In situ Preparation of Gold Nanoparticles of Varying Shape in

May 8, 2008 - The supramolecular template itself has the structure-directing ... metal salt solution and external reducing agent and sometimes .... Th...
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J. Phys. Chem. C 2008, 112, 8159–8166

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In situ Preparation of Gold Nanoparticles of Varying Shape in Molecular Hydrogel of Peptide Amphiphiles Rajendra Narayan Mitra and Prasanta Kumar Das*,† Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700032, India ReceiVed: December 27, 2007; ReVised Manuscript ReceiVed: March 17, 2008

We have synthesized the gold nanoparticles (GNPs) of different shaped by in situ reduction within the structuredirecting low molecular weight gel template of tryptophan-containing peptide amphiphiles in water without using any external reducing or capping agents. Gold nanocrystals with sheet, wire, octahedral, and decahedral shapes were modulated with the help of the different morphology of the supramolecular gel network. The gel morphology at minimum gelation concentration is the prime requirement to prepare such different nanocrystals of gold. We also elucidated that the seeding growth approach did not have any stimulating affect to control the shape of GNPs. The supramolecular template itself has the structure-directing capability. The formation of gold species in the aqueous solution was monitored by UV-vis spectroscopy, transmission electron microscopy, and X-ray diffraction studies. The tryptophan-based peptide hydrogelators are elegant hosts as structure-directing templates for in situ shape-controlled preparation and stabilization of GNPs. Introduction Metal nanoparticles (NPs) have been gaining enormous attention owing to their unique optical, electrical, and magnetic properties, as well as their potential applications in several frontier areas of nanotechnology and nanobiotechnology including catalysis, single-electron tunneling devices, nonlinear optical devices, biological labeling, surface-enhanced Raman Scattering (SERS), and so on.1–4 It is well-known that the inherent properties of such metal NPs like, gold, silver, platinum, and cadmium strongly depend on the shape and size of the particles.5–14 Thus, the synthesis of nanocrystals with controlled shape and size is of tremendous significance. To date, this has been primarily done in solution phase with different ratios of metal salt solution and external reducing agent and sometimes in the presence of structure-initiating ions.5–18 In this regard, the formation of small gold crystallites is known to be influenced by the photoresponsive cross-linking polymer gels, which guide the propagation of the metal NPs.19,20 The cross-linked polymers swell in alcohols with the formation of liquid-filled cavities and the NPs formed within these cavities is related to the degree of swelling of polymer gels.19–21 To this end, the low molecular weight gels, comprising a diversified supramolecular 3D network, such as fibers, thin sheets, helical, lamellar, etc., depending on the structure of the gelator, have found immense importance as templates for directing nanostructures.22,23 Synthesis and stabilization of NPs within such a hydrogel matrix is also increasing in recent dates.24 But most of these methods require external reducing agents. So, it is essential to have a template that itself can also provide active sites for reducing the gold solution to gold nanoparticle (GNP) in a single step. Very recently, Vemula and co-workers25,26 have reported in situ synthesis of GNPs in supramolecular gels where the gelator acts as a reducing and a capping agent. The preparation of NPs in * To whom correspondence should be addressed. Fax: +(91)-3324732805. E-mail: [email protected]. † Also at the Centre for Advanced Materials, Indian Association for the Cultivation of Science.

