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Nov 1, 2017 - Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States. •S Supporting Informati...
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Solution Effects on Peptide-Mediated Reduction and Stabilization of Au Nanoparticles Catherine J. Munro, and Marc R. Knecht Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01896 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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Solution Effects on Peptide-Mediated Reduction and Stabilization of Au Nanoparticles Catherine J. Munro1 and Marc R. Knecht1,* 1

Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida, 33146, USA.

ABSTRACT: Biomimetic methods for the preparation and application of inorganic nanomaterials represents a unique avenue to sustainably generate functional materials with long term activity. Typically, for the fabrication of these structures, the peptide is mixed with metal ions in solution prior to the addition of an exogenous reductant such as NaBH4, leading to nanoparticle nucleation and growth. In biological systems, strong reductants such as NaBH4 are not available, thus different metal ion reduction methods must be exploited. Recent work has shown that the AuBP1 peptide (WAGAKRLVLRRE), a Au binding peptide with an N-terminal tryptophan, can spontaneously reduce Au3+ without an exogenous reductant. Remarkably, this system demonstrated the formation of large Au aggregates initially that disassemble to form individual Au nanoparticles, stabilized by the peptide bound to the inorganic surface. In this contribution, we demonstrate the significant effects of aqueous solvent processing conditions (pH, ionic strength, and ion composition) on the rate of particle evolution. Understanding how such effects alter the metal ion reduction process and subsequent nanoparticle fabrication are important in controlling the final structure/function relationship of the resultant peptide-capped materials. This work identifies conditions that may enhance nanoparticle synthesis using biomimetic approaches where the peptide has complete control over complexation, reduction, nucleation, and growth of nanomaterials.

Peptide-based fabrication of inorganic nanomaterials has been extensively used over the past decade for a variety of applications including catalysis,1-3 nanoparticle assembly,4-7 optics/plasmonics,8 and biosensing.9-11 While these approaches generate the functional material, their production requires the use of an exogenous reductant such as NaBH4. Ideally, the peptide exploited to generate the structure should be able to control the fabrication process from metal ion reduction through nanoparticle nucleation, growth, passivation, and activation. In this sense, the biomolecule would possess the ability to control the fabrication process and activate the material for its eventual function, thus simplifying the production and use of the material. Such capabilities would more closely mimic the mechanisms exploited in biological systems, while also enhancing the sustainability of biomimetic synthetic approaches. Recent results have demonstrated enhanced capabilities of peptides for the fabrication of Au nanoparticles, controlling nanoparticle production, including metal ion reduction. These studies indicated that Au specific peptides with tryptophan residues could be employed to drive nanoparticle fabrication without the necessity of an exogenous reducing agent. The AuBP1 peptide (WAGAKRLVLRRE), previously identified by Sarikaya and coworkers through bio-combinatorial methods with the ability to bind Au surfaces,12 possesses an N-terminal tryptophan. This residue can drive the reduction of Au3+ to Au0, leading to nanoparticle nucleation and growth.13-14 Passivation of the final material subsequently occurs by the binding of the biomolecules to the inorganic surface. Other work identifying the unique capacity of tryptophan in short peptides was conducted by Si and Mandal.15 In this study, trimer peptides with a

C-terminal tryptophan were used for the synthesis of Au and Ag nanoparticles in the presence of methanol and NaOH with an approximate solvent-system pH of 11. Their proposed mechanism for tryptophan-based metal ion reduction suggested that the indole ring of the residue is first deprotonated by the basic solvent and then oxidized to the tryptophyl radical before further oxidation to kynurenine and other dimerized products. While the work of Si and Mandal suggests the necessity of a base to induce Au3+ reduction by tryptophan,15 research using AuBP1 indicated that Au3+ reduction can occur under acidic conditions.14 Taken together, these separate studies suggest that substantial effects of solvent conditions (i.e. pH, ionic strength, etc.) could play important roles in controlling peptide-driven metal ion reduction for nanoparticle production in addition to peptide residue composition and sequence. Herein an in-depth analysis of the solvent effects over peptide-driven metal ion reduction was examined using the wellstudied AuBP1 peptide. In this regard, the effects of solvent pH over a wide scale (1-13) were examined. Interestingly, while the solvent pH varied widely, the pH of the reaction system that demonstrated Au reduction was quite limited between 3.56 and 4.72 at a peptide:Au3+ of 3. Control studies indicated that both the peptide and Au3+ precursors worked together to modify the reaction pH, where the protonation state of the peptide and the ligands on the Au3+ species altered the rate of peptide-driven metal ion reduction. In addition, the effects of solvent ionic strength and the ionic compositions of these solvents were varied over four different metal salts (LiCl, NaCl, KCl, and MgCl2•6(H2O)). These results provide unique insights into the mechanism by which peptide-driven nanoparticle production

