Ultrathin Gold Nanoribbons Synthesized within the Interior Cavity of a

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Ultrathin Gold Nanoribbons Synthesized within the Interior Cavity of a Self-Assembled Peptide Nanoarchitecture Kin-ya Tomizaki,*,†,‡ Shota Wakizaka,† Yuichi Yamaguchi,† Akitsugu Kobayashi,† and Takahito Imai† †

Department of Materials Chemistry and ‡Innovative Materials and Processing Research Center, Ryukoku University, Seta, Otsu, Shiga 520-2194, Japan S Supporting Information *

ABSTRACT: There is increasing interest in gold nanocrystals due to their unique physical, chemical, and biocompatible properties. In order to develop a template-assisted method for the fabrication of gold nanocrystals, we demonstrate here the de novo design and synthesis of a β-sheet-forming nonapeptide (RU006: Ac-AIAKAXKIA-NH2, X = L-2-naphthylalanine) which undergoes self-assembly to form disk-like nanoarchitectures approximately 100 nm wide and 2.5 nm high. These self-assemblies tend to form a network of higher-order assemblies in ultrapure water. Using RU006 as a template molecule, we fabricated ultrathin gold nanoribbons 50−100 nm wide, 2.5 nm high, and micrometers long without external reductants. Furthermore, in order to determine the mechanism of ultrathin gold nanoribbon formation, we synthesized four different RU006 analogues. On the basis of the results obtained using RU006 and these analogues, we propose the following mechanism for the self-assembly of RU006. First, RU006 forms a network by the cooperative association of disk-like assemblies in the presence of AuCl4− ions that are encapsulated and concentrated within the interior cavity of the network architectures. This is followed by electron transfer from the naphthalene rings to AuIII, resulting in slow growth to form ultrathin gold nanoribbons along the template network architectures under ambient conditions. The resulting ribbons retain the dimensions of the cavity of the template architecture. Our approach will allow the construction of diverse template architectural morphologies and will find applications in the construction of a variety of metallic nanoarchitectures.

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INTRODUCTION

nanocrystals by capping surfaces on the gold crystals. The morphologies obtained using solution-phase synthesis of gold nanocrystals strongly depend upon surface energy because the surface area-to-volume ratio for nanoparticles is high, and the crystalline form tends to have the lowest surface energy per unit volume.23 However, the surfactants required in the synthesis of gold nanoparticles include harsh reagents, e.g., cetyltrimethylammonium bromide (CTAB), for particle morphology control. The development of more environment-friendly and lowtoxicity fabrication processes is thus desirable. To address this issue, a promising and powerful approach would be the use of biomolecules, including amino acids, peptides, and proteins, to control the anisotropic alignment of

There is increasing interest in gold nanocrystals due to their unique physical, chemical, and biocompatible properties as well as their promising application as catalysts, sensing/imaging systems, and photonic/plasmonic devices and in electronics, drug delivery, and photothermal therapy.1−9 The properties of gold nanocrystals are strongly dependent upon their size, shape, structure, surface decorations, and topology.10 Thus, the shapecontrolled synthesis of gold nanocrystals has been pursued over the past decade. The classical method for synthesizing spherical gold nanocrystals is the citrate reduction method.11 There has also been significant effort to synthesize shapes such as rods,12,13 wires,14 belts and combs,15 plates and prisms,16 polyhedra,17−19 cages and frames,20 and stars and flowers.21,22 The shape-controlled synthesis of gold nanocrystals has employed various surfactants to control the deposit of atoms over the nucleation site and to control the growth of the gold © 2014 American Chemical Society

Received: April 28, 2013 Revised: January 7, 2014 Published: January 16, 2014 846

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dimensions. One example is the use of ferritin and ferritin analogues to control the mineralization of atypical metal substrates within the protein cages, yielding encapsulated nanoparticles that retain the dimensions of the protein cavity.35 In this paper, we describe the de novo design, synthesis, and characterization of a β-sheet-forming nonapeptide, RU006 (AcAIAKAXKIA-NH2, X = L-2-naphthylalanine) and its selfassembled disk-like nanoarchitecture in ultrapure water. We also describe the fabrication and characterization of anisotropic, ultrathin gold nanoribbons encapsulated within the interior cavity of self-assembled RU006 structures. The ribbons are formed by the cooperative association of self-assembled disklike nanoarchitectures (50 μM) with HAuCl4 (50 μM) via molecular interactions. We also propose a mechanism for the formation of ultrathin gold nanoribbons by this peptidetemplating method by determining the relationship between the amino acid sequence and gold nanoparticle morphology by using four different RU006 analogues. We believe this approach will broaden the applicability of biomimetic self-assembly for the construction of metallic nanoarchitectures.