low molecular weight hydrogel without an external reducing agent notably increases its potential applications. This nanoscale material can be of great interest for biomedicinal application including drug delivery, labeling agents, sensors, etc. due to its comparable size scale with biomolecules.27 To achieve this goal, the gelator also needs to be compatible for biological systems. In this context, several amino acids like tryptophan, tyrosine, and arginine were found to reduce chloroaurate solution and also stabilize the GNPs.28,29 To this end, we have recently reported the hydrogelation of tryptophan-based cationic peptide amphiphiles in water at room temperature.30 The significance of such a supramolecular system could be distinctly increased if the shape of NPs also can be controlled within it. To the best of our knowledge there are no reports on the in situ shapecontrolled synthesis of GNPs in low molecular hydrogel matrix at room temperature. In the present paper, for the first time we have shown the in situ synthesis, stabilization, and growth of GNPs of varying shapes within supramolecular hydrogels of different tryptophan-based peptide amphiphiles (1-4, Figure 1). Importantly this method does not require any external reducing/capping agents to prepare the GNPs. Well-ordered twodimensional arrays of sheet, wire, octahedral, and decahedral gold nanocrystals were obtained in this straightforward method at room temperature. To control the shape of the GNPs further, we have used the widely known “seed”- or “germ”-mediated nanoparticles synthesis.31–36 However, the seeding growth method did not show any influence on controlling the structure of gold nanocrystals. It was also observed that the supramolecular template of the hydrogels at minimum gelation concentration (MGC) is essential to prepare different nanocrystals of gold. Experimental Section Materials. Silica gel of 60-120 mesh, L-tryptophan, Lphenylalanine, L-proline, n-hexadecylamine, N,N-dicyclohexylcarbodiimide (DCC), 4-N,N-(dimethylamino)pyridine (DMAP), N-hydroxybenzotriazole (HOBT), iodomethane, solvents, and all other reagents were procured from SRL, India. Water used

10.1021/jp712106d CCC: $40.75  2008 American Chemical Society Published on Web 05/08/2008

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Figure 1. Peptide amphiphiles, 1-5.

Figure 2. The UV-vis spectra of HAuCl4 solution (1.6 mM, at λmax ) 306 nm) and aqueous GNP seed solution (0.25 mM, at λmax ) 525 nm). The inset shows the corresponding HRTEM image of the seed solution. Scale bar ) 40 nm.

throughout the study was Milli-Q water. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. CDCl3 for NMR experiments was obtained from Aldrich Chemical Co. Amberlite Ira-400 chloride ion-exchange resin was obtained from BDH, UK. Mass spectrometric (MS) data were acquired by electron spray ionization (ESI) technique on a Q-tof-Micro Quadruple mass spectrophotometer, Micromass. Synthetic Procedure. Amphiphiles 1-4 (Figure 1) were synthesized and characterized following the previously reported protocol.30,37 Briefly; Boc-protected L-amino acids (10 mmol) were coupled with the methyl ester (11 mmol) of corresponding L-amino acids using DCC (11 mmol) and a catalytic amount of DMAP in the presence of HOBT (11 mmol) in dry DCM (10 mL). The reaction mixture was worked up in ethyl acetate (50 mL) and washed respectively with 2 M HCl (30 mL), brine (50 mL), 1 M aqueous sodium carbonate solution (50 mL), and brine (50 mL) to neutrality. The concentrated residue was purified by a silica gel 60-120 mesh column with ethyl acetate/ toluene as eluent. The column-purified -OMe-protected compound was saponified with 1 M NaOH aqueous solution (20 mL) at room temperature. After 6 h of stirring, MeOH was evaporated and the residue was added in 50 mL of water followed by washing with diethyl ether (75 mL). The pH of the aqueous part was adjusted to 2 with 1 M HCl and extracted with ethyl acetate (50 mL) and washed with brine (50 mL) to