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processes are driven, suggesting that different reaction mechanisms may be occurring under acidic versus basic conditions. They also demonstrate that the solvent ionic strength, as well as the ion identities, play significant roles in the metal ion reduction process as driven by peptides. Taken together, faster nanoparticle fabrication was observed using moderately basic solvents or by using solvents with low ionic strength that optimized both the peptide and Au3+ structure/composition for enhanced metal ion reduction. These results are important as they provide a key framework for the design of synthetic methods to generate functional materials under sustainable conditions that more closely mimic those exploited by nature.

EXPERIMENTAL SECTION Materials. HAuCl4 and NaOH were purchased from Acros Organics, while LiOH•H2O, trifluoroacetic acid (TFA), and triisopropyl silane (TIS) were purchased from Alfa Aesar. NaCl was purchased from EMD Millipore, while MgCl2•6H2O, KCl, and LiCl were purchased from EM Sciences. KOH, acetonitrile, methanol, and N,N-dimethylformamide (DMF) were purchased from BDH. All FMOC-protected amino acids, Wang resins, and coupling reagents were acquired from Advanced Chemtech. All reagents were used as received and ultrapure water (18.2 MΩ·cm) was employed in all experiments. Peptide Synthesis. Solid phase peptide synthesis was used following standard protocols on a TETRAS peptide synthesizer (CreoSalus).16 Peptides were cleaved from the Wang resins using a cocktail of TIS:H2O:TFA (25 μL: 25 μL: 950 μL) and purified using reverse-phase HPLC (Waters). The purified peptides were confirmed using ESI mass spectrometry. pH Modified Solvent. Initially the pH of 500 mL of ultrapure water was determined using a Fisher Scientific XL15 pH Meter. While monitoring the pH, standard solutions of HCl and KOH were titrated into the ultrapure water in 10 µL additions and allowed to thoroughly mix for the pH to stabilize. Solutions of pH 1, 3, 5, 7, 9, 10, 11, and 13 were made. Should the pH value go beyond the end point, 1 µL of HCl or KOH was added, as needed, to reach the anticipated end point pH. Substantial care was exerted to avoid titrating the water beyond the intended pH value. In all cases, it is assumed that there is some nominal concentration of HCl and KOH. Identical approaches were used when the base identity was varied over NaOH and LiOH to prepare the water. Concentrated Ion Solutions. Initially 10.0 mL of 1 M LiCl, NaCl, KCl, and MgCl2•6(H2O) were made using an appropriate mass of the salt in a 10 mL volumetric flask. From there, 100 or 1000 mL of 1.0, 0.1, and 0.01 mM salt solutions were made as dilutions from the 1.0 M stock. Peptide-Induced Au3+ Reduction Reaction. For each system, the peptide-driven Au3+ reduction reaction was processed using water that was pH adjusted or contained a specific salt concentration. Each reaction was processed at a constant Au3+ concentration, while the peptide concentration varied such that the peptide:Au3+ ratio ranged from 1-5. A description of the reaction using pH 7 water for a peptide:Au3+ ratio of 1 is described; however, changes to the solvent (i.e. different pH values or salt concentrations), the volume of peptide solution, and the volume of the solvent added were used to change the ratio

