Dendrites Fractal dimension
a b s t r a c t Highly ordered, multi-dimensional dendritic nanoarchitectures were created via self-assembly of diphenylalanine from an acidic buffer solution. The self-similarity of dendritic structures was characterized by examining their fractal dimensions with the box-counting method. The fractal dimension was determined to be 1.7, which demonstrates the fractal dimension of structures generated by diffusion limited aggregation on a two-dimensional substrate surface. By confining the dendritic assembly of diphenylalanine within PDMS microchannels, the self-similar dendritic growth could be hierarchically directed to create linearly assembled nanoarchitectures. Our approach offers a novel pathway for creating and directing hierarchical nanoarchitecture from biomolecular assembly. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hierarchical nanostructures are of growing interest due to their potential applications to photonics [1], energy devices [2], biosensors [3], separation media [4], catalysts [5], lithographic masks [6] and so on. The multilevel ordering of hierarchical structures enables the simultaneous incorporation of diverse functionalities. It is well known that the complex multi-functionalities of living systems are largely owing to their hierarchical structures [7,8]. Biomolecular self-assembly is considered a highly efficient route to hierarchical structures [9]. A wide spectrum of biomolecules such as peptides [10], proteins [11], DNA [12], and lipids [13] spontaneously assemble into hierarchical structures. Unlike synthetic self-assembly, which is usually dominated by undirectional weak intermolecular interactions [14–17], biomolecular self-assembly takes place via a subtle interplay of numerous highly specific interactions, such as hydrogen bonding, charged termini or sidechain interactions, and hydrophobic interactions, etc. [18,19]. As a consequence, the surrounding environment may significantly influence the outcome of biomolecular self-assembly. Recently, it has been reported that an aromatic peptide of diphenylalanine, which was introduced as the -amyloid fractal motif associated with Alzheimer’s disease, assembles into a variety of nanostructures such as nanotubes, nanowires, and nanoribbons according to its growth conditions [20–26]. Nonetheless, the organization of one-dimensional nanoscale objects into multi-dimensional struc-
∗ Corresponding author. Tel.: +82 42 350 3339; fax: +82 42 350 3310. E-mail address: [email protected] (S.O. Kim). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.05.003
tures and the theoretical analysis on this assembly behaviour have been rarely explored [19]. In this work, we present a hierarchical assembly method for generating multi-dimensional structures of diphenylalanine, which proves highly useful for advanced nanofabrication applications [27]. The aromatic peptide of diphenylalanine was found to spontaneously assemble into a highly ordered dendritic morphology during solidification from an acidic buffer solution. The assembly consisted of self-similar, multi-scale orderings, whose characteristic dimensions ranged from nanoscale to microscale. The dendritic growth was hierarchically directed into a well-ordered, linear array of nanoarchitectures by applying a physical confinement in a capillary [28].
2. Experimental 2.1. Materials The lyophilized form of the diphenylalanine (NH2 -PhePhe-COOH) peptide was purchased from Bachem (Bubendorf, Switzerland). KCl, citric acid, KH2 PO4 , glycine, and KCl were obtained from Sigma–Aldrich. All reagents were used as received without any further purification. Water used in all reactions was deionized by using Milipore MilliQ® . An aqueous buffer solution of pH 1 was prepared with 50 mM KCl. The pH 4, 7, 10, and 13 buffer solutions were prepared with 50 mM citric acid, KH2 PO4 , glycine, and KCl, respectively.
