Gold Nanoparticle Formation during Bromoaurate Reduction by Amino

May 24, 2005 - A matched pair of 1-cm-path-length quartz cuvettes (Starna, UK) was used ..... l-amino acid might reduce its complement of Au(0) and th...
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Langmuir 2005, 21, 5949-5956

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Gold Nanoparticle Formation during Bromoaurate Reduction by Amino Acids Suresh K. Bhargava,*,† Jamie M. Booth,† Sourabh Agrawal,† Peter Coloe,‡ and Gopa Kar† Advanced Materials and Catalysis Group, School of Applied Sciences (Applied Chemistry), Science, Engineering and Technology Portfolio, RMIT University, Melbourne, Australia, and School of Applied Sciences, Science, Engineering and Technology Portfolio, RMIT University, Melbourne, Australia Received February 1, 2005. In Final Form: April 19, 2005 The synthesis and characterization of water-soluble dispersions of gold nanoparticles by the reduction of a potassium tetrabromoaurate precursor solution using the amino acids L-tyrosine, glycyl-L-tyrosine, and L-arginine using alkaline synthesis conditions are reported. The particle sizes determined by smallangle X-ray scattering (SAXS) and high-resolution transmission electron microscopy (HRTEM) measurements are found to be inversely proportional to the rate of particle formation, which was determined by time-resolved UV-visible spectrophotometry measurements, and vary very slowly at intermediate gold concentrations and rapidly at the extremes. Dispersions produced with a mixture of the two amino acids glycyl-L-tyrosine and L-tyrosine showed particle sizes and particle size distributions which were directly proportional to the ratio of the two L-amino acids, thus offering the possibility for control over the properties of the gold nanoparticle dispersions.

Introduction The noble metal gold has been the subject of enormous interest and investigation since antiquity, from whence it has been used in glasses, pigments, and for both medical curative and diagnostic purposes.1 However, the prediction that nanoparticles formed in the range 1-10 nm would have electronic properties intermediate between those of the bulk metal and small molecules, offering exciting possibilities for novel devices or materials with applications on the basis of this phenomenon, has created an explosion of interest, the size of which can hardly be overstated. Gold nanoparticles, formed by various reduction techniques of an Au(III) precursor, offer not only the option of tailoring novel gold-based materials that will possess all of the inherent properties of gold materials (such as corrosion resistance), but also a useful “model system” for the investigation of nanoparticle specific phenomena because of the chemical stability of gold in the ground state form and its correspondingly facile reduction to Au(0). Thus, recent research has seen a vast number of studies reported, as illustrated by a recent review of the area,1 which draws the majority of its content from references published since the turn of the century. For the case of novel materials based on gold nanoparticles in which the size effects predicted by physicists are manifest, to facilitate wide application, it is obviously desirable to generate nanoparticles easily, cheaply, and with fine control over both particle size and the distribution of particle sizes within the samples. Typical examples of methods for the production of such samples are the * Author to whom correspondence should be addressed. E-mail: [email protected]. Telephone and Fax: +61 3 99253365. † Advanced Materials and Catalysis Group, School of Applied Sciences (Applied Chemistry), Science, Engineering, and Technology Portfolio. ‡ School of Applied Sciences, Science, Engineering, and Technology Portfolio. (1) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293.

reduction of Au(III) precursor solutions, which generate dispersions of gold nanoparticles in the appropriate solvent. However, a suspension of unstabilized gold nanoparticles, i.e., those with no surface charge stabilization or without ligand stabilization, will aggregate universally as their affinity for each other far outweighs their affinity for any solvent. A form of stabilization that has received much attention of late is that performed by capping the nanoparticles with an agent, such as an alkane thiol, which has at one extreme a functionality compatible with the gold nanoparticles and, at the other, compatible with the solvent in which the particles are to be dispersed. In this manner, postreduction capping by agents such as dodecanethiol has produced gold nanoparticles soluble in organic solvents such as toluene or hexane. Water-soluble dispersions have long been synthesized by processes such as reduction of tetrachloroauric acid with citrate;2 however, of potential interest for biological applications are biofunctionalized nanoparticles.3-7 Gold nanoparticles have been produced via the well-known reduction of chloroauric acid by sodium borohydride and subsequently capped with the amino acid L-lysine.4 This method successfully produced water-dispersible gold nanoparticles; however, studies have also been reported3,5,6 in which the reductant was the amino acid itself, which then proceeded to cap the particles produced and stabilize them with respect to aggregation. The method employed is essentially identical to the aforementioned production of gold nanoparticles by reduction and stabilization with citrate, with the amino acids L-tryptophan, L-tyrosine, (2) Turkevitch, J.; Stevenson, P.; Hillier, J. Discuss. Faraday. Soc. 1951, 11, 55. (3) Mandal, S.; Selvakannan, P. R.; Phadtare, S.; Pasricha, R.; Sastry, M. Proc. Indian Acad. Sci., Chem. Sci. 2002, 114, 513. (4) Selvakannan, P. R.; Mandal, S.; Phadtare, S.; Pasricha, R.; Sastry, M. Langmuir 2003, 19, 3545. (5) Selvakannan, P. R.; Mandal, S.; Phadtare, S.; Gole, A.; Pasricha, R.; Adyanthaya, S. D.; Sastry, M. J. Colloid Interface Sci. 2004, 269, 97. (6) Shao, Y.; Jin, Y.; Dong, S. Chem. Commun. 2004, 1104. (7) Swami, A.; Kumar, A.; D’Costa, M.; Pasricha, R.; Sastry M. J. Mater. Chem. 2004, 14, 1.