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and maintain a constant reaction volume of 200 µL. In this regard, to a reaction well in a clear 96-well plate, 20.0 µL of an aqueous 1.0 mM peptide solution in pH 7 water was diluted in 160.0 µL of the pH 7 water. To this mixture, 20.0 µL of 1.0 mM HAuCl4 in pH 7 water was added and the mixture was slightly agitated. The well plate was subsequently inserted into the plate reader (Synergy|Mx) to monitor the UV-vis absorbance of the material at 540 nm for 1500 min to ensure that the reaction reached completion. The nominal temperature of the reaction analysis in the plate reader was ~29 °C. As stated above, changes to the solvent (pH 7 water) were used to vary the pH, ionic strength, and ionic composition to probe such effects on the reaction. Reaction pH Analysis. The pH of the indicated reaction mixtures was studied throughout the course of the reduction reaction using a Metler-Toledo Seven Compact pH/Ion console with an InLab Micro Pro pH probe that can read the pH of small volume systems. Each reaction was set-up in an individual micro centrifuge tube using the established protocols. The pH of the micro centrifuge tube contents was measured first with just the solvent to confirm the pH, then with the diluted peptide, followed by the addition of the HAuCl4 solution from which the reaction commenced. Finally, the pH of the system was measured at the end of the reaction (1500 min). Characterization. TEM analysis was performed using a JEOL JEM-2010 microscope operating at 80 kV. The samples were prepared by drop-casting 5 µL of the nanoparticle solution onto a carbon-coated 200 mesh Cu grid (EM Sciences) and allowed to dry overnight. Sizing of the generated materials was performed on all grids over at least ten images for each grid, including at least 100 particles at their widest sections.

RESULTS AND DISCUSSION A single step biomimetic nanoparticle synthesis wherein the passivating molecule also drives metal ion reduction and nanoparticle nucleation and growth could find important uses in the fabrication of materials for a variety of applications. The effects of the reaction solvent (e.g. ionic composition and strength, as well as pH) on this process could be significant, especially for the reduction of Au3+ to Au0 by the peptide. To this end, changes in the peptide protonation state and/or the Au3+ ligand sphere could demonstrate dramatic changes in the reactivity of the system. Previous studies have presented conflicting information, suggesting that the process can be driven under both acidic and basic conditions,14-15 thus additional understanding is required. As such, we investigated the effects of solvent pH over a wide range (1-13), as well as solvent ionic strength, on AuBP1 peptide induced Au3+ reduction for nanomaterials production. To differentiate between the different reaction conditions, the solvent pH value is employed; however, this value does not necessarily reflect the reaction pH (vide infra). The effects of the solvent pH on peptide-driven Au3+ reduction and nanoparticle synthesis were initially studied using UVvis spectroscopy. For this, the plasmon band of the material (540 nm) was monitored as a function of time (every 10 min) for each reaction that varied the peptide:Au3+ ratio. Figure 1 presents the differences in plasmon evolution for the system studied at a peptide:Au3+ ratio of 3 under different pH conditions. All UV-vis spectra have been background subtracted and baseline corrected to follow the evolution of the plasmon absorbance in the absence of scattering effects. Note that the pH

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(AAGAKRLVLRRE), a mutation of AuBP1 where the N-terminal tryptophan has been replaced with an alanine residue, was used in the same manner as the AuBP1. Consistent with previous results,14 the AuBP1A showed no plasmon evolution, confirming that tryptophan is driving the peptide induced reaction (Supporting Information Figures S2-S11). For each reaction, the absorbance at 540 nm was monitored every 10 min (Figure 1f), from which a kinetic fitting analysis was performed to compare the pseudo-first order reaction rate constants (k) as a function of solvent pH.14 Note that these values are rough estimates as they conflate Au3+ reduction with particle nucleation and growth, but are used to compare the reactions. Using this approach with the reactions processed at a peptide:Au3+ ratio of 3, a rate constant of (1.57 ± 0.12) × 10-3 min-1 was determined for the reaction studied at a solvent pH value of 3. As the solvent pH increased to 5, the k value decreased to (0.7 ± 0.12) × 10-3 min-1. Remarkably, subsequent increases in the solvent pH to 7, 9, and 10, resulted in increased rate constants of (2.3 ± 0.06) × 10-3, (2.9 ± 0.54)× 10-3, and (3.5 ± 0.06) × 10-3 min-1, respectively. For all reactions studied at solvent pH values outside of this range (i.e. 10), no plasmon formation was evident, indicative of the lack of Au3+ reduction and nanoparticle growth. From this analysis, nanoparticle production was observed under both acidic and basic conditions; however, the reaction was clearly faster for solvents with higher pH values. Figure 1. UV-vis analysis of Au nanoparticle growth as a function of time using a peptide:Au3+ ratio of 3 at pH (a) 3, (b) 5, (c) 7, (d) 9, and (e) 10. For clarity, all spectra were background subtracted to emphasize the evolution of the plasmon band, where a full spectrum was recorded every 60 min. Part (f) presents the pseudo first order kinetic absorbance at 540 nm recorded every 10 min as a function of solvent pH.