T.H. Han et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 440–445
2.2. Methods 2.2.1. Diphenylalanine assembly into peptide nanostructures A predetermined amount of diphenylalanine was completely dissolved in the buffer solution to form a transparent solution (peptide concentration: 0.5–3 mg/mL). To prepare dendrites, an aliquot (20 L) of transparent peptide solution (pH 1) was dropped onto a cleaned silicon wafer and allowed to dry at room temperature. After complete drying, the dendrites remained on the substrate surface. In other buffer solutions, the diphenylalanine was also completely dissolved at 2 mg/mL. After leaving the vial at room temperature for several hours, nanostructures were formed in the vial in contrary with pH 1 condition. An aliquot (20 L) of peptide solution containing peptide nanostructures was dropped onto a cleaned silicon wafer and allowed to dry at room temperature. 2.2.2. Characterization The dendritic morphology was analyzed using a field emission scanning electron microscope (Hitachi S-4800, Japan). A thin layer of osmium was coated on the sample surface to enhance image contrast and sample stability. The dendritic assembly behaviour of diphenylalanine was observed under an optical microscope (Leitz Laborlux 12 Pols, Germany). The turbidity of the diphenylalanine solution was measured with a UV-Vis 1201 spectrometer (Shimadzu, Japan). The measuring sample was prepared by dissolving diphenylalanine in a pH controlled aqueous solution (concentration: 1–20 mg/mL) and kept for 5 h at an ambient condition prior to measurements. The fractal dimension of the dendritic morphology was determined by the box-counting method using Image J software version 1.40 (National Institutes of Health, USA). The SEM images of dendrites were converted into 8-bit binary format images, which were covered by square box arrays. The number of boxes occupied by the underlying dendritic morphology
441
(N) and the side length of boxes (s) were plotted in logarithmic scale to determine the fractal dimension of the dendrites (D). 2.2.3. Directed dendritic assembly in microchannel Directed dendritic assembly was achieved by physically confining the dendritic growth in a polydimethylsiloxane (PDMS) microchannel. The micromold for confining the peptide solution was prepared as follows: A PDMS prepolymer (Sylgard 184, Dow Corning) was mixed with a curing agent and poured in a pattern master having parallel grid relief structure and cured at 75 ◦ C. 3. Results and discussion The dendritic assembly of diphenylalanine was achieved through a simple process of solution casting from an acidic buffer solution. Diphenylalanine was completely dissolved in a buffer solution with the pH value of 1. A drop of the prepared solution was dispensed onto a clean silicon substrate and left to dry under ambient conditions. After complete drying, a highly ordered hierarchical morphology was obtained on the substrate surface. Fig. 1a shows the scanning electron microscopy (SEM) image of a self-assembled dendrite that resembles the ice crystal structures in snowflakes. The dendrite extends over several hundreds of micrometers. Fig. 1b is a magnified image of a dendrite edge, which reveals the rod-shaped nanoscale primary frameworks. The frameworks have an average diameter of approximately 840 nm. The growth of each linear framework was nucleated from the previously generated frameworks and preceded laterally at an angle of 45◦ or 90◦ with respect to previous frameworks. In the early stage of growth, the frameworks had a 4-fold symmetry. As growth continued, the dendrites formed highly branched structures with a specific branching angle of 45◦ . The nanoscale frameworks grew in both the out-of-substrate
Fig. 1. SEM images of dendrites self-assembled from an acidic buffer solution of diphenylalanine. (a) SEM image showing the morphology of an entire dendritic structure, (b) SEM image of a magnified view of a dendrite edge. (c and d) Magnified SEM images of the central region of a dendrite.
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Fig. 2. The fractal dimension analysis of dendrites using the box-counting method. (a and b) The black and white binary images of dendrites converted from SEM images. (c and d) The number of boxes needed to cover the dendritic patterns (N(s)) in figures (a) and (b), plotted as a function of the side length of boxes (s). The slopes in the logarithmic scale demonstrate the fractal dimensions (D) of 1.71 and 1.73 for (a) and (b), respectively.