10.1021/la050283e CCC: $30.25 © 2005 American Chemical Society Published on Web 05/24/2005

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L-arginine, L-lysine, and L-aspartic acid assuming the role of citrate. Such syntheses are far simpler than the BrustSchiffrin method,8 which has also found wide application and, therefore, perhaps more easily adaptable to large scales, provided the relevant criteria of size control, stability, and functionalization can be addressed. Turkevich9 reported that the production of gold nanoparticles via the reduction of chloroauric acid with citrate ions exhibits a well-defined temperature-dependent induction time in the particle growth regime, identified as the conversion of the citrate into acetone dicarboxylate. The author also found that the nucleation stage for citratesynthesized gold nanoparticles does not occur via the classical concentration fluctuation mechanism. Rather, the system forms a polymeric species of Au(III) complexed by the acetone dicarboxylate. When the polymers formed become large enough, such that enough gold is contained in them to form a particle whose lattice energy contribution overcomes the disruptive effect of the surface energy contribution, reduction occurs and a stable nucleus forms, which then catalyses particle growth by diffusional deposition. Therefore, the citrate reduction method requires: (i) the conversion of citrate into the more reactive acetone decarboxylate species and (ii) polymerization via complexation into species large enough to form stable nuclei upon reduction. However, small gold hydroxide polymers may also form spontaneously at elevated pH,9 which may satisfy condition two above. Burke10 has also stated that Au(III) hydroxide “apparently behaves as an acid, i.e., it tends to accept, rather than donate, hydroxide ions,” and Au(III) hydroxides at electrode interfaces have been reported as being of the form [Au2(OH)9]3+, which involve face-sharing octahedra that can link further to form low-density strand structures, in agreement with Turkevich.9 While Zhou et al.11 reported that adjusting the pH of solutions of silk fibroin to the range 9-11 is likely to enhance the electrondonating properties of tyrosine residues by rendering them negatively charged, it may also lower the reduction potential of the Au(III) by forming the hydroxide, and the aforementioned hydroxide polymer formation may also play an important role by forming incipient clusters, thus further favoring nanoparticle formation. Therefore, given the similarities of aqueous solutions of citrate and L-amino acids, such as the presence of electron donating carboxyl groups, the production of gold nanoparticles by the reduction with an L-amino acid would be expected to proceed similarly. Thus, the spontaneous formation of gold hydroxide polymeric species at high pH, which may, to some extent, fulfill the nucleation energy stabilization requirements, coupled with the enhancement of the electron-donating properties of the L-amino acids due to deprotonation of the molecules, and the enhancement of the reduction potential of the Au(III) species by hydroxide formation suggests that while gold nanoparticle syntheses may be problematic at low pH, it may be possible to produce nanoparticles at high pH from a wide range of L-amino acids. However, the different structures of the various amino acids, such as type and number of functional groups and aromaticity, may produce differing kinetics and, therefore, different particle morphologies. The aforementioned study of ref 6 reported varying morphologies of

(8) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D.; Whyman, W. Chem. Commun. 1994, 801. (9) Turkevich, J. Gold Bull. (Geneva) 1985, 18, 86. (10) Burke, L. Gold Bull. (London, U.K.) 2004, 37, 125. (11) Zhou, Y.; Chen, W.; Itoh, H.; Naka, K.; Ni, Q.; Yamane, H.; Chujo, Y. Chem. Commun. 2001, 2518.

