Morphosynthesis of Gold Nanoplates in ... - ACS Publications

May 29, 2009 - Department of Chemical and Biomolecular Engineering, National .... Singapore-MIT Alliance, 4 Engineering Drive 3, National University...
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J. Phys. Chem. C 2009, 113, 10887–10895

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Morphosynthesis of Gold Nanoplates in Polypeptide Multilayer Films Yen Nee Tan,‡ Jim Yang Lee,*,†,‡ and Daniel I. C. Wang‡,§ Department of Chemical and Biomolecular Engineering, National UniVersity of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore-MIT Alliance, National UniVersity of Singapore, Singapore 117576, and Department of Chemical Engineering, Massachusetts Institute of Technology, Room 16-429, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed: February 17, 2009; ReVised Manuscript ReceiVed: May 5, 2009

Multilayer thin films formed from the layer-by-layer (LbL) assembly of cationic linear polyethyleneimide (LPEI) and anionic polyaspartic acid (pLAA) were used as reactiVe templates for in situ morphosynthesis of gold nanoplates. Size- and shape-controlled growth was made possible by the specific morphogenic action of the polypeptide pLAA and growth in a spatially confined environment. LPEI, in addition to serving as the counter polyelectrolyte for pLAA in the LbL assembly, also provided the facility for chloroaurate anion (AuCl4-) binding through its protonated amine groups (NH3+). The availability and accessibility of these binding sites were important to particle size control and could be varied by the pH of the pLAA solution used in the LbL assembly. Size-controlled synthesis of gold nanoplates was accomplished by controlling the secondary structure of pLAA in the multilayer film. In particular, an increase in the fraction of pLAA R-helices was found to increase the availability and accessibility of the reducing carboxyl side groups of pLAA, resulting in more facile reduction kinetics and smaller particle size in the product. The polypeptide-based reactive templates offered a finer level of kinetic control, enabling the formation of gold nanoplates in a size range (i.e., 50 nm) not possible in a solution phase synthesis (i.e., 500 nm), using reaction mixtures with the same overall composition (i.e., pLAA, LPEI, and HAuCl4) and under the same environmental conditions. The key parameters affecting the polypeptide film formation and their effects on the size-controlled synthesis of gold nanoplates were identified and discussed in detail in this report. Introduction The control of material morphology (i.e., shape and size) at the nanometer length scale has been the centerpiece of materials research in the past decade. This is because the shape and size dependent properties of nanomaterials, metal nanoparticles in particular,1-7 can be used to advantage in a number of technological applications. The synthesis of anisotropic metal nanoparticles is an involved process but can nonetheless be accomplished by a number of techniques.8-17 One of them is using “hard” templates of inorganic mesoporous materials to constrain the material growth to follow the contour of the templates.8-10 Although “hard” templates can produce nanoparticles with a well-defined morphology; they are costly and the variety of available shapes is limited. More importantly, the template removal procedure can be detrimental to the nanoparticles formed. Templated synthesis can also be based on microstructured fluids such as micelles or vesicles.11-13 These “soft” templates have more versatile shapes and are easier to remove. However, they are not robust against possible exchange reaction with the metal precursor leading to the deconstruction of the soft template structure. Polymers, biomolecules, or specifically adsorbed ions which interact selectively with specific crystallographic planes of the nanoparticles have also been used * To whom correspondence should be addressed. Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. Fax: 65 6779 1936. Telephone: 65 6516 2899. E-mail: [email protected]. † Department of Chemical and Biomolecular Engineering, National University of Singapore. ‡ Singapore-MIT Alliance, 4 Engineering Drive 3, National University of Singapore. § Massachusetts Institute of Technology.

to induce anisotropicity.14-17 The effective decoupling of size and shape control is however not possible in this case. This is because the morphosynthesis of nanoparticles is a kinetically controlled process where a slow growth environment conducive to the development of anisotropy also leads to the formation of large particles.15 The biological systems are known to produce some of the very sophisticated inorganic nanostructures known to date.18-23 Therein, morphology control is accomplished either by growth in constrained environments, such as membrane vesicles,23 or through the selective binding of specific biomolecules (e.g., polypeptides) on inorganic surfaces.21,22 Hence, the controllability of particle morphology in a chemical synthesis may be improved by using a designed reactive polymeric matrix that constrains physical growth as well as providing a built-in tunable reactivity. The polymer matrix may be constructed by a variety of methods including Langmuir-Blodgett deposition,24-26 sol-gel entrapment,27 and self-assembly techniques such as self-assembled monolayers (SAMs)28,29 and layer-by-layer (LbL) constructions.30-33 Among them, the LbL technique can be implemented the most cost-effectively. The technique is simple and is able to assemble a multilayer film from polyelectrolytes in aqueous solutions through a diverse range of interactions, including hydrogen bonding, electrostatic interaction, and hydrophobic interaction.34-36 The properties of a LbL film may be tuned in many different ways through the selection of the polyelectrolytes, the solution composition, and the adsorption sequence. These variables may be used singly or in combination to produce a multitude of differently assembled nanostructures with the desired functionalities. Many polyelec-