and the in-substrate planes. Fig. 1c and d shows the resulting framework structures with a typical branching angle of 90◦ , which were frequently observed in the central part of a dendrite. The average diameter of a primary framework was approximately 530 nm, and the distance between two neighboring branches of a framework was approximately 3 m. Unlike the dendritic morphology of other soft materials [27,29,30], the dendrite of diphenylalanine exhibited a highly ordered regular morphology. Fig. 2 shows a dimensional analysis of a dendritic morphology. This analysis provides a useful basis for investigating the selfsimilarity of hierarchical structures [31,32]. To date, a few methods such as the box-counting method [29], the divider method [27], and the perimeter-area method [33,34], have been developed to determine fractal dimensions from an image analysis. In this work, the box-counting method has been used. A dendritic morphology was covered by square box arrays. When more than half of a square box area is occupied by underlying dendritic morphology, the box is considered occupied [35]. The number of boxes required to completely cover a dendritic morphology (N(s)) was counted as a function of the side length of the square box (s). The relation between s and N(s) provides the fractal dimension of a dendritic morphology (D) according to the following relationship: D = lim − s→o
log N(s) log(s)
(1)
We applied this straightforward method to determine D of the dendritic morphologies using a computer-aided image processing. Fig. 2a and b are black and white binary images of a dendrite transformed from the digitized SEM images of Fig. 1a and b. For each s, N(s) was counted using an automatic image analysis and plotted in Fig. 2c and d. Linear-regression was used to determine D, which was 1.71 and 1.73 for Fig. 2a and b, respectively. The dimensional consistency at different length scales verifies the self-similarity of the dendritic structures [29]. Moreover, the determined D value was
close to the ideal value of 5/3 for the structure generated by ‘diffusion limited aggregation’ (DLA) in two-dimensions [36–38] (see Supplementary information, Fig. S2). Fig. 3 shows a series of optical micrographs taken in succession at the various stages of dendrite growth. A drop of transparent diphenylalanine solution was dispensed onto a glass substrate. As water evaporated from the acidic peptide solution, convective flow occurred at the edge of the droplet, and this flow was visible as the movement of air bubbles (Fig. 3b). Water evaporation increased the local concentration of the diphenylalanine, particularly at the edge of a droplet, and eventually caused the nucleation of a dendrite at the position indicated by a white arrow in Fig. 3c. (The critical concentration for assembly was found to be approximately 7 mg/mL from the turbidity test [39], as shown in Fig. 4.) The growth of dendrites proceeded very rapidly after this nucleation event. Initially, the dendrites grew laterally over the substrate plane and eventually they grew out of the substrate plane (see Supplementary information, Fig. 3d and e). Our experimental observations of the dendritic assembly process along with the fractal dimension D value determined by the box-counting method suggest that the dendritic growth of diphenylalanine should occur via DLA on two-dimensional substrate surfaces [40–42]. In general, DLA occurs in a dilute solution or suspension, in which particles diffuse by Brownian motion until they contact or adhere to another particle. The dendritic patterns are formed only when the pattern formation is highly anisotropic. In the early stages of assembly, peptide molecules associate to form oligomeric aggregates. The oligomeric aggregates rapidly grew in a specific direction to form a rod-shaped nanoscale framework. This anisotropic assembly repeats at various length scales and leads to growth of a multi-dimensional dendritic architecture. Dendritic assembly of diphenylalanine was observed only at strong acidic conditions with the pH value around 1. Otherwise, diphenylalanine assembly generated completely different mor-
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Fig. 3. Mechanism of hierarchical dendritic growth. (a) An aromatic diphenylalanine molecule. (b and c) Nucleation of dendrites at the edge of peptide solution. (d) Growth of dendrites spreading over the substrate surface. (e) Multi-dimensional growth of dendrite architecture. The sizes of all images are 1000 m × 750 m.
phologies, as shown in Fig. 5. In the pH range of 4–7 nanotubular structures were observed (Fig. 5a and b). In a basic condition of a pH value around 10 irregularly aggregated short peptide nanowires were obtained (Fig. 5c and d). The generation of similar patterns by the pH-dependent hierarchical self-assembly of a cross-linkable coiled-coil peptide has been recently reported [27]. In general, the assembly of peptide molecules results from the subtle interplay of noncovalent interactions, such as ionic interaction, van der Waals interaction, hydrophobic interaction and hydrogen bonding [19,26]. Tamamis et al. [19] also revealed through both experiments and theoretical study that the self-assembly of diphenylalanine and triphenylalanine (FFF) into ordered discrete nanotubes and planar nanostructures, respectively, occurred through diverse molecular interactions. Diphenylalanine used in this work undergoes the tran-
Fig. 4. Variation of peptide solution turbidity as a function of peptide concentration, measured at the wavelength of 600 nm. The critical concentration for diphenylalanine assembly was found to be 7 mg/mL in acidic buffer solution (pH 1).