remove any traces of acid. The organic part was then dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to obtain the -OMe deprotected peptide. This C-terminal free peptide (8 mmol) was coupled with n-hexadecylamine (8.8 mmol) in a similar way with DCC (8.8 mmol), DMAP (catalytic amount), and HOBT (8.8 mmol) in dry DCM (10 mL). The purified BOC-protected long-chain dipeptide by column chromatography was subjected to deprotection by trifluoroacetic acid (TFA, 32 mmol) in dry DCM (8 mL) at room temperature. After 2 h of stirring, solvent was removed and the residue taken in ethyl acetate (50 mL) was thoroughly washed with aqueous 1 M sodium carbonate solution (50 mL) followed by brine (50 mL) to neutrality. The concentrated free amine (7 mmol) was quaternized with excess iodomethane, using anhydrous potassium carbonate (7.7 mmol for 2 and 15.4 mmol for 1, 3, and 4) and a catalytic amount of 18-crown-6-ether in dry DMF (5 mL) for 4-6 h. After the reaction mixture was concentrated, residue was taken in ethyl acetate (50 mL) and washed with aqueous 5% thiosulphate solution (50 mL) and brine (50 mL) to neutrality. The concentrated ethyl acetate part was crystallized from methanol/ether to obtain solid quaternized iodide salt, which was subjected to ion exchange on Amberlite Ira-400 chloride ion-exchange resin column to obtain the pure colorless chloride salts (1-4, 5.2 mmol). The overall yield was 70-80%. Synthetic Procedure for [1-(1-Hexadecylcarbamoyl-2phenylethylcarbamoyl)-2-(1-methyl-1H-indol-3-yl)ethyl]trimethylammonium Chloride (5, Indole N-Me Substituted 3). This amphiphile was synthesized from the iodide salt of 3. Iodide-3 (0.5g, 0.67 mmol) was treated with sodium hydride (0.74 mmol) and excess iodomethane in 5 mL of dry dimethyl sulfoxide (DMSO) under inert condition at ∼55 °C for 4-5 h. The reaction mixture was quenched with water and extracted with chloroform (20 mL). The chloroform part was concentrated and dried under vacuum. The desired compound was purified by column chromatography in a 230-400 mesh silica-gel column with 4% MeOH/CHCl3 as eluent. The concentrate of the column-purified fraction was crystallized from methanol/ ether. The material was then loaded on an Amberlite Ira-400 chloride ion exchange column to obtain the corresponding chloride salt (5) of the indole N-Me substituted 3. The overall yield was 0.2 g (0.3 mmol, yield 40%). 5: 1H NMR (300 MHz, CDCl3) δ 7.54-6.98 (m, 10H), 5.49-5.46 (m, 1H), 4.36-4.41 (m, 1H), 3.74-3.69 (m, 2H), 3.42 (s, 9H), 3.23-3.17 (m, 2H), 2.99 (s, 3H), 2.64-2.60 (m, 2H), 1.71-1.69 (m, 2H), 1.24 (br, 26H), 0.88-0.83 (t, 3H). EA calculated for C40H63ClN4O2 (3 mol % crystal water): C,

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Figure 3. The time-dependent UV-vis spectra of Au-nanoparticles in direct in situ reduction method (a-d) and seeding method (e-h) within the hydrogels of 1-4, respectively. [HAuCl4] ) 23, 10, 1.2, 0.77 mM for 1-4 at their MGC (116, 51, 6.1, 3.8 mM, respectively). The [Au-seed] ) 2.3, 1.0, 0.12, 0.077 mM for 1-4, respectively.

Figure 4. TEM images of directly synthesized nanoparticles (a, b in gel 1; d, e in gel 2; g, h in gel 3; j, k in gel 4) at MGC and the corresponding SAED pattern (c in gel 1; f in gel 2; i in gel 3; and l in gel 4). GNP-dispersed particle suspension (2 µL) was placed on a 300mesh Cu coated TEM grid and dried under vacuum for 4 h before taking TEM images.