gold nanoparticles along self-assembled architectures as well as to fabricate shape-controlled gold nanocrystals using a biomimetic approach. For instance, a small peptide (AYSSGAPPMPPF, A3) was identified from a phage-display library to have a high affinity for gold surfaces.24 A3 and its analogues have also been demonstrated to act as templates for the synthesis of highly ordered gold nanoparticle double helices25 and gold nanoparticles tethered to biomolecular recognition motifs.26 These methods provide predominantly spherical (isotropic) gold nanoparticles. Another gold surface-binding peptide was separately identified using a phage-display method. The peptide, Midas2 (TGTSVLIATPYV),27 and Midas-2 analogues were employed to fabricate coshell nanocables by sequentially guiding the formation of silica insulating shells on metallic gold nanoribbons.28 One of the Midas-2 analogues, Midas-11, afforded gold nanostructures with shapes and sizes strongly dependent on both the solution pH (pH 3.0−9.0) and HAuCl4 concentration (0.5−30 mM). The observed shapes included polycrystalline and polyhedral gold nanoparticles, peanutshaped nanoparticles, large two-dimensional hexagonal or trigonal single crystalline platelets, single crystalline nanofibers, and extended long single crystal nanoribbons consisting of trigonal segments joined together at the vertices and sides. Although it was concluded that the Midas peptide-mediated synthesis of gold nanostructures described in the literature28 provides a versatile and environmentally benign method for the production of shape-specific nanostructures for a variety applications, the mechanisms of gold nanostructure formations assisted by the Midas peptides remain unclear. It is important to understand the relationships between features of the template peptides and the morphologies of the product gold nanocrystals in peptide-directed gold nanocrystal synthesis. In order to diversify the utility of the template-assisted gold nanocrystal synthetic method, we need to understand the relationship between the morphology of the template molecule and the gold nanocrystals obtained. The self-assembly of small molecules into nanostructures is an attractive bottom-up approach for the fabrication of nanoscaled functional materials.29−32 From the viewpoint of template-assisted inorganic material synthesis, we are developing strategies for employing peptides designed de novo with well-defined β-sheet secondary structures to fabricate wellorganized inorganic materials. For instance, zinc oxide (ZnO) nanoparticles have been successfully aligned along the outer surfaces of peptide nanofibers composed of a β-sheet-forming amphiphilic nonapeptide (RU003: Ac-AIEKAXEIA-NH2, X = L-2-naphthylalanine) at room temperature by reacting with Zn(OH)2 sol.33 The use of RU003 as a template resulted in the predominant formation of straight fibrous structures and induced the nucleation of ZnO crystals at its surface. We also demonstrated the synthesis of silica nanofibers by using a self-assembled nonapeptide nanofiber as a template (RU019: Ac-KIEATXKIE-NH2, X = L-2-naphthylalanine) via hydrolysis/condensation of tetramethoxysilane as a silica source at ambient conditions followed by calcination of the peptide− silica hybrids.34 However, to date, the decoration of the outer surfaces of selfassembled peptide nanoarchitectures with inorganic materials has been predominantly demonstrated, whereas the availability of both the outer surface and an interior cavity of a selfassembled template nanoarchitecture for decoration would allow the fabrication of inorganic materials with controlled

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EXPERIMENTAL SECTION

General. All solvents and reagents, unless otherwise noted, were purchased from Wako Pure Chemical Industries (Japan) and used as received. Fmoc-amino acid derivatives and reagents for peptide synthesis were purchased from Watanabe Chemical Industries (Japan). Acetonitrile (HPLC grade) was purchased from Nacalai Tesque Inc. (Japan) and used for HPLC analysis and peptide purification. Ultrapure water (water) was purchased from Wako Pure Chemical Industries. Peptide Synthesis. The target peptides, RU006 and the RU006 analogues, [Ant6]-RU006, [Phe6]-RU006, [Arg4]-RU006, and [Ala4]RU006, were synthesized by Fmoc chemistry on Rink amide resin with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole monohydrate (HOBt) as coupling reagents.36 The amino acid side chain of arginine (Arg) and lysine (Lys) was protected with 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf) and tert-butyloxycarbonyl (Boc), respectively. Final deprotection and cleavage of the resin-bound peptides was performed by treatment with trifluoroacetic acid (TFA)/thioanisole/ m-cresol (90/7.5/2.5, v/v/v) at room temperature for 60 min. The crude peptides obtained were purified on a Hitachi LaChrom Elite HPLC system (Japan) using Cosmosil 5C18-AR-II packed columns (4.6 × 150 mm and 10 × 250 mm, Nacalai Tesque) with a linear gradient of acetonitrile/0.1% TFA at a flow rate of 1.0 mL min−1 for analysis and 3.0 mL min−1 for preparative purification. The purified peptides were lyophilized and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS, Shimadzu AXIMA-CFR Plus, Japan). Peptide Self-Assembly. Peptide stock solution was prepared by dissolving the purified peptide powder in 2,2,2-trifluoroethanol (TFE) to prevent self-assembly during storage. The concentrations of the stock solutions for RU006, [Arg4]-RU006, and [Ala4]-RU006 were determined by absorption spectroscopy (Shimadzu UV-3100 spectrophotometer, Japan) using an extinction coefficient of 5500 M−1 cm−1 for the 2-naphthylalanine residue (Nal) in aqueous solution containing 1% TFE (v/v).37 The concentrations of the stock solutions of the other peptides were determined by HPLC based on the peak area for RU006. The peptide stock solution in TFE was transferred into a microtube, dried with a N2 gas stream, and then dried in vacuo for 1 h. Water was added to the microtube, and the aqueous solution obtained was sonicated (Kaijo Sono Cleaner CA-44800, Japan) at 50 °C for 2 min to break up peptide aggregates and then filtered through a Cosmospin filter G (pore size 0.2 μm, Nacalai Tesque). The filtrate was incubated in a dry block heater (As One EB-303, Japan) at 40 °C for 1 day and then in a low-temperature incubator (Fukushima FMU0531, Japan) at 25 °C for 6 days for maturation. 847