10.1021/jp9014367 CCC: $40.75  2009 American Chemical Society Published on Web 05/29/2009

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trolytes are suitable for LbL assembly, including DNA37,38 and polypeptides.39-42 Polypeptides, in particular, are significantly different from common polyelectrolytes because of their ability to form secondary, tertiary, and quaternary structures and to undergo conformational changes depending on the environment. Control of peptide adsorption through the control of peptide structure or assembly conditions adds versatility to the preparation of polypeptide multilayer films with different properties.38-40 Polypeptides may also be designed with different compositions and amino acid sequences to impart different physical, chemical, and biological properties to the films.41,42 Recently, Rubner and co-workers have demonstrated the use of polyelectrolyte multilayers as nanoreactors for the growth of spherical gold nanoparticles.43 In their work, free amine groups were generated under specific post-assembly conditions to bind to the anionic gold precursor. The reduction to gold was based on the UV photoreduction of the gold precursor. On the other hand, the use of multilayer films for the morphosynthesis of anisotropic nanostructures has not been demonstrated. The main challenge lies with the difficulty in maintaining the shape-controlling functionality of the polyelectrolyte when the latter is assimilated into a multilayer film, especially when an external strong reducing agent (e.g., H2, NaBH4, and UV light) and post-treatment of the film are required for the nanoparticles synthesis. Previously, we have reported the use of aspartic acid as a multifunctional (i.e., reducing, shape-directing, and particlestabilizing) agent for the synthesis of anisotropic gold nanoplates in aqueous solutions.15 In this study, we incorporated the intrinsic multifunctional properties of aspartic acid into a multilayer film through the LbL assembly of polyanionic poly-L-aspartic acid (pLAA) and polycationic polyethyleneimide (LPEI). The polypeptide multilayer film was then used as a reactive template for the in situ morphosynthesis of gold nanoplates by contacting it with a gold precursor solution (HAuCl4). No further treatment was needed. The nanoparticles formed were retained by the polymer matrix but could be easily released by dissolving the polymeric matrix in an alkaline solution. A 10-fold decrease in the size of the gold nanoplates synthesized was made possible compared to the synthesis of gold nanoplates in pLAA solutions under otherwise identical conditions. The key variables affecting the film growth and the size of the gold nanoplates formed inside the film were identified. A mechanism was then proposed to rationalize the experimental observations. Presented below are the details of this investigation. Experimental Section Materials. Poly-L-aspartic acid (pLAA) (MW ) 25600) and hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 · 3H2O) (g99.5%) were purchased from Sigma-Aldrich Chemicals. Linear polyethyleneimide (LPEI) (MW ) 25000) was supplied by Polysciences. All chemicals were used as received without further purification. Ultrapure water (18 MΩ, prepared from a Millipore Elix 3 purification system) was used as the universal solvent and for rinsing. Preparation of LPEI/pLAA Films. LPEI/pLAA films were assembled on silicon substrates (from Silicon Quest International) and quartz microscope slides (from Electron Microscopy Sciences). Before deposition, a substrate first underwent 15 min of air plasma treatment in a Harrick PDC-32G Plasma Cleaner. LbL assembly was carried out on an automated Mircom DS50 HMS slide stainer. LPEI and pLAA aqueous solutions were adjusted to the desired pH values ((0.1) by 1 M HCl or NaOH.