that is indicated is of the water solvent used to prepare the reaction mixtures and not the pH of the actual reaction. Here, Au3+ ions were co-mixed with a peptide where both reagents were prepared in the same pH solvent. Sufficient volumes of the Au3+ and peptide were added to reach a peptide:Au3+ ratio of 3. The study was performed for solvents with pH values of 1, 3, 5, 7, 9, 10, 11, and 13; however, a plasmon band, indicative of Au3+ reduction and nanoparticle formation, only appeared for solvents with a pH between 3 – 10 (Figure 1 a-e) over the course of the 1500 min reaction period. For those systems that did not present a plasmon band, the data is presented in the Supporting Information, Figures S1 and S12-21. For the reactions that evolved a plasmon, the peak slowly grew in intensity over the lifetime of the experiment, eventually saturating when the reaction was complete. In addition, the solution color turned from pale yellow to deep pink, further indicating nanoparticle production. In general, the spectra demonstrated a blue-shift and/or a narrowing of the plasmon band as a function of time with no visible precipitate formed in the reaction wells. It is interesting to note that plasmon growth and blue shifting is enhanced using basic solvents as compared to acidic solvents, suggesting that different reaction processes may be occurring. To confirm that the tryptophan residue is the driving force behind the Au3+ reduction, the AuBP1A peptide

Figure 2. TEM analysis of the materials generated as a function of time for the reaction processed using the pH 3 solvent with a peptide:Au3+ ratio of 3. Part (a) presents the materials after 280 min at a low (left) and higher (right) magnification. Parts (b and c) present the same analysis for the materials after 655 and 1080 min of reaction, respectively.

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Figure 3. TEM analysis of the materials prepared using a peptide:Au3+ ratio of 3 using a solvent with a pH of (a) 3, (b) 5, (c) 7, (d) 9 (e) and 10. The time point at which the sample was taken was indicated in the image. Insets display higher magnification images of the sample.

TEM analysis of the materials prepared using the conditions of Figure 1 was completed. Three time points for each solvent pH were analyzed: the onset of plasmon formation, the halfway point of plasmon growth, and the terminal point of plasmon growth (determined by saturation in plasmonic growth by UVvis). Since the reaction rate generally increased with the pH of the solvent, different actual time points for each reaction were selected. Consistent with previous studies using the AuBP1 peptide for metal ion reduction,14 an intriguing disaggregation process was observed for particle synthesis regardless of the solvent pH. When considering the materials prepared using a peptide:Au3+ ratio of 3 at a solvent pH of 3 (Figure 2a), at the initial time point (280 min) formation of a mixture of large dendritic Au structures interspersed with smaller individual Au nanoparticles (overall average size = 53.5 ± 64.4 nm) were observed. Note that while many of the particles in the sample were 50 nm in size) were present in the mixture, thus resulting in the observed average size with a large standard deviation. In general, these larger dendritic structures appear to decompose to form smaller spheroidal Au nanomaterials as the reaction proceeds (23.3 ± 21.0 nm at 1080 min – Figure 2c) demonstrated by the change in the UV-vis plasmon as well as the shift

of the average particle size as a function of time. Note that larger Au structures do remain in the sample, but their fraction of the total number greatly diminishes, resulting in the smaller average particle size. Such effects were generally observed for all the materials imaged (Figure 3), regardless of the solvent pH. Note that the materials generated at pH 7 did possess large aggregated structures at the initial time point; however, they were very low in number compared to the smaller particles. As a result, a smaller initial average particle size was noted with a large standard deviation. Additionally, substantially broad particle sizes were observed in all the samples due to the anticipated breakdown process as mixtures of larger and smaller particles are commonly observed. It is interesting to compare the particle sizes when the reaction process was complete. While the average particle size is roughly similar (varied over a range of 12.1 to 23.3 nm in the solvent pH range of 3 to 10), the distribution of the particle size generally decreased with increasing solvent pH. This may be an effect of the reaction rate at which the particles were formed, wherein faster reaction processes facilitated enhanced particle stabilization through peptide binding; however, additional molecular level insights are required to confirm this hypothesis.