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Gold Nanocrystal Synthesis Using the Peptide-Templating Method. According to the standard procedure described in the Peptide Self-Assembly section, an aqueous solution containing peptide monomers/oligomers ([peptide] = 100 μM in water, 100 μL) was filtered through a Cosmospin filter G (pore size 0.2 μm, Nacalai Tesque) and subsequently mixed with an aqueous solution containing HAuCl4 ([HAuCl4] = 100 μM in water, 100 μL). The reaction mixture was incubated at 40 °C for 1 day and then at 25 °C for 6 days. If necessary, five portions of 5 μL of an aqueous solution containing NaBH3CN (800 μM) as a reductant were added to the reaction mixture at 60 min intervals with slow rotation. The obtained mixture was used for the following characterization. Transmission Electron Microscopy (TEM), Selected-Area Electron Diffraction−TEM (SAED-TEM), and Energy Dispersive X-ray Spectroscopy−Scanning TEM (EDS-STEM). Droplets of the reaction mixture (10 μL) were applied to a TEM grid (Cu 200 mesh covered with a collodion membrane, Nisshin EM, Japan) for 1 min and dried with a filter paper. If necessary, TEM samples were negatively stained with 2% phosphotungstic acid for 1 min, washed with water, and dried with a filter paper. All TEM samples were dried in vacuo before TEM measurements. TEM and SAED-TEM measurements were conducted at an accelerating voltage of 200 kV (JEOL JEM-2100, Japan). EDS-STEM measurements (samples on Cu 200 mesh covered with a microgrid, Nisshin EM) were conducted on a JEOL JEM-3000F (Japan) at an accelerating voltage of 300 kV. Measurements of Absorption Spectra of Gold Nanocrystal Formation. According to the procedure outlined in the Gold Nanocrystal Synthesis section, a mixture of peptide monomers/ oligomers and HAuCl4 in water was prepared, and absorption spectra of the reaction mixture were recorded on a Shimadzu UV-3100 spectrophotometer. Atomic Force Microscopy (AFM). A silicon wafer [p-Si(100), Mitsubishi Materials Co., Japan] was cut into 7 × 7 mm pieces and subjected to ultrasonic cleaning in acetone for 5 min (3 times), followed by photocleaning with an ultraviolet ozonizer (Kenix KUV100, Japan) at room temperature for 10 min. Droplets of the reaction mixture (10 μL) were applied to the silicon substrates for 1 min, washed with water, and dried with a filter paper. All AFM samples were dried in vacuo before AFM measurements were conducted (MFP-3D-SA-J, Asylum Technologies, Japan) using a cantilever (Olympus OMCL-AC240TS-C3, Japan). Tomographic Reconstruction of Three-Dimensional TEM Images of Gold Nanocrystals. In accordance with the procedure described under TEM measurements above, a sample grid was prepared. TEM images of the sample were acquired by electron beam irradiation at different incident angles ranging from −75° to +75° at 1° intervals on a JEOL JEM-2100 equipped with a high tilt specimen retainer (JEOL) and a goniometer (JEOL). Tomographic reconstruction of the TEM images was conducted according to the literature.38,39

Figure 1. Amino acid sequences of nonapeptide RU006 and the RU006 analogues, [Ant6]-RU006, [Phe6]-RU006, [Arg4]-RU006, and [Ala4]-RU006, studied in this investigation.