Tan et al. LbL assembly began by submerging a plasma-treated substrate in the LPEI solution (40 mM by repeat units) for 15 min followed by three 1.5 min rinses in ultrapure water under agitation. The substrate was then transferred to the pLAA solution (7.7 mM by repeat units) and was equilibrated there for 15 min, followed by the same rinsing steps. A bilayer is defined as the film formed from a complete cycle of polycation (LPEI) adsorption followed by polyanion (pLAA) adsorption, and the final assembly is called a multilayer. These are operational definitions which do not necessarily convey any information regarding the structure of the assembled films. The process was repeated until the desired number of bilayers was obtained. The film was then dried and stored in vacuum. Synthesis of Au Nanoplates in LPEI/pLAA Films. A solid substrate coated with the polypeptide multilayer film was immersed in a 0.3 mM HAuCl4 solution (10 mL) until the end of the metal ion reduction reaction. The substrate was then removed and washed copiously with ultrapure water, before it was blown dry with nitrogen and stored in a vacuum. Film Characterizations. The thickness of the film on the silicon substrate was measured with a J.A. Woollam V-Vase variable angle spectroscopic ellipsometer. The reported value was the average of at least 10 different sampling points on the film surface. The Cauchy model was used to fit the experimental data. The effects of underlying Si and SiO2 on film thickness were duly corrected. The secondary structures of the polypeptide films on quartz microscope slides were characterized by a Jasco J-810 circular dichroism (CD) spectropolarimeter. The instrument was set for 100 mdeg sensitivity, 1 nm response time, 1 nm data pitch, and 50 nm min-1 scan rate. A total of 3 scans were accumulated and averaged for each run. A blank quartz slide was used to generate the baseline spectrum, which was then subtracted from the sample spectra. Far UV CD spectra (190-250 nm) were deconvoluted into contributions from R helices, β sheets, β turns and random coils by the CONTINLL program in the public domain CDPro software suite. Particle Characterizations. The UV-visible spectra of assynthesized Au nanoparticles inside the film on quartz slides were recorded on a Shimadzu UV-2450 spectrophotometer at 1 nm resolution. The Au nanoparticles could be released from the polypeptide films by 1 mM NaOH solution. The recovered gold nanoparticles were imaged by a JEOL JEM-2010 transmission electron microscope operating at 200 kV. All samples for microscopy were prepared by dispensing 10 µL of the nanoparticle solution onto a 3-mm carbon coated copper grid, followed by drying in vacuum at room temperature. The shape and size distribution of the as-synthesized gold nanoparticles were determined from counting over 100 particles in randomly selected regions on the TEM copper grid. The surface topography of the samples was examined by atomic force microscopy (AFM; on a DI NanoScope-III(a) MultiMode microscope) operating in the tapping mode to minimize damage to the surface structure. A single-crystal silicon probe was used for the measurements. Results and Discussion Film Growth Behavior and Degree of Intermixing. Linear polyethyleneimide (LPEI) and poly-L-aspartic acid (pLAA) are weak polyelectrolytes with degrees of ionization varying considerably with pH. These are factors which could affect the LbL film growth and the structure of the resulting film. It was found experimentally that LPEI/pLAA multilayers could only be formed in the pH range of 3-6. When either one of the

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Figure 1. Growth behavior of LPEI/pLAA films assembled in LPEI solution at pH 5.5 and pLAA solution at pH 3.5 (rectangles), pH 4.0 (spheres), pH 4.5 (triangles), pH 5.0 (stars), and pH 5.5 (asterisks).

polyelectrolytes was uncharged (see Supporting Information, Figure S1), the lack of electrostatic interaction between the two polyelectrolytes resulted in no film growth, even with 50 cycles of attempted “deposition”. For this study, the multilayer LPEI/ pLAA films were assembled at a fixed LPEI solution pH of 5.5 and five different pLAA solution pH values (3.5, 4.4, 4.5, 5.0, and 5.5) to investigate the effects of pH on film thickness and film organization. Figure 1 shows the average dry film thickness as a function of the number of bilayers. A quick survey reveals that film thickness grew exponentially in all pLAA solutions. Such growth behavior could be understood in terms of the diffusion-based film build-up mechanism proposed by Picart and co-workers.44,45 In this mechanism, polyelectrolyte A, which is present in large excess in the assembly solution, diffuses toward the film interior while the freely diffusible components of the counter polyelectrolyte B in the film diffuse outward, forming complexes at the outer layer of the film with polyelectrolyte A arriving from the assembly solution. This mechanism implies that the amount of newly adsorbed polyelectrolyte is proportional to the amount of diffusible polyelectrolyte already present in the film, resulting in exponential growth. Film buildup was also more facile at the lower pH of the pLAA solution (Figure 1). For example, at the pLAA pH of 5.5, 24 bilayers were required to build a 670 nm film whereas only 7 bilayers were needed to reach a thickness of 624 nm at pH 3.5. This can be explained by the difference in charge densities between LPEI and pLAA under the prevailing deposition conditions. At the low pH of the pLAA solution, the charge density of the pLAA chains is lower; consequently, charge compensation of LPEI required a larger amount of the weakly dissociated pLAA. The reduced interaction (per unit volume basis) between the two polyelectrolytes led to a smaller enthalpic gain in adsorption. Although adsorption could still be spontaneous, the adsorbed species adopted a looplike configuration to lower the entropic penalty (Figure 2A). This also led to a thicker film buildup per deposition. At high charge density, the entropic penalty for spreading the adsorbed polyelectrolyte chains flat on the surface (Figure 2B) was compensated by the enthalpic gain in adsorption, and a more compact film was formed per deposition. Although the “multilayer” films were assembled by the sequential deposition of alternating species on the surface, the