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gold nanoparticles produced by auric ion reduction with the amino acids L-arginine, L-tyrosine, L-tryptophan, L-lysine, and L-aspartic acid. In particular, the platelike morphologies produced by L-aspartate reduction were attributed to specific binding of the amino acid to faces other than the {111} set, although this differs with previous findings.12 However, the relatively recent interest in the production of gold nanoparticles means that extant phenomenological studies of the formation of gold nanoparticles by auric ion reduction by simple L-amino acids, in the manner of the intense study of citrate systems, are limited to essentially this single work. Accordingly, the following study investigates the particle sizes and morphologies of gold nanoparticles produced using the amino acids L-tyrosine, glycyl-L-tyrosine, and L-arginine. Materials and Methods Chemicals. Potassium tetrabromoaurate was prepared by the direct bromination of gold powder in the presence of potassium bromide. Potassium hydroxide (laboratory reagent grade) was obtained from Merck. All amino acids used were of laboratory reagent grade or better and of the L- stereochemical configuration, and all distilled water used in the preparation of the nanoparticle dispersions was purified with a Millipore filter (18.2 MΩ). Synthesis of Gold Nanoparticles. The required amount of amino acid was weighed out on an analytical balance, to which was added 10 mL of 0.1 M KOH solution. After dissolution, the required amount of a 50 mM solution of KAuBr4 was measured out with either a macro- or micropipet, its volume made up to 10 mL, and then added dropwise with stirring to avoid as much as possible large local concentrations and thus provide homogeneity to the amino acid/KOH solution at room temperature. All samples prepared were therefore 0.5 mM in KOH. Characterization. Transmission Electron Microscopy. Samples for TEM analysis were prepared by placing a drop of the gold suspension on a clear, dry 200 mesh copper grid coated with a carbon film. The sample deposited on the grid was allowed to dry in air for a few minutes, following which the excess solution was removed using filter paper. The size, morphology and microstructure of the gold nanoparticle samples prepared as above were studied using a JEOL 2010 transmission electron microscope equipped with a Gatan CCD camera (MSC SI0031) using Gatan Digital Micrograph software. The TEM operated at an accelerating voltage of 200 kV. Small-Angle X-ray Scattering. All small-angle X-ray scattering analyses reported were performed using a Bruker Nanostar SAXS camera using point-collimated copper KR radiation, equipped with a position-sensitive 2D “Hi-Star” detector. All experiments were performed under vacuum to minimize background scattering. Sample transmissions were estimated using a glassy carbon standard, which avoided the need to remove the instrument beam-stop and thus protect the detector. The nanoparticle solutions were analyzed in a vacuum-stable quartz capillary provided by Bruker. Data acquisition was performed at a sample-to-detector distance of 106 cm, which allowed analysis of intensity behavior over the wavevector range: 0.0996 Å-1 e q e 0.235 Å-1. The scattering profile of the empty sample geometry was subtracted from the final scattering profile of the sample (per-pixel subtraction), after first correcting for the intensity of the X-ray beam. UV-Visible Spectrophotometry. The UV-vis spectrophotometric analyses reported were all performed on a Varian Cary 1C UV-vis spectrophotometer at ambient temperature. A matched pair of 1-cm-path-length quartz cuvettes (Starna, UK) was used for all measurements. SAXS Analysis. The scattered intensity of a sample as a function of the wavevector, q, where q ) (4π/λ) sin θ and 2θ is the scattering angle, can be written as the square of the form factor, F(q): (12) Chiang, Y.-S.; Turkevich, J. J. Colloid Sci. 1963, 18, 772.

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1 I(q) ) 〈|F*(q)F(q)|〉 V

(1)

For the central part of a symmetric scattering entity, F(q) can be written:

F(q) ) (∆F)

∫ cos qr dV

(2)

By expanding cos qr into its corresponding power series in Cartesian coordinates and neglecting terms of second order or higher, we arrive at the “Guinier Approximation”:

[

I(q) ≈ (∆F)2V exp -

]

q2R2g 3

(3)

For spherical particles, the radius of gyration Rg is related to the geometric radius of the particle, r, by:

R2g )

x35r

2

(4)

Thus, a plot of ln I versus q2 gives a straight line in the “Guinier Region”, the gradient of which can be used to determine the average radii of the particles. For homogeneous spheres, the form factor from (2) can be derived analytically, and is given by:

sin qr - qr cos qr F(q) ) 3 (qr)3

(5)

Therefore, the scattering from a polydisperse system of spherical particles can be written:

I(q) ) (∆F)2

∫

r max

r min

[

]

sin qr - qr cos qr (qr)3

DV(r)m(r) 3

2

dr (6)

where m(r) ) (4π/3)r3 and DV(r) is the volume distribution function. The solutions of the above integral equation presented in this work were obtained using the program GNOM.13 The volume distribution can be easily converted to a number distribution; however, in practice, this is not always reliable, as the weighing by the volume of the particles in the volume distribution means that small errors in DV(r) at low r result in large errors in frequency.

Results and Discussion Particle Synthesis and Characterization. Figure 1a-c shows HTREM images and the insets show electron diffraction patterns obtained from samples prepared with approximately 0.15 mmol of either L-tyrosine (1a), glycylL-tyrosine (1b), or L-arginine (1c) reacted with approximately 0.034 mmol of aqueous potassium tetrabromoaurate in alkaline solution at room temperature. All three samples are obviously nanoparticulate, and the corresponding electron diffraction images index to face-centered cubic (fcc) structures, characteristic of gold in the bulk form. The L-tyrosine image of Figure 1a reveals a distribution of large particles that are roughly spherical in shape, having diameters falling in the range 5-40 nm and with smooth surfaces at the length scale resolved. Also of note is the presence of extremely small particles with diameters in the range 0.7-1.5 nm, which correspond to the sizes reported for gold nanoparticle nuclei.9 The images suggest that the particles themselves consist of multiply twinned crystals, in agreement with the previous findings reported by Turkevich,9 who proposed that nanoparticle growth occurs via an initial nucleation step, followed by a coagulation stage, in which the nuclei form small clusters (13) Semenyuk, A. V.; Svergun, D. I. J. Appl. Crystallogr. 1991, 24, 537.