Alanine (Ala) residues were placed at the first, third, fifth, and ninth positions to make RU006 a nonapeptide. Two reference peptides, [Ant6]-RU006 and [Phe6]-RU006, were designed to replace the Nal residue at the sixth position in RU006 with an L-2-anthrylalanine (Ant) and a phenylalanine (Phe), respectively, and used to investigate the role of aromaticity in gold nanocrystal synthesis. Additionally, the RU006 analogues, [Arg4]-RU006 and [Ala4]-RU006, were designed to have an L-arginine (Arg) or an Ala residue instead of the Lys residue at the fourth position in RU006, respectively, to allow the comparison of morphologies in self-assembled peptides and gold nanocrystals. Peptides were prepared by standard solid phase peptide synthesis using Fmoc chemistry,36 purified by reverse-phase HPLC, and characterized by MALDITOFMS (Figures S1−S10). Conformational/Morphological Analysis of Self-Assembled Peptides. First, conformational analyses of RU006 and the RU006 analogues were conducted by measuring the ATR-FTIR spectra of peptide films prepared from aqueous solutions containing the matured peptides (Figure S11). The amide I region, originating from the amide carbonyl stretching frequencies between 1600 and 1700 cm−1, is often used to assess the amide mode. Each ATR-FTIR spectrum exhibited a strong amide I band around 1625−1630 cm−1 and a weak band around 1670 cm−1, indicating that an antiparallel β-sheet conformation was predominant within the self-assembled peptide nanoarchitecture.40,41 We further characterized the nanostructures of selfassembled RU-006 by TEM on a collodion membrane-covered Cu grid (Figure 2) and by tapping-mode AFM on an Si(100) surface (Figure S12). Figure 2A shows the presence of roundshaped assemblies of RU006 with a width of approximately 100 nm. AFM analyses of self-assembled RU006 are shown in Figure S12; the geometries of the nanostructures, which are approximately 100 nm wide and 2.5 nm high, suggest that RU006 tends to form a disk-like assembly with the thickness of a bilayer composed of two-dimensional β-sheets. [Ant6]-RU006, in which an aromatic Ant residue was incorporated in place of the Nal residue in RU006, showed a network architecture made of disk-like assemblies which were smaller than RU006-based assemblies (Figure 2B). Replace-

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RESULTS AND DISCUSSION Design and Synthesis of Template Peptides. An amphiphilic nonapeptide, RU006, was designed to have an Lisoleucine (Ile) at the second and eighth positions and an aromatic amino acid, L-2-naphthylalanine (Nal), at the sixth position, on one face to provide the driving force for selfassembly into a β-sheet conformation via hydrophobic interactions and π−π stacking (Figure 1). An L-lysine (Lys) was placed at the center of the hydrophobic face (the fourth position) to improve the water solubility of the peptide and to accommodate AuCl4− ions within the hydrophobic interior of the self-assembled peptide nanoarchitecture during selfassembly. Another Lys residue was placed on the hydrophilic face (the seventh position) to make the peptide water-soluble and to favor an antiparallel orientation of the β-strands within the β-sheet structure (and/or destabilize a parallel alignment by charge repulsion among the positively charged side chains). L848

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aggregation states of the peptides in the solution phase (Figure S13). RU006 apparently formed particles with an RH value of around 100 nm and tended to associate into larger aggregates (Figure S13A). Replacement of Nal with Ant in [Ant6]-RU006 resulted in a narrow distribution in RH between 150 and 500 nm (Figure S13B). [Phe6]-RU006 appeared to form monodisperse particles with an RH value around 100 nm (Figure S13C). [Arg4]-RU006 showed a tendency similar to that of RU006 (Figure S13D). [Ala4]-RU006, which forms a fibrous structure (Figure 2E), showed predominantly large particles with an RH value of 1 μm in water (Figure S13E). These DLS data agree reasonably well with those for the self-assembled peptide nanostructures determined by TEM and AFM measurements and suggest that the peptides stably form characteristic nanoarchitectures in an aqueous environment. With these β-sheet-forming template peptides in hand, we attempted to synthesize gold nanocrystals using a peptidetemplating method. Gold Nanocrystal Synthesis by a Peptide-Templating Method. Figures 3A and 3B show TEM images of gold nanocrystals synthesized by a peptide-templating method in which RU006 monomers/oligomers were mixed with HAuCl4, incubated for 7 days in water, and then reduced with NaBH3CN. Surprisingly, meandering gold nanoribbons 50− 100 nm wide and micrometers long were observed (Figure 3A). A close-up TEM image of an edge of a gold nanoribbon (Figure 3B) shows a periodic stripe structure with intervals of ca. 1.5 Å. According to the featured structure of face-centered cubic (fcc) crystals, the gold nanoribbon was highly crystalline and partially arranged in the ⟨110⟩ direction of elongation. In order to investigate the role of an external reductant, we used the SAED-TEM technique to analyze the differences in crystallinity of gold nanoribbons formed before and after the addition of NaBH3CN. Figures 3C and 3D show dark-colored materials evident in the TEM images, even though the TEM samples were not negatively stained. These results indicate that the obtained gold nanoribbons are metallic. We further investigated the electron diffraction patterns of the samples before and after the addition of NaBH3CN and found significant spots distributed in a ring shape, indicating that the obtained gold nanoribbons were highly crystalline and had polycrystallinity. We next conducted absorption spectroscopy measurements of the reaction mixture before and after the addition of NaBH3CN. Each spectrum has an absorption band around 270 nm from the naphthalene ring and a broad absorption band in the visible light region (Figure 3E). No significant differences between the absorption spectra of the reaction mixtures before and after the addition of NaBH3CN were observed. These results clearly suggest that template RU006 has the potential to reduce AuIII to Au0 slowly without the need for an external reductant in order for gold nanoribbons to grow. We therefore demonstrated that the peptide-templating method, incorporating a lengthy incubation of RU006 and HAuCl4 without the addition of an external reductant, NaBH3CN, provides reproducibly ribbon-like, flattened gold nanocrystals, as seen in Figure S14. Furthermore, in order to confirm such gold nanoribbon formation in solution, we conducted in situ FE-SEM analysis of the reaction mixture of RU006 and HAuCl4 (Figure S15).43,44 The direct observation of gold nanocrystal formation allowed the clear detection of backscattered electrons from a ribbon shape, indicating that gold nanoribbons certainly formed in the