66.59; H, 9.64; N, 7.77. Found: C, 66.93; H, 9.23; N, 7.73. ESI-MS m/z 631.4760, calculated 631.4951. [R]20D -15.3 (c 0.014 g cm-3 in MeOH). Preparation of GNPs within Molecular Hydrogel. The required amount of gelators (140, 60, 8, and 5 mg of 1-4, respectively) was added to 2 mL of water and the mixture was heated at ∼40 °C to produce a homogeneous solution. To this gel, HAuCl4 (final concentrations in 2 mL of gel were 23, 10, 1.2, and 0.77 mM respectively for 1-4) was added very slowly over 10 min at room temperature. The concentration ratio was maintained as gelator:HAuCl4 ) 5:1 (to avoid precipitation of gelator molecules due to displacement of Cl- counterion by AuCl4-). After addition of the HAuCl4 solution, the yellow solution immediately became colorless (Au3+ to Au+), which turned to ruby red (Au+ to Au0) within a few minutes. Preparation of Seed-Mediated GNPs in Hydrogel. GNP seeds were prepared according to the previously reported method.32–36 Typically in a 10 mL aqueous solution containing 0.25 mM HAuCl4, ice cold NaBH4 (0.3 mL of 0.01 M) was added all at once with stirring. Stirring was continued for another 30 s. The solution immediately turned orange-red, indicating formation of gold nanoparticles. The seed solution showed a band centered at 525 nm, and the particle size is measured to be around 10-15 nm, confirmed by the UV-vis spectra and

Figure 5. XRD diagrams of the gold nanocrystals: (a) sheet, (b) wire, (c) octahedron, and (d) decahedron in directly (nonseed) synthesized nanoparticles. The dried GNPs embedded gels (1-4) were placed on the glass slide and the X-ray diffraction patterns were obtained with Cu KR (λ ) 0.15406 nm).

transmission electron microscopy (TEM) study, respectively (Figure 2). The solution was used as a gold seed stock solution after 3-4 h of its preparation, allowing all the excess borohydride to degrade. The solution should be used within 24 h of its preparation to avoid thin gold film formation by particle aggregation. The required amount of gelators (140, 60, 8, and 5 mg of 1-4, respectively) was added to 2 mL of water and the mixture was heated at ∼40 °C to produce a homogeneous solution. To these homogeneous gels, a gold seed stock solution was added under constant stirring to obtain final GNP seed concentrations of 2.3, 1.0, 0.12, and 0.077 mM for 1-4, respectively. HAuCl4 (final concentrations in 2 mL of gel were 23, 10, 1.2, and 0.77 mM respectively for 1-4, HAuCl4:seed ) 10:1) was then added very slowly to this GNP-seeded hydrogel at room temperature. A successive three-step method was used for the seed-mediated nanoparticles preparation. The step-by-step particle enlargement is more effective than a onestep seeding method to avoid secondary nucleation. Two milliliters of hydrogel (1-4) was taken in three sample vials (labeled A, B, and C). From A to B to C, for each vial 0.2 mL of seed-doped gel having HAuCl4 was transferred to the next

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Figure 8. The UV-vis spectra, TEM image, and corresponding SAED pattern (shown in inset) of the GNPs (0.077 mM HAuCl4 in water) in 4 below MGC (0.025% w/v).

Figure 6. The TEM images of the GNPs prepared in hydrogel of 1-4 (a-d, respectively) by the seeding growth method. The inset shows the single gold nanocrystals from the bulk of the corresponding images.

diffraction patterns were obtained from a Bruker D8 Advance diffractometer with Cu KR (λ ) 0.15406 nm). Fluorescence Spectroscopy. The emission spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer by exciting the probes for tryptophan (Eex ) 280 nm), ditryptophan (Eex ) 320 nm), and kynurenine (Eex ) 365 nm) at room temperature. The excitation and emission slit widths were maintained at 20 nm. Fourier Transform Infrared Spectroscopy (FTIR) Measurement. The FTIR measurements of the nanoparticle-embedded dried gel (1-4) from D2O were carried out in a Shimadzu FTIR-8100 spectrophotometer, using a silicon wafer. Result and Discussion

Figure 7. The TEM image of decahedral GNPs embedded in hydrogel 3 (at MGC ) 0.4% w/v).