Figure 1. (a) HRTEM of L-tyrosine-prepared gold nanoparticles with inset electron diffraction image. (b) HRTEM image of glycyl-L-tyrosine-prepared gold nanoparticles with inset electron diffraction image. (c) HRTEM image of L-arginine-prepared gold nanoparticles with inset electron diffraction image.

and, subsequently, the particles formed catalyzed diffusional/depositional growth by addition of single gold atoms. The morphologies of the particles of Figure 1a suggest that this is a good approximation for the formation of gold nanoparticles by tyrosine reduction of Au(III). The multiply twinned structures suggest a coagulation regime of the nuclei apparent in the image, and the smooth surfaces of the particles suggest that the particles formed during this stage are then “filled out” by deposition of atomic gold. The final particle shape would depend to a large extent on the structure formed during the coagulation step. This step would be expected to proceed via a diffusionlimited aggregation mechanism with some rearrangement during the attachment step to align the crystal planes of the two nuclei.9 The paradigm of diffusion is the random walk,14 and the aggregation of species following such trajectories is expected to produce highly disordered structures14-16 unless substantial rearrangement occurs upon contact. Thus, the morphologies of each particle formed by nucleus aggregation are not expected to be identical. The subsequent depositional growth stage would be concentrated at the junctions of the coalesced nuclei, where the surface curvature is expected to be large,17 as the particles attempt to reduce the positive surface-energy contribution from the particle surface tension.18 Given a sufficient supply of auric ions, spherical particles would then be expected to result. However, if the auric ion concentration is depleted before a spherical structure is formed, anisotropy will result. Figure 2 presents a higher magnification of the same sample, which clearly exhibits a cluster that is most likely the result of the coagulation process, consisting of 3-4 nuclei that have aggregated, forming a highly anisotropic structure in which some (14) Witten, T. A., Jr.; Sander, L. M. Phys. Rev. Lett. 1981, 47, 1400. (15) Meakin, P. Phys. Rev. Lett. 1983, 51, 1119. (16) Kolb, M.; Botet, R.; Jullien, R. Phys. Rev. Lett. 1983, 51, 1123. (17) Eggers, J. Phys. Rev. Lett. 1998, 80, 2634. (18) Moody, M.; Attard, P. J. Chem. Phys. 2002, 117, 6705.

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Figure 2. Zoomed perspective of the l -tyrosine-prepared gold nanoparticles, showing both coagulation and interparticle neck formation.

deposition is apparent at the junctions of the aggregated nuclei, which however has not filled out to a significant extent by further accretion of nuclei or single gold atoms. The manifestation of the depositional growth stage is apparent from the formation of necks between some of the particles illustrated in Figure 2, suggesting that the small cluster observed in Figure 2 formed at a later time, in which the auric ion concentration was significantly depleted, and thus growth was inhibited. Given the smoothness of the surface at these length scales, it is reasonable to assume that the species deposited have a length scale that is roughly atomic. The glycyl-L-tyrosine-produced particles (Figure 1b) exhibit similar structural characteristics to the L-tyrosineproduced particles insofar as the image clearly shows particles comprised of multiply twinned crystals with diameters falling in the range 5-30 nm and which have smooth surfaces. However, absent from the image is the large number of nuclei present in the L-tyrosine sample. The particle size distribution also appears narrower, although the percentage deviation may well be similar, as the average particle size is smaller. Thus, while the particle morphologies indicate a similar growth mechanism consisting of nuclei forming small aggregates, which then fill out by a diffusional deposition process, the absence of the nuclei and the smaller particle size indicate a subtle difference in growth kinetics. However, while the two samples of Figure 1a and b are only subtly different, the morphologies of the L-arginineprepared particles illustrated in Figure 1c differ considerably. The most obvious difference is the highly anisotropic particle structures. The particles, like the L-tyrosine and glycyl-L-tyrosine samples, appear to be comprised of multiply twinned crystals resulting from the coagulation of nuclei formed at early reaction times; however. analogously to the glycyl-L-tyrosine sample, none of the nuclei could be found using the microscope, and in contrast to both other samples, the particle surfaces are extremely rough. The shapes also hint at platelike morphologies, which are indicative of repressed growth,12,19 during the depositional stage. This suggests that the growth process involves nucleation and subsequent coagulation, followed by limited depositional growth. The L-arginine-produced particles also appear to be larger than those prepared by reduction with L-tyrosine, with diameters falling in the range 15-50 nm; however, the size distribution appears to be much narrower. Particle Size and Solution Composition. Another phenomenon apparent from the HRTEM images is the variation in average particle size with the sequence: glycyl-L-tyrosine < L-tyrosine < L-arginine. Figure 3 presents average particle radii of gyration calculated from (19) Engelbrecht, J.; Snyman, H. Gold Bull. (Geneva) 1983, 16, 66.