J. Phys. Chem. C, Vol. 113, No. 25, 2009 10889 films should not be regarded as stratified heterostructures consisting of alternating layers of different polyelectrolytes. The two polyelectrolytes actually interpenetrated because of the electrostatic interaction between them. However, depending on the experimental conditions, the degree of interpenetration (or intermixing) may not be sufficiently high to produce a completely well-mixed homogeneous structure.46,47 In our study, the degree of intermixing of the two polyelectrolytes in the film was dependent on the pH of the assembly solution. In the case of deposition at low pLAA pH, the large volume of materials adsorbed per deposition step was not conducive to mixing. On the other hand, interdiffusion of the polyelectrolyte chains occurred with each deposition and hence the degree of intermixing should increase with the number of deposition cycles. The problem of a large deposition volume per cycle for films assembled at low pLAA pH was exacerbated by the reduced opportunity in inducing mixing because of the smaller number of cycles involved in building up to the same film thickness. Therefore, it is reasonable to assume that, for the same thickness, films synthesized at low pLAA pH were less well mixed (Figure 2C) than films fabricated at high pLAA pH (Figure 2D). Secondary Structures in LPEI/pLAA Films. Besides mixing with LPEI in the multilayer film, the polypeptide electrolyte pLAA could also form secondary structures because of its inherent chirality. Figure 3 shows that circular dichroism (CD) spectra of LPEI, pLAA, and LPEI/pLAA complexes in solution and in a multilayer thin film deposited on quartz slide at the same pH. The nonchiral LPEI in solution did not show any absorption difference between right- and left-circularly polarized lights. On the contrary, there were notable differences between the CD spectra of pLAA in solution and those of LPEI/pLAA complexes in solution and in the multilayer film. The CD spectra could be deconvoluted into contributions from R helices, β sheets, β turns, and random coils.48 The results in Table 1 show that the dominant secondary structure of pLAA in solution at different pH values was random coils. While the presence of LPEI with pLAA in the solution did not result in significant deviations from the random coil structure, layer-by-layer deposition of LPEI and pLAA had shifted the dominant secondary structure of pLAA from random coil (in solution) to R-helices (in the polymer matrix). The factors affecting the distribution of pLAA secondary structures in a multilayer film were investigated next. It was found that the fraction of R helices increased with the number of bilayers at the expense of the random coils for all pH values of the pLAA solution tested within the range appropriate for LbL assembly (Figure 4). The fractions of β sheets and β turns, on the other hand, remained relatively constant. This is an indication that LbL assembly had rendered the ordered states of pLAA more energetically favorable. The pLAA chains probably underwent reorientation at each deposition step in order to lower their energy. Unlike the R helix structure, which is stabilized by hydrogen bonding between CO and NH groups of the same strand, the formation of β-sheets requires stabilizing hydrogen bonds to be formed in the same plane between CO and NH groups on two adjacent strands. Hence, the formation of intrastrand R-helices is entropically favorable to the formation of stand-to-strand β-sheets. This is especially so in the presence of intervening LPEI chains which disrupted the more demanding hydrogen bonding that is required to organize pLAA into globally ordered β-sheets. Hence, the pLAA chains adopted the locally ordered R helix conformation. As pH had no apparent effect on the dominant secondary structures of pLAA in solution (Table 1), the changes in the secondary structure distribution within the film had to result

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Figure 2. Conformation of the adsorbed pLAA chains on the film surface at different charge densities: (A) loopy structure (thicker layer per deposition) and (B) flat chains (thinner layer per deposition). Degree of intermixing of LPEI/pLAA multilayer induced by the number of deposition steps to reach the same overall thickness: (C) a heterogeneous film structure from poor intermixing and (D) a homogeneous film structure from good intermixing.