vial at 30 min intervals. For all the cases, the solutions in the vials were stirred gently for a few minutes for homogeneous mixing, and after HAuCl4 addition, the seed-doped solutions turned red within 2-5 min. The seed concentration was 10 times diluted from the previous vials from A-C. UV-Vis-NIR Measurements. The UV-vis-NIR spectra were taken with a Perkin-Elmer Lamda 950 UV/vis/NIR spectrophotometer. GNP embedded gels were 10 times diluted before taking each spectrum. Preparation of TEM Grid. After the in situ reduction for 4 h, the GNP-embedded gels were centrifuged and the precipitate was thoroughly washed with water three times and finally with ethanol and acetone to confirm the removal of gelators. GNPs were redispersed in acetone. Two microliters of this redispersed particle suspension was placed on a 300-mesh Cucoated TEM grid and dried under vacuum for 4 h before taking TEM images. TEM measurements were performed on a JEOL JEM 2010 microscope. As we have placed diluted GNP solutions on the TEM grid, very few nanocrystals were seen in each mesh. X-ray Diffraction (XRD) Measurement. The dried GNPs embedded gels were placed on the glass slide and the X-ray

In our recently published work30on the structure and properties of low molecular weight amphiphilic peptide hydrogelators, we have found that gelator 1 (Figure 1) forms an opaque gel with MGC of 7% (w/v) and the gelators 2-4 (Figure 1) form transparent hydrogels with MGC of 3%, 0.4%, and 0.25% (w/v), respectively. These stable transparent hydrogels are thermodynamically reversible in nature and have different threedimensional (3D) supramolecular networks, induced from the varied headgroup architecture of 1-4, as seen in their SEM images (Figure S1, Supporting Information). The presence of tryptophan moiety in the gelator molecules motivated us to utilize the four peptide amphiphiles for shape-controlled in situ synthesis of GNP. Tryptophan reduced the chloroaurate ion to Au0 (GNP)38 within such a structure-directing 3D-network without any additive or foreign element. That could possibly lead to the formation of GNPs of different shapes and sizes, which were immediately stabilized by the other tryptophan moieties within the gel network. Importantly, these hydrogelentrapped GNPs showed remarkable stability in aqueous solution for months under ambient conditions. Gold nanocrystals exhibit strong surface plasmon resonance (SPR) absorption, depending on the size and shape of the particles.1–11,39,40 GNPs of varying shapes embedded within the four hydrogels clearly demonstrate different surface plasmon resonance bands. The observed peak in time-dependent UV-vis spectra at 559, 688 nm for sheets, 595 nm for wire, 515 nm for octahedral, and 585 nm for decahedral shaped GNPs respectively in gel 1-4 (Figure 3a-d) are fairly consistent with previous experimental and theoretical results.41–44 The weaker band at 688 nm is usually taken as an indication of either elongation or

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Figure 9. Emission spectra of (a) GNPs (0.077 mM HAuCl4 in water) in 4 (0.025% w/v) of tryptophan (Eex ) 280 nm) with time (0 min-4 h) and (b) ditryptophan (Eex ) 320 nm) with time (0 min-4 h) (the inset shows the enlarged view of ditryptophan spectra in neat gel at 0 min).

Figure 10. FTIR transmittance bands of amide stretching frequencies of GNPs embedded dried gels from D2O at their MGC (7%, 3%, 0.4%, and 0.25% w/v for 1-4, respectively).

aggregation of NPs. All peaks broaden with time from 4 h onward presumably due to the increase in the nanoparticle size as well as the weak reducing power of the tryptophan residue. To improve the particle size distribution, the in situ reduction of HAuCl4 in molecular hydrogel was carried out by a wellknown seeding method. We observed that the UV-vis spectra of GNPs, embedded in gels 1-4, improved (Figure 3e-h) and the peaks became sharp in nature. However, in all the cases the peaks appeared (Figure 3e-h) at wavelengths greater than that of the seed solution (525 nm, Figure 2 and the TEM image of the seed solution were shown in inset) indicating the growth of NPs on the seed surface. The sharp peaks of GNPs (558 and 660, 568, 571, and 583 nm for gel 1-4, respectively, Figure 3e-h) observed in the seeding method were red-shifted compared to those observed in the nonseed mediated approach.

