Methionine-stabilized nonclassical growth within self-assembled de novo gold nanoparticles in conjunction with secondary nucleation inhibition

The key steps for seed mediated growth of noble metal nanoparticles involve primary and secondary nucleation, which depends upon the energy barrier and ligand supersaturation standards of the medium. Herein we report the unique case of methionine (Met) controlled growth reaction, which rather proceeds via impeding secondary nucleation in presence of citrate stabilized gold nanoparticle (AuNP). The interaction between freshly generated Au and thioether group of Met in the medium restricts the secondary nucleation process involving further Au reduction. This incomplete conversion of Au results in a significant enhancement of the zeta (z) potential even at low concentration of Met. Furthermore, the aurophilic interaction of Au controls the selfassembly process of the in situ generated emissive nucleated particles. Nucleation of primary particles on seed surface, their segregation and time dependent conversion to larger particles within self-assembly confirm the nonclassical growth, which has further been explored with Met containing bio-inspired peptides.

Growth reactions for gold nanoparticles as seed or biomolecules such as amyloid fibrils involve secondary nucleation as one of the major steps. 1,2 Small molecule could suppress the secondary nucleation in the growth medium for amyloid beta peptides. 3 However, appropriate small molecules as capping agents in the growth reaction for noble metals follow secondary nucleation leading to a variety of final shapes. 4 Weak or strong binding efficiencies of such small molecules play an important role in the growth process in general, including the growth mechanism of anisotropic gold nanoarchitecture. 5 Compared to the high energy barrier involved in classical growth process by addition of atom to the crystal lattice, oriented attachment among nanoparticles involves low defect-free energy pathway for nonclassical growth. 6 Intermediate synthesis at the molecular level has been traced for time dependent seed mediated gold nanocluster growth. 7 Fluctuations of surface atoms successfully demonstrated the coalescence behavior of gold nanoparticle at 873 K within 1 hour time span on a silicon surface. 8 Very recently, atomic scale mechanism of nonclassical nucleation of gold nanoparticle has been demonstrated by liquid-phase scanning transmission electron microscopy. 9 The ligand's functional group and concentration are highly important in the pre-nucleation stage during gold nanoparticle synthesis. 10 A wide range of shape evolution from spherical gold nanoparticle seed has been reported in presence of Ag + and halides through kinetic and surfacecontrolled growth. 11,12 The combination of icosahedral gold seed and alkylamines shows growth into highly symmetric gold nanostars. 13 Not only the small molecules, but also the biomolecules such as peptides or nucleic acids play an important role in the nucleation process for nanoarchitectures. [14][15][16][17][18][19][20][21][22] The growth reaction of octahedral and cubic gold nanoparticle seed in presence of amino acids and peptides containing cysteine ended up in asymmetric evolution. 23,24 A handful of recent reviews have been reported focusing on the precisely defined anisotropic gold nanoparticle formation through growth reactions. [25][26][27] Furthermore, a wide range of capping and reducing agents used for the selective growth along different facets of anisotropic gold seeds have been found to give a broad variety of final shapes. [28][29][30][31][32][33][34] In our earlier report on the growth reaction involving AuNP as seed and hydroxylamine as reducing agent studied for all the amino acids, the positive z potential has been observed solely in presence of Met. 35 In this current effort, we have focused on Met as small molecule for the stabilization of Au + in secondary nucleation step. The seed catalyzed Au + to Au 0 conversion in the secondary nucleation step 1 is significantly inhibited through the stabilization of Au + -S(thioether, Met) interaction 36 . X-ray photoelectron spectroscopy (XPS) analysis after the Met controlled growth reaction confirms the stabilization of Au + species. The transmission electron microscopic (TEM) images with Met variation show stepwise formation of smaller sized nucleated gold nanoparticle (nAuNP) on parent AuNP seed surface, their detachment from seed and nonclassical growth of smaller individual to larger particles with time within the self-assembly of nAuNP (Scheme 1).
The unique behavior from Met has been explored in the growth reactions with three bio-inspired peptides having variable Met residues.
Role of spectator seed in growth reaction. Citrate stabilized AuNP (15.0 ± 2.0 nm, Figure S1) showing surface plasmon resonance (SPR) peak at 522 nm has been synthesized as seed for the growth reactions in the following steps. An additional small hump around 610 nm appears in absorbance spectra during 30 min incubation of 1.20 nM AuNP with 9 mM Met ( Figure S2).
Addition of 300 µM Au 3+ salt for the growth reaction in presence of excess hydroxylamine as reducing agent turns red-violet color solution immediately into blue and shows dual peak in the absorption ( Figure S3). The time dependent absorbance spectra up to 30 min after growth reaction Scheme 1: Schematic illustration of the Met controlled partial inhibition of secondary nucleation followed by time dependent nonclassical growth. a Blue and yellow spheres for Au + and Au 0 have been used respectively to represent the 3:1 ratio between Au + and Au 0 within nAuNP. These spheres do not represent the size of Au + and Au 0 .
show the red-shifted SPR peak at 540 nm and the additional peak at 696 nm ( Figure 1A and S3).
TEM image after 30 min of this growth reaction reveals spherical assemblies of average diameter 170 nm ( Figure 1B). This observation is distinctly different from our earlier observations made regarding the remaining amino-acids. 35 Closely focusing on one such assembly, highly dense self-assembled nAuNPs has been observed (inset Figure 1B). Interestingly, TEM image reveals the maximum dimension (~11 nm) of individual particle within the self-assembly; noticeably smaller than the parent seed. In addition to the self-assembled nAuNPs, presence of particles of parent seed dimension ( Figure S4) in the same TEM grid confirms the role of seed as spectator only. The same growth reaction without parent seed shows no SPR peak, which endorses the important role of the parent seed in the Met controlled nAuNP synthesis.  Figure 1C). On contrary to the anticipated classical growth of seed, monitoring the same growth reaction with 1 mM Met results in the development of ~6.5 nm dimension segregated nAuNP ( Figure S6). Growth reaction at 3 mM Met concentration produces small self-assembly of nAuNPs ( Figure S7) along with the existence of the spectator seeds in the TEM images.