Figure 3. CD spectra of LPEI and pLAA in solution and in a multilayer thin film. (A) LPEI solution at pH 5.0; (B) pLAA solution at pH 5.0; (C) a LPEI/PLAA solution mixture (1:1) at pH 5.0; and (D) a LPEI/ pLAA film (20 bilayers) at pH 5.0.

from the reorganization of the polypeptide structure during film buildup rather than from the charge density on pLAA. Identification of AuCl4- Binding Sites and Reducing Groups in LPEI/pLAA Films. Figure 5 shows the evolution of UV-vis spectra with time during the synthesis of Au nanoparticles in a 20 bilayers LPEIpH5.0-pLAApH5.5 film. Two processes may be identified in the synthesis. One is the accumulation of bound AuCl4- within the film, as shown by the increase in AuCl4- absorbance in the 300 to 400 nm spectral region with time.49 The other is the formation of Au nanoparticles which absorbed strongly in the 500-600 nm range due to surface plasmon resonance (SPR).15,49-51 Since there was no extraneous reducing agent present, the bound AuCl4- had to be reduced by some specific functional groups in the films, which may or may not be the same as those giving rise to the AuCl4- adsorption. The same phenomenon was observed for the LPEIpH5.0-pLAApH5.5 films with different number of bilayers (Figure S2, Supporting Information). A more subtle implication from the UV-vis study is that AuCl4- binding and AuCl4-

reduction were fulfilled by two different functional groups within the film. In the example of Figure 5, during the initial stage of the reaction (i.e., induction period from 0-1 h), only AuCl4binding to the film was observed with no sign of formation of the gold nanoparticles. This indicates that AuCl4- binding and Au nanoparticle formation were different processes and the latter was slower. The SPR peak of nanogold at λmax ) 560 nm first appeared at 2 h. The absorbance then increased with time monotonically to a steady-state value when particle growth was terminated due to the exhaustion of the reducing functionality of the film. At the same time, the absorbance of AuCl4- in the 300-400 nm spectral region continued to grow monotonically. The uncorrelated growth patterns of AuCl4- adsorption and Au nanoparticle formation suggest strongly that the reducing groups for AuCl4- reduction are different from the binding groups, which continued to function even after reduction was no longer possible. If AuCl4- binding and reduction were fulfilled by two different functional groups within the film, the identification of these functional groups and their polyelectrolyte parentage would be the natural next order of business. To answer this, we also conducted the solution phase synthesis of gold nanoparticles using the same two polyelectrolytes and gold precursor at the same concentrations. The experimental results showed that LPEI was unable to reduce AuCl4- in the solution (no reduction was detected for a week). However, when pLAA was used for the synthesis, large gold nanoplates (∼500 nm) were formed (Figure S3A, Supporting Information) in the solution within 24 h. This is not surprising, since the reduction capability of aspartic acid (i.e., the monomer of pLAA) has been demonstrated before in our previous study on gold nanoplate synthesis.15 In another control experiment, we used a mixture of LPEI and pLAA with the same concentrations as those used in the LbL film assembly, for the synthesis of gold nanoparticles. Large gold nanoplates were again obtained in the (mixed) solution in a day (Figure S3B, Supporting Information). This indicates that LPEI did not interfere with the kinetics of AuCl4reduction and hence had no apparent influence on the size of the gold nanoplates formed. Considered together, it may be deduced that the reducing functionality of the multilayer film

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TABLE 1: Secondary Structures of LPEI, pLAA, and Their Complexes in Solution and in a Multilayer Thin Film secondary structure (%) LPEI pH5.0 pLAA pH3.0 pLAA pH5.0 pLAA pH7.0 LPEI pH5.0/PLAA LPEI pH5.0/PLAA

pH5.0 pH5.0

solution complexes LBL film (20 bilayers)

R helix

β sheet

β turn

random coil

dominant structure

NA 18.3 16.9 23.6 18.7 46.0

NA 6.7 7.2 4.1 6.0 3.3

NA 28.5 26.3 2.3 30.8 20.7

NA 46.6 49.7 50.0 44.4 30.0

NA random coil random coil random coil random coil R helix

was based on the carboxyl side groups of pLAA. As the gold precursor exists primarily as AuCl4- anions in aqueous solution, it is reasonable to assume that LPEI, being a polycationic electrolyte, participated in AuCl4- binding through electrostatic interaction. During Au nanoparticle synthesis, the amine groups of LPEI protonated by the acidic HAuCl4 solution would induct AuCl4- ions into the film interior for reaction. After reduction of AuCl4- to zerovalent gold by proximal reducing sites, the positively charged amine groups were regenerated and were available for the next round of binding with AuCl4- from the solution. This explains the observation that the absorbance of AuCl4- continued to grow even after the formation of Au nanoparticles had ended. It was found experimentally that pLAA

had lost its ordered secondary structure after the formation of gold nanoparticles in the multilayer film (Figure S4, Supporting Information). The conformational change was one of the consequences of the oxidation of carboxyl groups on the pLAA side chains and indicated the involvement of pLAA in the chloroaurate reduction reaction. Size-Controlled Growth of Au Nanoplates in LPEI/pLAA Films. The synthesis of gold nanoplates in the LPEI/pLAA films was investigated by varying the reaction conditions. Parameters such as the secondary structure of pLAA and the extent of intermixing between LPEI and pLAA in the film were found to be the most important to particle size control. The following is a summary of the size-controlled growth of Au nanoplates

Figure 4. Distributions of secondary structures in LPEI/pLAA films assembled in a LPEI solution of pH 5.0 and pLAA solutions of pH (A) 5.5, (B) 4.5, (C) 4.0, and (D) 3.5, as functions of the number of bilayers.