This may be due to the rapid formation of GNPs on the seed surface leading to irregular growth as reflected in their TEM images (discussed later). The morphology of GNPs was characterized by the TEM and related selected area electron diffraction (SAED) patterns (Figure 4). The TEM image of GNPs developed in gel 1 of lamellar morphology (SEM image of gel 1, Figure S1a, Supporting Information) showed the formation of triangular 2D-sheets (Figure 4a,b) of 1-µm dimensions (the SAED pattern, Figure 4c). The observed sharp peaks at 2θ ) 38.22° and 81.71° in the XRD pattern correspond to (111) and (222) planes of face centered cubic (fcc) gold (JCPDS File No. 04-0784, Figure 5a). Diffraction corresponding to other crystal faces was sufficiently weak and scarcely discernible in pattern. This clearly indicates the preferential growth of nanocrystals oriented along high-

8164 J. Phys. Chem. C, Vol. 112, No. 22, 2008 intensity, (111) planes.41 The reduction of chloroauric acid within the intertwined fibrous 3D matrix of gel 2 (SEM image, Figure S1b, Supporting Information) produced wire-shaped GNP (Figure 4d,e) having an average diameter of 10 nm together with some tiny NPs of 10-20 nm dimension. Probably the fusion between these tiny NPs directs the formation of wireshaped gold nanocrystals (marked arrow, Figure 4e) consistent with the recent observation by Pei et al.42 XRD patterns showed that the wire-shaped gold nanocrystals were composed of (111), (200), (220), and (311) (at 2θ ) 38.25°, 44.46°, 64.65°, and 77.64°, respectively) crystalline facets (Figure 5b), and the corresponding SAED patterns were shown in Figure 4f. To examine the organization pattern of GNPs in the hydrogel matrix of 2, the TEM images were taken 30 min after in situ reduction. It was observed that the closely packed NPs (average size of 4-6 nm, Figure S2, Supporting Information) self-assemble to form the wire-shaped GNP of 50-120 nm width and approximately 1 µm of length. We believe that chloroaurate ions are first entrapped into the intertwined fibrous 3D-matrix and then reduced in situ by the tryptophan moiety of host peptide amphiphiles and stabilized by the gelators. Again the difference between the porous network structures of hydrogels 3 and 4 (Figure S1c,d, Supporting Information) is expected to develop GNPs of varying shapes. Controlled growth of GNPs in gel 3 generates majorly octahedral nanoparticles43,44 of 60 ( 10 nm dimension (Figure 4g,h, SAED pattern shown in Figure 4i). GNPs formed within the porous network of gel 4 were mainly decahedral41 in shape with a uniform size of 210 ( 15 nm (Figure 4j,k, SAED pattern shown in Figure 4l). The XRD patterns for octahedral Au-nanoparticles formed in 3 (Figure 5c) and decahedral Au-nanoparticles formed in 4 (Figure 5d) showed the preferential growth along (111) relative to (200), (220), and (311) planes. The size of the decahedron matches the pore diameter (100-200 nm, as observed in the SEM image of the dry gel in Figure S1d, Supporting Information, as well as from the morphology of the corresponding wet gels by brightfield and fluorescence microscopy, please see ref 27) indicating the growth of the NPs within the gel network. It is obvious that various hydrogels acting as a capping agent will alter the surface energies of different facets of fcc metal nanocrystals. Such variation in surface potential along the several crystal facets is expected to tune the shape of the resulting particles as described in previous reports.18,41 In our case also, hydrogelating capping agents (1-4) probably adsorb on the gold nanoparticles and the varying adsorption ability of gelators along the different crystal facets leads to the development of the crystallographic plane (111) with lowest surface free energy. A considerable difference was observed in the intensity ratio of (111) and (200) planes (at 2θ ) 38.2° and 44.5°, respectively) for each hydrogelator. This variation probably tunes the surface free energies of different facets of gold nanocrystals and thus ultimately leads to the variation in shapes of the gold nanoparticles with different growth rate along (111) and (200) planes. Hence, it may be suggested that the gel morphology substantially influences the size and shape of GNPs, which is also consistent with the UV-vis study as described in the preceding paragraph. As demonstrated in the UV-vis study, GNPs were also prepared by in situ reduction, using the seed-mediated method with the objective of better control in the shape and size of NPs. In addition to the direct wet chemical synthesis, seedmediated growth of NPs is a well-known method of controlling the shape and size of GNPs for various technological importances.1,16,31–36 In this process, Au nanoparticles with a