Figure 2. TEM images of the peptide nanostructures self-assembled in water (A) RU006, (B) [Ant6]-RU006, (C) [Phe6]-RU006, (D) [Arg4]RU006, and (E) [Ala4]-RU006. TEM samples were negatively stained with 2% phosphotungstic acid.

ment of the aromatic Nal residue in RU006 with an aromatic Phe residue had little effect on the morphology of selfassembled [Phe6]-RU006, but its diameter was a bit larger than that of RU006-based assemblies (Figure 2C). Replacement of the hydrophilic Lys residue at the fourth position in RU006 with a more basic Arg residue ([Arg4]-RU006) likely caused the formation of disk-like assemblies with diameters larger than those of RU006 and with a tendency to stick to each other (Figure 2D). Surprisingly, fibrous formations were obtained with [Ala4]-RU006 (Figure 2E), even though the only Lys residue on the hydrophobic face was replaced with a neutral Ala residue. It is important to understand the aggregation states of the peptides in the solution phase, even though the nanostructures of the self-assembled peptides in the solid state were already characterized using TEM and AFM techniques. Dynamic light scattering (DLS) analysis can provide the hydrodynamic radius (RH) of macromolecules following application of the Stokes− Einstein equation, but not their structures and shapes.42 Therefore, it is often necessary to combine DLS technique with alternative techniques such as TEM and AFM in order to determine the aggregation states of molecules in the solution phase. Thus, we performed DLS analysis of the aqueous peptide solutions matured for 7 days to understand the 849

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Figure 4. (A) AFM height image and (B) AFM sectional analysis of gold nanocrystals synthesized by RU006 templating.

ribbon-like architectures approximately 50−100 nm wide and micrometers long accompanied by nanoscale spheres. Figure 4B shows that the observed ribbon-like architectures have an ultrathin, uniform thickness (ca. 2.5 nm) corresponding to the self-assembled RU006 nanoarchitectures shown in Figure S12. It was also found that the nanospheres observed in Figure 4 have diameters ranging from several nanometers to 10 nm, which is greater than the thickness of the ribbon-like architectures. This indicates that the nanospheres might be gold nanoparticles formed without the assistance of the template peptides. We attempted to determine the localization of characteristic atoms such as gold, chlorine, nitrogen, oxygen, and carbon within the gold nanoribbons synthesized by RU006 templating using EDS-STEM (Figure 5). Black ribbons in the panel of the bright field (BF)-TEM image were similar to those observed in Figure 4A. The black ribbons and characteristic X-ray signals for Au atoms superimposed well, showing that the ribbonscattered electrons originated from gold nanocrystals. Notably, characteristic X-ray signals corresponding to nitrogen, oxygen, and carbon generated by the template peptides were also superimposed on the Au signals as well as signals from chlorine. If AuCl4− ions were associated with the Lys side chains on the outer surfaces of the peptide nanoarchitectures, gold nanospheres might form at the outer surfaces facing the bulk aqueous medium due to favorable surface energy. However, very few gold nanospheres were evident in the BF-TEM image (Figure 5). These results may suggest that template RU006 is closely associated with the surface of the gold nanoribbons and that the chloride ions are associated with the positively charged Lys side chains as counterions. In order to characterize the ultrathin gold nanoribbons in more detail, we conducted tomography-TEM measurements with electron beam irradiation at different incident angles ranging from −75° to +75° at 1° intervals. Figure 6A shows representative TEM images obtained at incident angles of −75°, −45°, 0°, +45°, and +75°. The TEM image at 0° incident angle indicates a ribbon-like gold crystal partly stacked by

Figure 3. (A) TEM image for gold nanocrystals synthesized by RU006 templating with the addition of an external reductant, NaBH3CN. (B) An expanded view of the circled area in panel A. (C, D) TEM images for gold nanocrystals synthesized by RU006 templating before and after the addition of NaBH3CN as a reductant, respectively. Insets: SAED patterns. (E) Absorption spectra of the reaction mixtures for gold nanocrystal synthesis by RU006 templating before and after the addition of NaBH3CN. The TEM samples were not stained.