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Figure 3. Radii of gyration determined from SAXS measurements of three samples identical in composition, however, prepared with either L-tyrosine, glycyl-L-tyrosine, or L-arginine.

SAXS data of the three samples using Guinier’s approximation. This disparity prompted the investigation presented of Figure 4a-c, which exhibits time-resolved UV-vis spectrophotometric data of three samples prepared with 0.2 mmol of L-tyrosine, glycyl-L-tyrosine, and L-arginine, with 0.05 mmol of Au(III) for the L-tyrosine and glycyl-L-tyrosine samples, and 0.08 mmol for the L-arginine sample. The higher gold concentration for the L-arginine sample was necessary to provide a measurable response within a reasonable time, and conversely, the lower gold concentration of the L-tyrosine and glycyl-L-tyrosine samples was used to extend the reaction time to facilitate measurement. As expected, all three samples develop a prominent peak at approximately 530 nm, characteristic of the surface plasmon resonance of gold nanoparticles and thus consistent with the HRTEM images of Figure 1a-c. The major apparent trend, however, is that the time-dependence of the intensity of the plasmon peaks follows the sequence L-arginine > L-tyrosine > glycyl-L-tyrosine. Throughout this study, it was noted that the ruby-red color, resulting from the surface plasmon resonance developed more quickly in samples prepared with glycyl-L-tyrosine, than those samples prepared with L-tyrosine. However, both were reasonably facile with respect to dispersions produced using L-arginine, which, as Figure 4c illustrates, took several days to form. Figure 4c demonstrates that the gold nanoparticle formation kinetics differ considerably for the L-arginine system under the reaction conditions utilized, and Figure 1c indicates that this difference in kinetics manifests as a significant difference in particle size and morphology. The highly anisotropic structures suggest that the particles form by an extensive coagulation mechanism of nuclei formed at early reaction time, and Figure 4c indicates that this mechanism is extremely slow. However, of chief interest is the lack of any significant effects of depositional growth. This, therefore, suggests that while the L-arginine molecules are able to form nuclei, and therefore reduction is possible, the deposition at later time is significantly inhibited. This indirectly supports the mechanism reported2 for the production of colloidal gold by reduction with citrate mentioned previously. Gold nanoparticle formation using citrate proceeds via the formation of polymeric Au(III)/acetone dicarboxylate species, a mechanism by which depositional growth cannot proceed. Thus, the nucleation step and the depositional growth step may differ in manifestation from L-amino acid to L-amino acid. That is, while for some L-amino acids both steps may be facile, for some one may not proceed to the same extent as the other. Obviously, for L-arginine, the data suggest

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Figure 4. (a) Time-resolved UV-vis spectrophotometry of a sample prepared using glycyl-L-tyrosine. (b) Time-resolved UV-vis spectrophotometry of a sample prepared using L-tyrosine. (c) Time-resolved UV-vis spectrophotometry of a sample prepared using L-arginine.

that the nucleation step, while extremely slow, is at least far more facile than that of the depositional growth step. The particle growth process, therefore, most likely consists of a nucleation step, followed by a coarsening stage during which the nuclei aggregate. Shao et al.6 reported the formation of platelike crystals of gold by L-aspartate reduction of tetrachloroaurate without pH adjustment, which was attributed to the binding mode of the L-aspartate molecules preferentially directing growth of the crystal. However, such structures were not observed in samples prepared with L-tyrosine, L-tryptophan, L-lysine, and L-arginine under the conditions utilized. Given the disparity in reaction kinetics between the arginine sample and the other two, in which no platelike structures are observed, the presence of these platelets would seem more in line with the repressed growth theory.12 The SAXS data for the L-arginine sample also showed significantly lower intensity than the L-tyrosine and glycyl-L-tyrosine samples, suggesting that a much lower percentage of the auric ions were converted to bulk Au(0) in the reaction, supporting the hypothesis of inhibited deposition. The dominance in intensity of the sample prepared with glycyl-L-tyrosine (Figure 4a) over that of the L-tyrosine sample (Figure 4b) at the same reaction time suggests that the reduction process is proceeding more rapidly in the sample prepared using glycyl-L-tyrosine. While the exact redox mechanisms for the reduction of Au(III) ions at elevated pH are not apparent in the literature for the three amino acids used in this study, and efforts are currently being made to determine them, a simple inspection of the functionalities of the amino acids in light of tryptophan studies5 suggests the trends can be reconciled as follows. Because the formation of gold nanoparticles requires extensive coordination to the Au(III) species, i.e., for incipient nucleus formation creating favorable conditions for reduction, this is likely to be more facile for glylcyl-L-tyrosine as it contains an amide, an amine, a