Mitra and Das diameter of 10-15 nm were prepared in an aqueous solution by the reduction of HAuCl4 with sodium borohydride. When seed particles are introduced in the growth solution, they act as nucleation centers, autocatalyzing the reduction of Au+ to Au0 on their surfaces to grow the NPs. The reducing agent used in the second stage of “seed”-mediated growth is usually a weaker one. In the direct synthetic approach tryptophan is a mild reducing agent. If the rate of GNP synthesis can be accelerated, the shape and size of GNPs may further improve in the tryptophan-based template. The morphology of in situ prepared GNPs by seed-mediated growth was also characterized by TEM study. However, the seeding method did not improve the shape of GNPs and rather showed NPs of irregular shapes with very few regular ones (Figure 6a-d) for 1-4, respectively. The doping of seed into gel 1 produced triangular sheets with NPs clusters (Figure 6a). In case of 2-4, the preoccupancy by the Au-seed into the void volume of the 3D-network probably leaves a space of irregular architecture for the next growth of GNP on the seed surface (Figure 6b-d). The TEM images of these GNPs showed a twin boundary in the particles suggesting the formation of polyhedral Au nanoparticles in gels 2-4. The growth of gold nanocrystals of varying shape in the nonseeding method possibly avoids these disturbance and preoccupancy issues of seeding protocol within the supramolecular hydrogels. The hydrogelators are able to swell with water and the swollen gels having liquidfilled void space are expected to control the growth and organization of the nanoparticles. In the absence of Au seed, a very slow addition of the HAuCl4 solution to the native gels yielded GNPs, which are presumably used as seed immediately in the next growth. For instance in the case of gelator 4, the TEM image of GNPs grown after 5 min showed an average diameter of 2-4 nm (Figure S3, Supporting Information), which might have been the nucleation center for consequent shapecontrolled growth of GNP. These GNPs of 2-4 nm are likely to direct the varying shapes of nanocrystals inside the gel where the void volume is probably related to the degree of hydrogel swelling as reported earlier in the case of cross-linked polymer gels.19–21 Shape-controlled metal NPs in the solution phase were synthesized by ceasing the additional nucleation with capping agents at the end of the reaction.45,46 The observed TEM images in Figure 4 are of the gold nanocrystals, which were extracted from the supramolecular hydrogel network after in situ reduction of HAuCl4. To confirm whether the same morphology of the nanocrystal was present within the hydrogel, we have taken the TEM images of the GNP-gel 3 composites (Figure 7). This image showed that the shape and size (55 ( 10 nm) of gold nanocrystals stay almost intact as that was found for GNPs extracted from gel 3 (Figure 4g,h). Hence, judicious variation in the headgroup geometry would lead to the development of shape-controlled NPs within the supramolecular templates. Under similar reaction conditions, we have carried out the GNP synthesis below the MGC using gelator 4 (0.025% w/v) to find out how important is the presence of the supramolecular gel template in controlling the shape of NPs. From the UV-vis spectra it could be seen that the peak appears at 542 nm corresponding to that of spherical NPs (Figure 8).1,45 Also the TEM image and SAED pattern (inset of Figure 8) showed irregularity in GNPs shapes and formation of spherical nanostructures, indicating that the gel template at gMGC is the prime requirement for directing the shape of different gold nanocrystals. Toward understanding the crucial role of indole N-H of the tryptophan moiety in the in situ reduction, it has been reported recently that the oxidation of the tryptophan moiety results in