Methionine and Au
The smaller sized nAuNP formation prompts us to monitor the emission properties of these assemblies. The luminescence spectrum confirms a weak emission at 430 nm from the solution after 30 min growth reaction by exciting the solution at 412 nm ( Figure 1D). This weak emission is either due to the aggregation induced quenching within the self-assembly or due to the larger dimension (~11 nm) compared to the reported emissive gold nanoclusters. 37 In order to achieve better luminescence behavior from the nAuNPs, 10 µM Au 3+ salt has been introduced in the growth reaction (Scheme 1, condition 1). The dual absorbance nature after the growth reaction in presence  This trend confirms two different origins for z potential and 1 mM Met concentration has been observed as a critical concentration, which echoes the Met dependent absorbance data and TEM images procured for this study.
In order to correlate the z potential trend and the self-assembly of the nAuNPs, XPS measurements ( Figure S12) have been carried out for the mixture containing spectator seeds and self-assembled nAuNPs obtained from the growth reactions in presence of 9 mM Met.
Deconvolution studies of binding energy for Au 4f !/# and 4f $/# show the formation of Au + after the growth reaction ( Figure 1I). Similar negative control studies on binding energies for the parent AuNP seed show no formation of Au + ( Figure S13). The unusual stability of Au + in presence of Met in this growth reaction makes it extraordinary. At the initial nucleation step, hydroxylamine as a mild reducing agent reduces the added Au 3+ ions to Au + ions. In the standard secondary nucleation process, seed AuNP participates in the reduction of Au + to Au 0 followed by growth of the parent seed. 1 In this Met controlled growth, secondary nucleation process is partially inhibited due to the unusual stability of freshly generated Au + by the available Met in the solution. 36 The weak emission from this Au + /Au 0 combination in water is similar to the previous report on weakly emissive Au 0 @Au + -thiolate-based core-shell nanocluster in 75% ethanol. 38 XPS study further confirms the presence of 3:7 ratio for Au + :Au 0 after the growth reaction, while 4:6 ratio for Au 3+ :Au 0 has been used in the growth reaction. The secondary nucleation step involving Au + conversion to Au 0 has thus been restricted by 75% in presence of Met. The self-assembly process of nAuNPs in this study has been observed after the growth reaction due to the aurophilic interaction 39 between Au + -Au + present in these nucleated particles (Scheme 1).
The formation of Au + within nAuNPs is responsible for the significant change in the z potential measurements at low concentration (0.1 to 1 mM) of Met. The overall trend of z potential and Met concentration ( Figure 1H) follows equation (1) where, z' (-39.5 mV) is the z potential before addition of Au 3+ , c (4.5) is coefficient and k (0.40) is the Met dependent conversion factor from Au 3+ to Au + and Au 0 .
In addition to the Au + -thioether interaction, absence of the Met carboxylate stretching 41 frequency at 2116 cm -1 in infrared spectrum confirms the interaction between Au + and carboxylate anion in the self-assembled nucleated particles ( Figure S17). The presence of a less intense peak at the same position detected in the infrared spectrum confirms weak interaction prevailing between Au 0 and Met carboxylate after the 30 min incubation of Met to the parent AuNP seed. In anticipation of behavior analogous to the Met selective secondary nucleation inhibition process followed by self-assembly, we have investigated a few other sulfur containing molecules and inorganic salts such as 6-mercaptohexanoic acid (1), 3-mercaptopropionic acid (2), ethyl 4-amino-2-(methylthio)pyrimidine-5-carboxylate (3), lipoic acid (4), oxidized and reduced glutathione (5,6), sodium sulfate (7) and sodium thiosulfate (8). We have observed the classical growth of the parent AuNP seed producing the spherical gold nanoparticle of 16-18 nm diameter (Figure 2A, S18 and S19) after the growth reactions in presence of 1-8 without any positive z potential data.
In the cases of oxidized and reduced glutathione, aggregations of the particles after growth have been observed without any self-assembled geometry. Worth mentioning, the thiol containing amino acid cysteine behaved completely different in the similar growth reaction. 35 Time dependent nonclassical crystal growth within self-assembled nAuNPs. In order to monitor the effect of the reaction time on the nAuNP formation and their size, incubation time of