sition of ionic forms at three pH values including the pK of the carboxylic group (2.2), the pK of amine group (9.3) and the isoelectric point of free phenylalanine (5.7). At a very low pH below the pK of the carboxylic group, the carboxylic acid groups lose their negative charge, which eliminates the electrostatic repulsion between their C-terminal ends. Additionally, the increase in ionic strength of the medium by acidification with HCl could generate the intercalation of Cl− ions between positively charged N-terminals to form the FF+ · · ·Cl− · · ·FF+ salt bridges [43,44] (see Supplementary information, Fig. S1). We investigated the evolution of the dendritic morphology under physical confinement in a soft mold. The strong selfsimilarity of the dendritic growth of diphenylalanine allows for the production of hierarchically directed nanoarchitecture. The procedure for creating the physically confined dendritic structures is schematically illustrated in Fig. 6a. An acidic peptide solution was dispensed onto a silicon substrate. A PDMS soft mold engraved with microchannels was placed over the solution and left until drying was complete. We note that the acidic peptide solution (pH 1) oxidized the surface of the microchannels of the PDMS mold such that the peptide solution completely wet the walls of the microchannels (see Supplementary information, Fig. S3). Upon drying, the peptide solution was concentrated along the sidewalls of the PDMS channel. The nucleation and growth of dendrites occurred in this confined solution. Fig. 6b shows a large field view of a linear array of dendritic structures created using the PDMS mold with the channel width of 25 m. Fig. 6c is a magnified image of Fig. 6b, and demonstrates that lateral confinement of the solution at the sub-micrometer scale (600 nm) resulted in a highly regular nanoarchitecture of “zig-zag” shaped linear arrays. Fig. 6d and e are SEM images of the dendrites formed in a PDMS channel with the channel width of 50 m. The dendritic structure laterally confined in a width of 5.5 m, showed linearly organized primary peptide frameworks connected to one another at right angles. These highly regular, linear arrayed morphologies clearly demonstrate that physically confined den-
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Fig. 5. Various morphologies of diphenylalanine assemblies according to pH conditions. Nanotubes were formed at the pH values of 4 (a) and 7 (b). Fibril like aggregation was observed at the pH value of 10 (c) and (d).
Fig. 6. Directed assembly of diphenylalanine. (a) Schematically illustrated procedure for hierarchically directed dendrite assembly in capillary confinement. (b–e) SEM images of linearly ordered dendritic morphologies. (c and e) Magnified images of (b) and (d), respectively.
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dritic growth may generate the hierarchically directed assembly of nanoscale frameworks. 4. Conclusions We have fostered the highly ordered dendritic assembly of diphenylalanine during its solidification from an acidic buffer solution. The hierarchical peptide structures were found to show self-similar fractal dimensions by dimensional analysis using the box-counting method. The dendritic assembly could be hierarchically directed into linearly arrayed nanoarchitectures by confining diphenylalanine solution within PDMS microchannels, and the assembled morphology could also be tuned. The large surface area and the tunability of the assembled morphology make the dendritic growth of diphenylalanine potentially useful for advanced nanofabrication. Furthermore, the fractal dimension analysis of the box-counting method used in this work would be useful for the analysis of various biomolecular assembled morphologies. Acknowledgements We thank Dr. S.-J. Lee for helpful discussions. This work was supported by the National Research Laboratory Program (R0A2008-000-20057-0) and the Fundamental R&D Program for Core Technology of Materials funded by the Korean government. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2010.05.003. References [1] [2] [3] [4]
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