Preparation of Gold Nanoparticles of Varying Shape the development of a number of byproduct.38,47 Each of these byproducts has a specific absorption and emission: for example, tryptophan, Eex ) 280, Eem ) 350-360 nm; ditryptophan, Eex ) 320, Eem ) 380-390 nm; and kynurenine, Eex ) 365, Eem ) 460-470 nm. According to the literature, the formation of these byproducts occurs via the tryptophyl radical intermediate.47 To elucidate the mechanism in the present case, fluorescence experiment was carried out using hydrogelator 4 (Figure 9). A red shift from 357 (in absence of metal ion) to 386 nm was observed during reduction of Au3+ to Au0 within the hydrogel of 4 in 4 h (Eex ) 280, Figure 9a). Upon excitation at 320 nm (Eex of ditryptophan), the emission intensity at 388 nm (for ditryptophan formation) increased with time up to 4 h (Figure 9b) and was almost absent in neat 4 (inset of Figure 9b). This confirms that the tryptophan moiety does not get oxidized in the absence of metal ions. It was shown in our previous study that the indole N-H did not play a significant role in hydrogelation.48 Hence the N-H proton can easily participate in the reduction of Au3+. The participation of indole N-H in the in situ reduction of auric acid to GNP was further confirmed with the use of gelator 5 (where the indole N-H of 3 was substituted by a -Me group, Figure 1). MGC of this gelator 5 was 1.0% w/v, which is a little higher than that of its precursor 3. Under the identical reaction conditions as described above (20 mg of 5 and 3.0 mM HAuCl4 were taken in 2 mL of water), no GNP formation was observed. Thus consistent with the previous observation by Sastry et al.,38 indole N-H plays the key role as reductant in the in situ synthesis of GNP. The presence of NPs in the low molecular weight gel is known to change the viscoelastic properties of the nanocomposites as recently reported by Bhattacharya et al.49 It also would be interesting to determine whether the formation of GNPs in the hydrogel of 1-4 has disturbed the network between the amphiphiles. To demonstrate that we have performed the FTIR study of the GNPs-embedded gels of 1-4 by preparing them in D2O, keeping all other conditions identical. FTIR spectra of these dried gels showed that the CdO stretching frequencies remain at 1647 cm-1 (Figure 10) for GNPs-embedded hydrogel of 1 to 4, which is comparable with that of neat gelators in D2O (∼1650 cm-1)30 in the absence of any NPs. This indicates that the intermolecular hydrogen bonding between amide moieties remains intact and holds the gel network. Otherwise the CdO stretching frequencies would have increased as observed in the nonself-assembled state of the gelators in CHCl3 (Table S1, Supporting Information). Conclusion In conclusion, for the first time, we have prepared the GNPs of varying shapes by in situ reduction of HAuCl4 within low molecular weight hydrogel matrix at room temperature in the absence of any foreign ion or external reducing/capping agent. The shape of GNPs was easily modulated in the varied morphology of the 3D-supramolecular hydrogel matrix simply by judicious alteration of the headgroup architecture of hydrogelating peptide amphiphiles. The seeding growth approach to further improve the shape of the GNPs within the hydrogel matrix did not have any inspiring effect. The presence of the gel template is essential for directing various shapes of NPs. Hence, the tryptophan-based hydrogelators are elegant hosts as structure-directing templates for in situ shape-controlled preparation and stabilization of GNPs. This present approach could have applications in the generation of organic-inorganic hybrid material in material science for various nanobiotechnological applications.

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