carboxyl, and a phenol group, whereas L-tyrosine contains only a phenol, an amine, and a carboxyl. Thus, glylcylL-tyrosine has an extra oxygen and an extra nitrogen in the amide group, and therefore, its potential for complexation would be much higher. The glycyl-L-tyrosine molecule contains an extra carbonyl group with respect to L-tyrosine in the form of an amide and, therefore, contains more π electrons than a molecule of L-tyrosine. These extra π electrons will also help stabilize an oxidized form of the L-amino acids. While L-arginine is the slowest of the three despite having five or six electron donating groups, it does not contain a phenyl group, unlike L-tyrosine and glycyl-L-tyrosine. Thus, electron transfer may not occur readily as the oxidized form may be energetically unfavored. A corollary of this is the fact that, in the literature studies using amino acids, L-tryptophan is the most effective at producing gold nanoparticles.5,6 It can actually reduce at moderate pH and room temperature, albeit slowly unless some heating is applied.5 However, it is highly aromatic, and thus contains many mobile π electrons that may help stabilize the oxidized form to a much greater extent than L-amino acids such as L-lysine and L-valine, which contain none despite having the potential to form complexes with Au(III) species.4 In fact, the authors state5 that, from their data, it is most likely that an aromatic proton is lost in the reduction process, suggesting that stabilization by mobile aromatic electrons is of importance. Therefore, from a simple consideration of the number of electron donating groups and the number of π electrons of each L-amino acid, it might be expected that nanoparticle formation would proceed more readily with glycyl-L-tyrosine than L-tyrosine, while L-arginine, containing only two π bonds that are well separated, would be the least favorable of the three. This differing reduction facility between glycyl-L-tyrosine and L-tyrosine has important consequences for particle growth. Figure 5a and b presents average particle diameters, calculated from SAXS data by assuming

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Figure 5. (a) Particle diameters (nm) produced by reduction of KAuBr4 with glycyl-L-tyrosine as a function of gold concentration. (b) Particle diameters (nm) produced by reduction of KAuBr4 with L-tyrosine as a function of gold concentration.

spherical shapes, for samples prepared using constant molar amounts of either L-tyrosine or glycyl-L-tyrosine (7 mM), but having varying concentrations of gold. The experiments were limited to these two L-amino acids because of the extremely long induction times of L-arginine-prepared samples. The difference in particle sizes implicit in the data of Figure 3 is apparent over the entire range of gold concentrations used. The data are therefore consistent with the experiments of Lei et al.,20 who aggregated latex spheres using an electric field in which the facility of aggregate nucleation was field dependent. Tuning the field to inhibit aggregation resulted in a disproportionate inhibition of nucleation, and thus the nuclei that did form had larger amounts of latex spheres to draw from and therefore grew larger. Given the disparity in intensities of Figure 4a and b, a similar effect would be expected to manifest. Classically, the nucleation step is rate determining, as the nuclei formed catalyzed growth by diffusion and deposition at later stages, evidenced by the autocatalytic growth behavior of gold nanoparticles produced by citrate reduction.9 Therefore, while a slight decrease in reduction facility might have a dramatic effect on nucleation, the catalysis of diffusional growth at later times by the formation of nuclei will mean that this stage of growth is less affected. Therefore, lowering the reduction facility will result in a disproportionate decrease in nucleation, and the diffusional growth stage may be expected to proceed to a similar extent because of autocatalysis. Thus, in the L-tyrosine sample, each nucleus will have a larger amount of Au(III) to bind during the diffusional growth regime and thus will grow larger than their glycyl-L-tyrosine counterparts. Also immediately apparent is the insensitivity of particle size to changes in gold concentration across a broad range. The particle sizes do, however, appear to diverge at low gold concentrations. This is consistent with the results obtained for citrate2 and the theory of von Weimarn.21 As precursor concentration is lowered, the facility of the nucleation step in the particle growth process drops disproportionately, similarly to the aforementioned nucleation dependence on reduction kinetics although, in this case, it is the result of a concentration variation. Therefore, fewer nuclei form with respect to the number of gold species present. This can be easily reconciled by the fact that the nucleation step requires the formation of a polymeric, complexed species, which would obviously be a concentration-dependent process, and as per the results of Turkevich et al.,2 a disproportionate amount of nuclei will form. This disproportion again results in the nuclei that do form (20) Lei X.-Y.; Wan, P.; Wei, Q.-H.; Zhou, C.-H; Min, N.-B. J. Cryst. Growth, 1996, 166, 909. (21) Weiser, H.; Milligan, W. J. Phys. Chem. 1932, 36, 1950.