reaction mixture. However, the in situ FE-SEM image of the gold nanoribbons was slightly out of focus, probably due to Brownian motion of the particles. Additionally, the treatment of HAuCl4 with template peptide RU006 in water in the dark did not affect the absorption spectrum compared with that for RU006, suggesting that reduction of AuIII to Au0 might occur by direct electron transfer from RU006 and not by a photoinduced process (Figure S16). Thereafter, we synthesized gold nanocrystals using the peptide-templating method without the addition of external reductants under the standard conditions. Characterization of Gold Nanoribbons Synthesized by the Peptide-Templating Method. In order to understand the mechanism by which gold nanoribbons form during peptide-templating, we characterized the morphologies of gold nanoribbons by AFM (Figure 4), EDS-STEM (Figure 5), and tomography-TEM techniques (Figure 6). Figures 4A and 4B show the AFM height image and AFM sectional analysis, respectively, of samples taken from a reaction mixture and mounted onto a Si(100) surface. Figure 4A shows meandering 850

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Figure 5. EDS-STEM analysis of gold ribbons synthesized by RU006 templating (sample not stained). White ovals in the six panels clearly indicate a superimposition of gold nanocrystals and template peptides.

Figure 6. (A) Representative TEM images obtained by electron beam irradiation at different incident angles ranging from −75° to +75° at 1° intervals (sample not stained) for a gold nanoribbon synthesized by RU006 templating. (B, C) Three-dimensional images of a gold crystal (different angles) obtained by tomographic reconstruction of the TEM images in panel A.38,39 Scale bars = 100 nm.

crystal at a −75° or +75° incident angle. This indicates that organic template molecules, which are less effective at scattering an electron beam, cover the surface of the gold nanoribbon. These results are supported by the three-dimensional images of the ribbon-like crystal shown in Figures 6B and 6C.38,39 Thus,

nanospheres. When the incident angle was tilted from 0° to −75° or +75°, an ultrathin ribbon-like gold crystal was observed. Interestingly, the aggregates of gold nanospheres that overlap with the end of the ribbon-like crystal at 0° incident angle appear to float on the surface of the ribbon-like 851

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Figure 7. (A) Time dependence of ultrathin gold nanoribbon formation by RU006 templating. Absorption spectra of the reaction mixture at 0, 1, 2, 3, 4, and 6 h after initiation of the reaction. Inset: time-dependent changes in absorbance at 510 nm. (B) NaCl concentration dependence of ultrathin gold nanoribbon formation by RU006 templating. (C) Absorption spectra of gold nanocrystals synthesized by templating with RU006, [Ant6]RU006, and [Phe6]-RU006. (D) Absorption spectra of gold nanocrystals synthesized by templating with RU006, [Arg4]-RU006, and [Ala4]-RU006. Absorption spectra in panels B, C, and D were acquired 7 days after the reactions started.

days reaction. The absorption band in the visible light region decreased as the NaCl concentration increased from 0 to 1.0 mM. We also analyzed aliquots of reaction mixtures containing NaCl (0, 0.5, and 1.0 mM) by TEM (Figures S18A and S18B, S18C and S18D, and S18E and S18F, respectively). The TEM samples were negatively stained with 2% phosphotungstic acid. The morphologies of the gold nanocrystals changed from ribbon-like architectures to aggregates of small spheres as the NaCl concentration increased from 0 to 1.0 mM, suggesting that NaCl inhibits AuCl4− ion uptake into the hydrophobic interior of self-assembled RU006 nanoarchitectures by acting as counteranions for the positively charged Lys side chains. We next attempted to identify which amino acids play important roles in the synthesis of ultrathin gold nanoribbons by the peptide-templating method. We employed two different RU006 analogue peptides, [Ant6]-RU006 and [Phe6]-RU006, for the synthesis of gold nanocrystals and measured the absorption spectra and TEM of the reaction mixtures. Figure 7C shows absorption spectra of the reaction mixtures containing RU006, [Ant6]-RU006, and [Phe6]-RU006 as templates after 7 days reaction. The absorption spectrum for [Ant6]-RU006 shows a strong absorption band at around 550 nm, characteristic of the surface plasmon resonance effect. TEM images of the sample taken from the reaction mixture of [Ant6]-RU006 and HAuCl4 do not show any ribbon-like (anisotropic) gold nanocrystals; instead, relatively small (isotropic) gold nanocrystals ca. 20 nm in diameter were synthesized (Figure 8A), comparable to the size of the selfassembled [Ant6]-RU006 nanostructures shown in Figure 2B. In contrast, the absorption spectrum of [Phe6]-RU006 did not show a strong absorption band in the visible light region even after 7 days’ incubation (Figure 7C). TEM images of the [Phe6]-RU006 reaction mixture (Figure 8B) show many disklike assemblies, the same as those in Figure 2C, but no gold