Supporting Information
The supporting information contains instrumental information and Figures S1-S23.

Growth reaction of AuNP with Met with variable concentration of gold salt
Different sets of 900 µL gold nanoparticles seed solution were incubated separately for 30 min with 90 µL of 100 mM Met. During the incubation period the color of the solutions gradually changed towards reddish violet. Thereafter, 9 µL of 200 mM NH2OH (pH 5 maintained with addition of NaOH) was added to the above solutions and stirred vigorously for 10 min followed by the addition of variable amount (0.5-50 µL) of 0.8% (w/v) HAuCl4 to induce the reduction reaction. In these cases, the final volume of the reactions was adjusted to 1020 μL by addition of deionized water. After addition of gold salt, the reddish violet color immediately changed to blue. The solution was analyzed up to 30 min by different characterization techniques. µL of 200 mM NH2OH (pH 5 maintained with addition of NaOH) was added to the above solutions and stirred vigorously for 10 min followed by the addition of 15 µL of 0.8% (w/v) HAuCl4 to induce the reduction reaction. In these cases, the final volume of each reaction was adjusted to 1020 μL by addition of deionized water. After addition of gold salt, the reddish violet or red color immediately changes to blue. The solutions were analyzed up to 30 min by different characterization techniques.

Growth reactions of AuNP with variable incubation time of Met
Different sets of 300 µL gold nanoparticles seed solution were incubated for 0 min, 5 min and 10 min separately with 30 µL of 100 mM Met. During the incubation period the color of the solution remained unchanged up to 10 min. There was gradually change in color towards reddish violet after 10 min. Thereafter, 3 µL of 200 mM NH2OH (pH 5 maintained with addition of NaOH) was added to the above solutions and stirred vigorously for 10 min followed by the addition of 5 µL of 0.8% (w/v) HAuCl4 to induce the reduction reaction. In each case, the final volume of the reaction was adjusted to 340 μL. After addition of gold salt, the red color immediately changed to blue. The solutions were analyzed up to 30 min by different characterization techniques.
Growth reactions of AuNP with Met containing peptides (M1, M3 and M5) 5 mM stock solutions of M1, M3 and M5 were prepared separately in DMSO due to hydrophobic nature of Met. Three sets of 300 µL gold nanoparticles seed solution were incubated for 30 min with 20 µL of 5 mM M1, M3 and M5 peptides. During the incubation period the color of the solutions gradually changed towards reddish violet for M3 and M5 peptides with high Met content. Thereafter, 3 µL of 200 mM NH2OH (pH 5 maintained with addition of NaOH) was added to the above solutions and stirred vigorously for 10 min followed by the addition of 5 µL of 0.8% (w/v) HAuCl4 to induce the reduction reaction. In each case, the final volume of the reaction was adjusted to 340 μL. After addition of gold salt, the reddish violet color immediately changes to blue. The solutions were analyzed up to 30 min by different characterization techniques.