Figure 6. Volume distributions of two samples prepared with (i) L-tyrosine and (ii) glycyl-L-tyrosine; oscillations are assumed to be artifacts of the indirect Fourier transformation.

having more Au(0) atoms to accrete. Takiyama22 and Chow and Zukoski,23 in agreement with the theory of von Weimarn,21 reported an exponential decrease in the sizes of gold colloids produced via the sodium citrate method with increasing initial concentration of auric ions, which the steep decrease in particle size with increasing gold concentration of Figure 5a and b supports. A slight increase in average particle diameter is also observed at high gold concentrations in accord with the experiments reported21 for gold particle formation in basic medium, further23 confirming that the two disparate trends, one predicted by von Weimarn21 (decreasing particle size), and one observed by Weiser and Milligan21 (increasing particle size), which resulted in some antipathy between the respective authors, are both characteristic of colloidal gold systems. Intermediate to these extremes, however, the particle sizes of the two systems are stable across a broad concentration range. While these results therefore indicate a robust synthesis, producing particle sizes that are relatively constant as a function of gold concentration, they do not offer much hope for tailoring particle size. When the particle sizes do diverge at low gold concentrations, the change is abrupt and dramatic, and thus to control particle size in this regime, fine control is needed over solution compositions, and the dilute samples which result are problematic to analyze reliably. While an increase in average particle diameter at high gold concentrations is observed, tuning particle size using such concentrations has its own inherent problems, not the least of which is the high reagent cost. The volume distribution functions, D(r), calculated from SAXS data using the program GNOM with two typical samples, one each prepared using L-tyrosine and glycylL-tyrosine, are presented in Figure 6. As expected, the L-tyrosine distribution peaks at a much higher particle (22) Takiyama, K. Bull. Chem. Soc. Jpn. 1958, 31, 944. (23) Chow, M.; Zukoski, C. J. Colloid Interface Sci. 1994, 165, 97.

Gold Nanoparticle Formation

radius than that of the glycyl-L-tyrosine sample, consistent with the average particle diameters calculated from Guinier’s approximation. However, also in evidence is the extremely broad distribution of the L-tyrosine sample with respect to the glycyl-L-tyrosine sample. The classical “burst nucleation” theory24 states that nucleation is a result of supersaturation, giving local concentration fluctuations large enough to form stable particles, which then catalyze depositional growth. Therefore, the formation of nuclei occurs at early reaction times in such systems and stops once the concentration falls below the supersaturation limit. If the concentration of precursor is high enough, then continued conversion into the aggregating species will mean that the supersaturation limit will also be reached at later times, and nucleation will again occur. Therefore, a decrease in the amount of precursor as a function of time due to growth means that the production of nuclei at various times results in these nuclei being exposed to varying concentrations of precursor. Thus, a nucleus forming at an early time will have a large amount of precursor to draw from, while a nucleus forming at later times will have a smaller amount to draw from. Therefore, the particles forming at early times will be larger than those forming at later times, and thus, for samples in which nucleation is spread out over a large time interval, a broad particle size distribution will result.24 The nucleation of gold nanoparticles by L-amino acid reduction, however, if assumed to be analogous to nucleation in citrate systems, is not a result of supersaturation, but rather the formation of polymeric incipient nuclei, and therefore the burst nucleation paradigm may not be entirely appropriate. If a species is less capable of forming such polymeric precursor particles, the nucleation step will be less facile. However, if this is simply rate limited, this nucleation will occur over a larger time interval. Therefore, the lower number of functional groups of the L-tyrosine peptide, coupled with the fewer π electrons stabilizing an oxidized species, may mean that nucleation events will be kinetically slower than for that of glycylL-tyrosine. Thus, for the same reasons given above for classical growth, the particle size distribution is expected to be broader. This is confirmed by the presence of nuclei in Figure 1a, as the fact that nuclei remained in solution and were not consumed suggests that they formed at late reaction times. Conversely, if binding and/or reduction is facile, then nucleation will be facile, Therefore, for a sample such as the glycyl-L-tyrosine sample whose volume distribution function is illustrated above, the higher rate of nucleation at early times, resulting in large numbers of nuclei, means that the nuclei will be forming over a short time interval and thus will be exposed to approximately the same concentration of Au(III) reducing to Au(0) for approximately the same amount of time. Thus, diffusional growth conditions are almost identical for each particle, and therefore the particle size distribution would be expected to be narrow, as illustrated in Figure 6, which is supported by the fact that no nuclei are observed in the HRTEM images. Figure 7 illustrates a similar phenomenon for two samples, both prepared using glycyl-Ltyrosine. However, one has five times the gold concentration of the other (0.086 mM compared to 0.017 mM), and thus nucleation rate is expected to be much higher. The samples, whose volume distribution functions are presented, produce particles whose mean diameters are relatively similar, although as explained above, the sample with less gold produces a slightly larger particle size. However, the size distributions are significantly different. (24) LaMer, V.; Dinegar, R. J. Am. Chem. Soc. 1950, 72, 4847.

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Figure 7. Volume distribution functions for two samples prepared with glycyl-L-tyrosine, identical in L-amino acid concentration, however, with [Au(III)]: 0.086mM or 0.017mM.

Figure 8. Particle diameters calculated from Guinier’s Approximation assuming a spherical geometry.