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A more comprehensive comparison of the reaction process wherein the peptide:Au3+ ratio was varied between 1-5 at all pH values using the HCl/KOH system can be seen in Figure 4. UVvis spectra plotted as a function of time show the growth of the plasmon band for the reactions at peptide:Au ratios of 1, 2, 4, and 5 (Supporting Information, Figure S12-S21). From the calculated k values for these reaction conditions (Figure 4), two trends were clear. First, in confirmation of our previous work,14 as the peptide:Au3+ ratio is increased, the rate of plasmon growth also increased, regardless of the solvent pH. Second, for all peptide:Au3+ ratios studied, the same trend in k values as a function of solvent pH is maintained. The rate constant at solvent pH 5 is lower than at solvent pH 3; however, as the solvent pH becomes more basic (>5), increasing rate constants were observed. For all systems, no reactivity was noted at extremely acidic or basic conditions, demonstrating that the solvent pH has a substantial effect on the reaction.

pH was initially determined using a standard pH meter. As anticipated, using the microprobe, the pH of the small solvent volume was nearly identical to the pH of the bulk solvent (black squares). When the peptide was added to the system (blue triangles), the pH deviated substantially from the anticipated values. Using solvents with a pH of 1 and 3, the pH of the solution with the peptide remained roughly the same as the solvent; however, when the pH of the solvent ranged between values >3 and ≤10, the pH of the peptide solution was roughly constant around 4. Interestingly, when water of pH 10 was used with the peptide added, the pH of the system increased to 5.98, while solvent with a pH >10 resulted in peptide solutions that mirrored the pH of the solvent.

Figure 5. pH analysis of the reaction solution at a peptide:Au3+ of 3 where the component added is indicated.

Figure 4. Comparison of the pseudo first order rate constants observed for plasmon growth as a function of peptide:Au3+ ratio and solvent pH.

Due to the small reaction volumes (200.0 µL), the reaction process was studied using the solvent pH; however, addition of the peptide and HAuCl4 to the reaction mixture is likely to have substantial effects on the actual pH of the reaction. As such, a microprobe was used to measure the pH of the reaction solution at different time points for the HCl and KOH pH adjusted systems. Following the same protocol as the study of Figure 1, each reaction using the solvents with selected pH values was set up in an individual micro centrifuge tube. The pH of the tube content was measured first with just the solvent, then with the appropriate amount of peptide added, followed by the addition of the HAuCl4. The reaction was allowed to proceed to completion, after which the pH of the system was measured. Figure 5 presents the actual pH of the system as a function of the reagent added. This study was completed for the reactions at a peptide:Au3+ ratio of 3. Initially, the pH of the solvent alone was measured using the microprobe. Note that the bulk solvent

In a second pH analysis, the pH of the solution at the selected solvent pH values was probed when just HAuCl4 was added at the appropriate concentration (Figure 5 – red circles). In this situation, a nearly identical trend as to the system with just the peptide added was observed where the pH of the HAuCl4 systems matched the anticipated values at the extreme pHs; however, with a solvent pH between 3-10, the pH of the solution was nearly constant at 4. When both the peptide and HAuCl4 were added to the reaction mixture and the reduction process commenced, (inverted pink triangles), the pH of the system mirrored the data for the peptide and HAuCl4 alone. Finally, when the reaction was completed (green diamonds), the same pH was observed as compared to the start of the reaction, suggesting that the pH of the reaction does not vary substantially throughout the chemical process. In the observed deviations from the solvent pH values between a pH range of 3-10, it is likely that structural/compositional changes to both the AuBP1 peptide and the HAuCl4 are occurring in the reaction prior to the reduction process. For the peptide itself, the sequence contains numerous ionizable residues, including one tryptophan, one lysine, three arginines, and one glutamic acid, as well as the N- and C-termini. As such, different protonation states of the peptide sequence must be oc-