the dimensions of the ultrathin gold nanoribbon formation are apparently dictated by the dimensions of the template architecture. Mechanism of Ultrathin Gold Nanoribbon Formation by the Peptide-Templating Method. We characterized the ultrathin gold nanoribbons synthesized by RU006-templating in order to understand the mechanism of formation of ultrathin gold nanoribbons using this technique. To this end, we conducted a time-dependent analysis of ultrathin gold nanoribbon formation by RU006 templating using absorption spectroscopy measurements and TEM. Figure 7A shows the changes in the absorption spectra of a mixture of RU006 and HAuCl4 in water; the absorption band in the visible light region increased as a function of time, plateauing 6 h after the reaction started. We analyzed aliquots (negatively stained with 2% phosphotungstic acid) of the reaction mixture after 2, 4, and 6 h reaction time using TEM (Figure S17). Figure S17A shows dark-colored aggregates of relatively spherical gold crystals on the edges of light gray-colored, ameba-like networks (t = 2 h). Figure S17B shows gold crystals joined together to form ribbon fragments within the ameba-like networks (t = 4 h). Figure S17C shows that gold nanoribbon formation is essentially complete along the ameba-like networks (t = 6 h). These results suggest that the template peptide RU006 likely forms ameba-like networks in the presence of HAuCl4. The results further suggest that AuCl4− ions are encapsulated via electrostatic interactions and are gradually reduced within the interior cavity of the self-assembled RU006 nanoarchitectures by RU006 itself, resulting in the growth of gold crystals. In order to understand how AuCl4− ions are encapsulated within the interior cavity of the self-assembled RU006 nanoarchitectures, we examined gold nanocrystal synthesis by RU006 templating in the presence of NaCl. Figure 7B shows changes in the absorption spectra of a reaction mixture after 7 852

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residues: the Ant residue is likely a faster reducing agent than Nal, leading to isotropic gold nanocrystals. We further employed the RU006 analogues, [Arg4]-RU006 and [Ala4]-RU006, to identify the role of the Lys residue at the hydrophobic face (the fourth position) in RU006. In Figure 7D, [Arg4]-RU006 showed a small absorption band at 550 nm and a broad band at long wavelengths, a combination of which might support the formation of aggregates made of plate-shaped gold nanocrystals (Figure 8C). These results indicate that gold nanocrystals underwent aggregation in the relatively large interior cavity of the loosely packed (due to charge repulsions by guanidino moieties) self-assembled [Arg4]-RU006. Furthermore, the absorption spectrum of [Ala4]-RU006 had a strong absorption band at 550 nm (Figure 7D), which was similar to that for [Ant6]-RU006 (Figure 7C). This might indicate relatively small and dispersed gold nanocrystal formation (Figure 8D). [Ala4]-RU006, lacking a Lys residue at the hydrophobic face, in the presence of AuCl4− ions inhibits fibril formation and instead results in the generation of irregular aggregates. Consequently, [Ala4]-RU006 aggregates might be less effective at AuCl4− ion uptake within the interior cavity. As a result, there may be insufficient AuCl4− ions to promote the synthesis of micrometer-long nanoribbons, so only relatively small, isotropic gold nanocrystals are generated. The lysine residue at the hydrophobic face of RU006 would be effective for concentrating AuCl4− ions within the interior cavity of a self-assembled RU006 nanoarchitecture. On the basis of the results above, we propose a mechanism for the formation of ultrathin gold nanoribbons by peptidetemplating, as described in Figure 9. Nonapeptide RU006 undergoes self-assembly to form a network structure (50−100 nm wide, 2.5 nm thick, and several micrometers long) by cooperatively gathering disk-like assemblies in the presence of AuCl4− ions. At this stage, AuCl4− ions are encapsulated and concentrated within the interior cavity of the network architectures as counteranions to the positively charged Lys

Figure 8. TEM images of gold nanocrystals synthesized by templating with the RU006 analogues (A) [Ant6]-RU006, (B) [Phe6]-RU006, (C) [Arg4]-RU006, and (D) [Ala4]-RU006. TEM samples for [Phe6]RU006 (panel B) and [Ala4]-RU006 (panel D) were negatively stained with 2% phosphotungstic acid.

structures. Surprisingly, replacement of the Nal residue with a Phe residue in RU006 drastically altered the results, as described above. This would suggest that the electron source to reduce AuIII to Au0 is an electron-rich naphthalene or anthracene ring. Differences in morphology between gold nanocrystals synthesized by RU006 and [Ant6]-RU006 could result from differences in aromaticity between Nal and Ant

Figure 9. Proposed reaction mechanism for the synthesis of ultrathin gold nanoribbons within the interior cavity of a self-assembled peptide nanoarchitecture. 853