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Figure 6. Kinetics of gold nanoplate formation in LPEIpH5.0/pLAApH5.5 films with different number of bilayers.

Figure 5. (A) Evolution of UV-vis spectra of gold nanoparticles synthesized in a LPEIpH5.0-pLAApH5.5 film of 20 bilayers. (B) Kinetics of gold nanoparticle formation as monitored by changes in the absorbance of the Au SPR peak with time.

in the LPEI/pLAA films prepared at different assembly solution pH values and with different number of bilayers. Effects of pLAA Secondary Structures. Kinetics of gold nanoparticle formation in LPEIpH5.0/pLAApH5.5 films with different number of bilayers was followed by measuring the changes in SPR absorption with time. Figure 6 shows the increase in the number of bilayers had greatly shortened the induction time (e.g., from 8 h for the 16 bilayers film to 0.5 h for the 20 bilayers film). This correlated positively with the increased presence of pLAA R-helices in the films with a larger number of bilayers (Figure 4A). As stated previously, pLAA was the source of reducing groups in the film. It is believed that the R-helix structure improved the accessibility of the reducing carboxylic groups on the pLAA side chains, better exposing them to the bound AuCl4- ions on the LPEI chains, thereby resulting in faster reduction. At the end of the reaction, gold nanoplates were found among the gold nanoparticles released from the film and dispersed in the solution (Figure 7A and B; more TEM images of the assynthesized gold nanoparticles can be found in Figure S5 of the Supporting Information). The planarity of the gold nanoplates was confirmed by atomic force microscopy (AFM), as shown in the inset of Figure 7B. Figure 7C shows the selectedarea electron diffraction (SAED) pattern obtained by directing

the electron beam perpendicularly to the flat triangular face of a nanoplate. The inner spots (circles) corresponded well with the formally forbidden 1/3{422} reflections, and the outer spots (boxed) could be indexed to the {220} reflections. The SAED pattern suggests that the gold nanoplates were single crystalline, and the 6-fold rotational symmetry of the diffraction spots indicates that the triangular faces were the {111} planes of fcc Au. All of these observations are consistent with previous studies on gold (or silver) nanocrystals bounded by atomically flat surfaces.15,51,52 An average particle size for the nanoplates was determined by counting no less than 100 gold nanoplates from random samples of the TEM images. The average size of the nanoplates was found to decrease with the increase in the number of bilayers (Figure 8), and the size distribution also became progressively narrower (Figure S6, Supporting Information). This is typical of kinetically controlled particle growth: The induction time is analogous to the time of nucleation. A shorter induction time implies that small gold nanoparticles serving as the centers of nucleation were readily formed due to a prevailing high Au0 supersaturation. Subsequently reduced gold atoms would find it energetically more favorable to accumulate on the existing nuclei rather than to form new ones. Particle growth ensued. With a large number of nucleation centers and a fixed number of reducing groups in the film, growth would be limited and many small particles were produced. On the other hand, a slow induction period suggests that nucleation was a difficult process. There were fewer gold nucleation centers, and continuing reduction led to larger particles being formed on these centers. An increase in the number of bilayers improved the availability of the reducing groups by increasing the fraction of pLAA in the more accessible R-helix structure, leading to faster reduction kinetics and a large number of smaller particles. Similar trends were also observed for films synthesized at other pLAA pH values with different number of bilayers (Table S1, Supporting Information). Effects of LPEI/pLAA Film Structure. Quite different from the synthesis in solution phase where the reducing and binding groups are homogeneously distributed and easily accessible, synthesis inside a polymer matrix is constrained by the distribution and arrangement of the various functional components (e.g., binding sites and reducing groups) required for the conduct of the reaction. Films assembled in LPEIpH5.0 and pLAA

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Figure 7. (A and B) Representative TEM micrographs of gold nanoplates synthesized by the reduction of AuCl4- ions in an LPEIpH5.0/pLAApH5.5 film (20 bilayers). The AFM image in inset 7B shows the planarity of gold nanoplates. (C) SAED pattern showing the single crystallinity of a gold nanoplate. The boxed spots and circled spots correspond to the {220} and 1/3{422} reflections, respectively.

Figure 8. Induction time and average particles size of gold nanoplates synthesized in LPEIpH5.0/pLAA films with different number of bilayers.