The sample prepared using a lower gold concentration in which particle formation is much slower has a much broader size distribution than that produced using a higher gold concentration, thus supporting the above hypothesis. An interesting question then, given the apparent differences in average particle sizes and size distributions produced by each L-amino acid in solutions of identical amounts of gold and L-amino acid is: what effect does performing the reduction step using a mixture of the two L-amino acids have on these two sample characteristics? Figure 8 presents average particle diameters, again calculated from Guinier’s approximation, for seven samples in which the amount of gold and the total amount of L-amino acid used remain constant, but in which the proportion of L-tyrosine to glycyl-L-tyrosine used to give the total L-amino acid amount is varied. The data clearly show a smooth variation of average particle diameter between the two extremes corresponding to 100% glycylL-tyrosine and 100% L-tyrosine. Thus, the indication is that, despite the two L-amino acids giving significantly different particle sizes due to their corresponding difference in electron transfer stabilization and complexing options, this difference in kinetics can be exploited to produce particles with average diameters intermediate to these two extremes, and as Figure 8 illustrates, the variation is smooth and monotonic. The volume distribution functions also vary smoothly between that characteristic of each sample. Figure 9 illustrates this phenomenon using five of the samples from Figure 8. As the percentage of L-tyrosine is increased (i.e. the molar ratio of L-tyrosine-to-glycyl-L-tyrosine multiplied by 100), the volume distribution shifts to higher particle radii and broadens. Thus, the continuous variation observed in average particle diameter is also manifest in the variation of particle volume distribution.

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each L-amino acid “blend” together and intermediate properties are produced in samples produced using a mixture of the two. Attempts are currently underway to test this hypothesis using L-amino acids with significantly different reduction potentials. Conclusion

Figure 9. Volume distribution functions obtained from samples prepared with differing ratios of L-tyrosine-to-glycyl-L-tyrosine, converted to percentage L-tyrosine.

Extending the previous discussion of the variation of the rate of nucleation and growth for samples prepared with L-tyrosine and glycyl-L-tyrosine, a broad particle size distribution indicates a relatively large nucleation time interval.24 If the two L-amino acids were to be mixed, obviously the polymerization/nucleation step will be affected and will be a result of the formation of complexed species containing a mixture in the same proportion of L-amino acids as that of the solution. The data of Figure 9 suggest that the facility of the conversion of these species is directly proportional to the ratio of the two L-amino acids, and thus as the ratio is varied from 100% L-tyrosine to 100% glycyl-L-tyrosine, the facility of nucleation increases, resulting in a shorter time interval for nucleation and therefore a more homogeneous particle size distribution. While Figure 4a and b illustrate that the reduction kinetics of the two L-amino acids are slightly resolved, the data of Figure 9 confirm that the resolution is not significant enough to allow the resolution of particle characteristics characteristic of the individual L-amino acids in mixtures of the two. It may be expected that, if the reduction facilities of the two L-amino acids was sufficiently different, then one L-amino acid might reduce its complement of Au(0) and then cap the aggregated particles before the process occurring because the other L-amino acid proceeded to any significant extent. This then might be expected to produce a bimodal size distribution, as the reduction, aggregation, and capping processes are essentially occurring in isolation for each L-amino acid. The fact that this is not observed in Figure 9 suggests that the process kinetics are sufficiently similar, such that the characteristics of the samples produced with

Syntheses of gold nanoparticles via reduction of a Au(III) precursor solution (potassium tetrabromoaurate) using the peptides L-tyrosine, glycyl-L-tyrosine, and Larginine were reported. HRTEM images clearly show the existences of nanoscale particulates, and the electron diffraction patterns obtain index to the fcc structure characteristic of gold in the bulk form. Particles produced using L-tyrosine were found to be larger on average than those produced in identical conditions using glycylL-tyrosine, which is consistent with the relative kinetics of the two processes, as are the larger sizes and different morphologies of the L-arginine-prepared particles. Small-angle X-ray scattering measurements were performed on samples produced containing varying amounts of precursor, and for both L-amino acids L-tyrosine and glycyl-L-tyrosine, particle size was found to be stable across a wide range of composition. Sharp increases in particle size were observed at low bromoaurate concentrations, again consistent with nucleation and aggregation theories of particulates in solution; however, the abrupt changes inhibit tuning particle size, and the low precursor concentrations required inhibition-reliable analysis. Particle synthesis using a mixture of L-tyrosine and glycyl-L-tyrosine, however, showed a smooth variation in particle size that was in proportion with the ratio of the L-amino acids used. Therefore, performing the synthesis using a mixture of two L-amino acids with different reduction potentials allows the fine-tuning of particle size between those characteristic of the individual L-amino acids for the synthetic conditions used. The particle size distributions also showed smooth variation between those distributions characteristic of the individual L-amino acids, and thus, in both respects, the properties of the particles produced using mixtures of L-amino acids are intermediate to those expected for the single L-amino acids. This variation is achievable at any precursor concentration, and thus avoids the dilution problems associated with particle size variation via precursor concentration variation. LA050283E