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fabricated anisotropic, ultrathin gold nanoribbons approximately 50−100 nm wide and micrometers long encapsulated within the interior cavity of the self-assembled RU006 nanoarchitecture ([RU006] = [HAuCl4] = 50 μM in water). NaCl competitive assays and gold nanocrystal synthesis using the RU006 analogues [Ant6]-RU006, [Phe6]-RU006, [Arg4]RU006, and [Ala4]-RU006 revealed the mechanism of ultrathin gold nanoribbon formation by RU006 templating. It appears that RU006 forms a network (50−100 nm wide, 2.5 nm thick, and micrometers long) by cooperative association of disk-like assemblies via AuCl4− ions that are encapsulated and concentrated within the interior cavity of the network architectures as counteranions for the positively charged Lys side chains. This is followed by electron donation from the naphthalene rings to AuIII to slowly crystallize ultrathin gold nanoribbons along the template network architectures in ambient conditions. Our findings will help diversify the morphologies of template architectures and widen applications for diverse metallic nanoarchitectures.

side chains at the hydrophobic face of RU006. Electrons are donated from naphthalene rings inside the cavity to AuIII to afford seed crystals of gold. Gold crystals grow slowly along the template network architectures at 40 °C over 6 h. The use of RU006 as a template results in the formation of ultrathin gold nanoribbons whose width and thickness reflect the dimensions of the template architecture. Recently, the formation of gold nanoparticles by short peptides was characterized by two basic functionalities of the amino acid residues: reducing capability for precursor ions and capping (binding) capability for the nanoparticles formed.45 The authors summarized that peptides containing the aromatic amino acids, L-tryptophan (Trp) and L-tyrosine (Tyr), were fast reducing agents, but amino acid mixtures of Trp and Lcysteine (Cys), L-methionine (Met), or L-histidine (His), all of which are good binders of metal ions, showed exceptionally slow reduction rates. They also proposed a model for the synthesis of gold nanoparticles using Trp-interdigitated peptides, XWXWXWX (W = Trp, X = other amino acids) in aqueous solution: (i) the formation of peptide−AuCl4− complexes, noting that strong complexes inhibit the subsequent reduction to Au0; (ii) reduction facilitated by peptides to release Au atoms for nucleation; (iii) gold crystal growth by the addition of more Au atoms from the solution or by fusion with other nuclei. This proposed mechanism supports our mechanism well: briefly, the hydrophobic face of RU006 has a Nal residue (at the sixth position) as a reducing agent and a Lys residue (at the fourth position) as a binder to AuCl4− ion through electrostatic interactions, especially within the hydrophobic cavity of the self-assembled nanoarchitecture. Antiparallel β-sheet formation-based peptide self-assembly might contribute to cooperatively fix spatially the naphthalene and ammonium moiety in close proximity within the cavity. Lastly, we examined the catalytic activities of gold nanocrystals synthesized by RU006, [Ant6]-RU006, [Arg4]-RU006, and [Ala4]-RU006 in the reduction of 4-nitrophenol to 4aminophenol in the presence of NaBH4 according to the literature.46 However, gold nanocrystals synthesized by the RU006-based peptide-templating method showed too low a catalytic activity to measure, probably resulting from their relatively large particle size and/or the decoration of the outer surfaces of the gold nanocrystals with template peptides. In future work we will modify the amino acid sequences of RU006-based template peptides to provide gold nanocrystals with controlled shapes, especially gold nanorods applicable for photothermal therapy.47 The present peptide-templating method, employing the interior cavity of a self-assembled template architecture to control the morphology of inorganic materials, represents a new direction in peptide-directed metallic nanoarchitectures. Further investigations are required to improve the peptidetemplating method by understanding the relationship between the self-assembled template nanoarchitectures and the morphologies of the inorganic materials obtained.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC and MALDI-TOFMS profiles, ATR-FTIR spectra, and DLS data for all new peptides; AFM data for RU006; in situ FESEM image and TEM images for gold nanocrystals synthesized by RU006 templating; absorption spectra for gold nanocrystal synthesis in the dark. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel +81-77-543-7469; Fax +81-77-543-7483 (K.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research (K.T.) from the Japan Society for the Promotion of Science (JSPS). Electron microscopy images were obtained at the Ryukoku University Electron Microscope Laboratory. Chimera, used for topographic reconstruction of TEM images, was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41GM103311). We thank Professor Hisakazu Mihara (Tokyo Institute of Technology, Japan) for assistance with ATR-FTIR and DLS measurements.

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REFERENCES

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CONCLUSIONS We have described the de novo design, synthesis, and characterization of the β-sheet-forming nonapeptide, RU006, and the formation of self-assembled disk-like nanoarchitectures in ultrapure water. RU006 predominantly forms an antiparallel β-sheet conformation within these nanoarchitectures. These self-assembled structures are approximately 100 nm wide and 2.5 nm high and tend to form a network structure. We also 854

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