Figure 9. Average particle size of gold nanoplates synthesized in LPEIpH5.0/pLAA films of similar film thickness: 650 nm (squares) and 350 nm (triangles), assembled in pLAA solutions of different pH.

solutions of different pH values with comparable thicknesses were found to form nanoparticles with a variety of sizes (Figure 9). As discussed previously, films synthesized at a high pLAA pH required more bilayers to build up to the same thickness than films prepared at a lower pLAA pH. The increase in the number of bilayers also led to a more interpenetrated network

of the two polyelectrolytes as each deposition cycle introduced additional mixing. In such a film, the pLAA chains and LPEI chains were better intermixed. Since LPEI supplied the AuCl4ions binding sites, the improved intermixing between LPEI and pLAA increased the accessibility of the reducing functional groups to the bound AuCl4- ions. The more homogeneous overall reaction environment promoted the formation of a larger number of smaller nucleation centers. This explains the decrease in the size of the gold nanoparticles with increasing pH in the range pH 4.5 to 5.5. However, the trend was reversed for films assembled at pLAA pH 4.5 and lower. This could be rationalized by the decrease in the number of charged binding sites for AuCl4- in films assembled at low pLAA pH. The low pH pLAA solution used for the LbL assembly would extensively protonate the amine groups of the LPEI chains, which in turn facilitated the deposition of pLAA through increased electrostatic interaction. The film prepared under such conditions would have relatively fewer free amine side groups left on LPEI to be protonated subsequently by the acidic HAuCl4 solution (and used for AuCl4- binding) during gold nanoparticles synthesis. As a result, the binding functionality of the film was diminished. The number of AuCl4- binding sites was determined by the number of free (unprotonated) amine groups left over from pLAA deposition. Therefore, even though the degree of intermixing was not high inside a pLAA pH 3.5 film compared to a pH 4.0 film, a smaller number of gold nanoparticles were formed because of the scarcity of the bound AuCl4- (due to the scarcity of the binding sites) during reaction with HAuCl4. Hence, it can be seen that the difference in particle size produced by films with similar film thickness but different assembly pH may be traced to the accessibility and availability of AuCl4- binding sites. Proposed Mechanism. Aspartic acid (Asp) and its homopolymer, i.e. polyaspartic acid (pLAA), were both reducing and capping agents in the gold nanoparticles synthesis. Anisotropy in the nanoparticle product was caused by the asymmetric adsorption of capping agent on the different crystallographic planes of the nuclei. Independent control of size and shape is difficult with the Asp (or pLAA) concentration, since concentration affects both the shape and the size of the nanoparticles formed. For example, a high Asp concentration for fast kinetics and formation of smaller particles also promotes the nonspecific adsorption of Asp on the nuclei as a side effect, which is against anisotropy development. On the contrary, pLAA synthesis of Au nanoplates in a multilayer film offers additional parameters (e.g., film composition, pLAA secondary

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Figure 10. Schematic illustration on the growth of gold nanoplates in the LPEI/pLAA film. Step 1: Submerging the film in an acidic HAuCl4 solution causes the protonation of the amine groups of LPEI left unused during the LBL assembly. The expanded view above the film shows the film internal structure. Step 2: Diffusion of AuCl4- into the film and binding to the protonated amine groups through electrostatic interactions. Step 3: Exposing the bound AuCl4- to the reducing carboxylic groups of pLAA results in the formation of gold nuclei. Step 4: Regeneration of positively charged amine groups to bind another batch of AuCl4- diffused into the film. Step 5: Formation of gold nanoplates. The process of binding and reduction would continue until no more reducing groups are available in the film.

structure content) which may be used to decouple size and shape control. In this work, we were able to improve the reduction kinetics by bringing the AuCl4- ions closer to the reducing groups of pLAA in the multilayer film (hence smaller nanoparticle products) instead of increasing the pLAA concentration, which has the side effect of losing shape selectivity. A mechanism for size-controlled nanoplate formation in the LPEI/ pLAA films based on our experimental observations is illustrated schematically in Figure 10. In essence, there are two independent parameters that can be used to tune the size of nanoplates formed in a multilayer: (1) the pH of the polyelectrolyte solution used in the LbL assembly, which determines the number of amine groups left unprotonated after film formation and is available for chloroaurate binding, and (2) the number of bilayers in the film, which determines the R-helix content of pLAA and thus the accessibility of the reducing carboxylic group of pLAA for reaction kinetic control. The major contribution of this study is that we were able to produce gold nanoplates using a simple biomolecule (pLAA) to ensure biocompatibility in a size range (e100 nm) that is much smaller than what is possible with the solution phase

synthesis. This was accomplished by carrying out the synthesis in a multilayer and by varying the physical conditions of the film as described above. The small gold nanoplates synthesized as such may find potential application in photothermal cancer treatment; similar to the gold nanoshells.53 The flat surface of the nanoplates could establish better thermal contact between the particles and the tumors cells to shorten the NIR exposure time. Conclusion In conclusion, we have successfully applied polypeptide multilayer films (LPEI/pLAA) as the reactive templates for the in situ size-controlled synthesis of gold nanoplates. Through the use of weak polyelectrolytes and simple pH adjustments of the processing solutions, it was possible to create films with a controlled content of free amine groups. These groups could be protonated in HAuCl4 solution to form binding sites for the chloroaurate anions. The bound gold precursor was then converted into gold nanoplates by the reduction and shapedirecting action of pLAA. The intermixing between LPEI and

Morphosynthesis of Gold Nanoplates in Polypeptide Films pLAA during LbL assembly enabled the bound gold precursors to access the reducing carboxylic groups of pLAA for reduction. The rate of reduction was determined by the availability of the reducing carboxylic groups and was promoted by the R-helix content of pLAA, which could be increased by increasing the number of bilayers used in film formation. As the reducing groups were oxidized after reacting with the gold precursor, they depleted progressively with time. The formation of the charge-neutral elemental gold also released the positively charged amine groups for binding with further gold precursor ions. However, the synthesis of Au nanoplates ended with the exhaustion of the nonregenerative reducing groups. The gold nanoplates synthesized as such could be stored dry in the film as a nanocomposite or be released from the film by dissolving the polymer matrix in aqueous alkaline solution. Acknowledgment. This work was supported by SingaporeMIT Alliance. Y.N.T. would like to acknowledge the Singapore-MIT Alliance for her research scholarship. The authors would also like to thank Prof Paula Hammond (MIT) and Dr. Pil. J. Yoo (Seoul National University) for their helpful discussions and comments on the layer-by-layer assembly method. Supporting Information Available: Figures showing calculated net charges of LPEI and pLAA at different solution pH values, the time-course of UV-vis spectra for gold nanoparticle synthesis in LPEIpH5.0-pLAApH5.5 films with different number of bilayers, TEM images of gold nanoparticles synthesized by the reduction of AuCl4- in (A) pLAA solution and (B) LPEI/ pLAA solution mixtures, CD spectra of a LPEIpH5.0/pLAApH5.0 film before (hollow triangles) and after (solid triangles) formation of gold nanoparticles, representative TEM micrographs of gold nanoplates synthesized by the reduction of AuCl4- ions in a LPEIpH5.0/pLAApH5.5 film (20 bilayers), and particle size distributions of gold nanoparticles synthesized in LPEIpH5.0/ pLAA pH5.5 films with different number of bilayers; and a table showing the induction time and average particle size of gold nanoplates synthesized in films assembled at different LPEI/ pLAA pH values and with different number of bilayers. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chem. ReV. 2005, 105, 1025–1102. (2) Narayanan, R.; El-Sayed, M. A. Chim. Oggi-Chem. Today 2007, 25, 84–86. (3) Kelly, K. L.; Coronado, E.; Zhao, L. L.; Schatz, G. C. J. Phys. Chem. B 2003, 107, 668–677. (4) Xiong, Y. J.; Wiley, B.; Xia, Y. N. Angew. Chem., Int. Ed. 2007, 46, 7157–7159. (5) Huang, X. H.; Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Nanomedicine 2007, 2, 681–693. (6) Orendorff, C. J.; Sau, T. K.; Murphy, C. J. Small 2006, 2, 636– 639. (7) Murphy, C. J.; Sau, T. K.; Gole, A. M.; Orendorff, C. J.; Gao, J.; Gou, L.; Hunyadi, S. E.; Li, T. J. Phys. Chem. B 2005, 109, 13857–13870. (8) Han, Y. J.; Kim, J. M.; Stucky, G. D. Chem. Mater. 2000, 12, 2068– 2069. (9) Keating, C. D.; Natan, M. J. AdV. Mater. 2003, 15, 451–454. (10) Mayya, K. S.; Gittins, D. I.; Dibaj, A. M.; Caruso, F. Nano Lett. 2001, 1, 727–730. (11) Steigerwald, M. L.; Alivisatos, A. P.; Gibson, J. M.; Harris, T. D.; Kortan, R.; Muller, A. J.; Thayer, A. M.; Duncan, T. M.; Douglass, D. C.; Brus, L. E. J. Am. Chem. Soc. 1988, 110, 3046–3050.

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