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curring in the system as a function of the solvent pH. Unfortunately, identification of the protonation state of each residue within the peptide sequence at specific solvent pH conditions remains a grand challenge and cannot be readily ascertained at present. While the individual amino acids have known pKa values, these can dramatically change when incorporated into a peptide sequence, thus the exact protonation state of the peptide cannot be readily determined. When considering the HAuCl4 reagent, it is further likely that this metal precursor is also structurally changing as a function of solvent pH. As identified by Wang et. al,17 when HAuCl4 is dissolved in solutions of low pH (pH = 2.91), it spectates from AuCl4- into AuCl2.91OH1.09-. As the system pH increases to 10.35, the structure of the species changes to predominate as AuCl0.10OH3.90-.17 As such, it is clear that as the pH of the system increases, the number of Cl- ligands on the Au3+ ion decrease concomitant with an increase in the number of OH- ligands. Wang et al. ultimately observed changes in the morphology of gold colloids made using the Au3+ species with differing Cl- and OH- ligands in the presence of ascorbic acid and sodium benzenesulfonate.17 To this end, for materials made under acidic conditions, a more uniform spherical material was prepared than for the colloids made under basic conditions that resulted in the production of dendritic-like Au morphologies.17 Taken together, these results demonstrate unique effects of the solution conditions on the overall reduction reaction driven by the AuBP1 peptide. Clearly the solvent pH, the protonation state of the peptide, the structure of the Au3+ reagent, and the peptide:Au3+ ratio play critical roles in the reaction. While the solvent pH was selected to range from 1-13, the changes in peptide protonation state and structure of the Au3+ reagent caused the reaction solution to vary substantially from the initial solvent pH. While under extreme solvent pH conditions (pH 11), the reaction mixture had generally the same pH as the solvent, while at solvent pH values between 3-10, the pH of the reaction remained generally constant at a value of ~4. To maintain this pH of 4, the protonation state of the peptide and the structure of the AuCl4- must change to bind or release protons or to bind OH- ions from the solution, the culmination of which resulted in the reaction pH of 4. As such, the structure/protonation state of the two reagents could differ drastically when the solvent pH ranged from 3-10. These changes are likely the cause of the changes in the reduction rate. In this regard, the rate of reduction was substantially enhanced by solvents with a basic pH. Under these conditions, the protonation state of the peptide and the AuClxOHy- structure are likely optimized to facilitate peptide-driven Au3+ reduction. A variety of evidence is known to support reagent structural effects altering the metal ion reduction process. For instance, our previous results have demonstrated that when the AuBP1 peptide sequence was randomly rearranged, the rate of Au3+ reduction was significantly altered.14 In this situation, the tryptophan residue responsible for Au3+ reduction was sequestered at an interior position of the peptide sequence, rather than at the N-terminus. Due to changes in the electronic environment of the residue, changes in the reduction rate were observed, which likely correlate to the present system where changes in the protonation state are anticipated to alter the reaction process. While it is not possible to accurately determine the protonation state of the peptide at each solvent pH value, it is likely that at high

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solvent pH (i.e. 9-10), the peptide is more fully deprotonated, thus facilitating the reduction process. Furthermore, changes to the metal ion structure are known to affect the ability of the ion to interact with organic ligands in solution for nanoparticle production. For instance, when using Pt2+ metal ions for the production of Pt dendrimer-encapsulated nanoparticles (DENs), the PtCl4- precursor must undergo a ligand substitution process with H2O to generate a PtCl(H2O) complex that can bind the amines of the dendrimer prior to reduction.18 Additionally, Somorjai and coworkers have previously shown that the binding of the Pt2+ salts to different organic ligands can dramatically change the reduction potential of the metal ion for nanoparticle production.19 As previously indicated above, Wang et. al. also demonstrated changes to nanoparticle morphology for Au nanoparticles prepared using the different AuClXOHy- precursor.17 As such, different degrees of interaction between the organic ligands (e.g. peptides or dendrimers) with the metal salts are possible, based upon the metal precursor structure, where these changes could provide altered reduction reactivity. In the present AuBP1 AuCl4- system, the fastest reduction reactivity was observed at higher pH values where AuCl0.10OH3.90- likely dominated the system, thus suggesting that this Au3+ complex is likely the most reactive for peptidedriven metal ion reduction for nanoparticle formation. Based upon changes in the reaction mechanism due to peptide protonation state and the AuCl4- structure as a function of solvent pH, attempts were made to probe the reaction mechanism in more depth; however, due to the low peptide concentration, they were ultimately unsuccessful. First, degradation of the final Au nanoparticles using NaCN was studied to release the bound peptides from the nanoparticle surface. Once released, ESI mass spectrometry analysis was processed to identify changes in the molecular weight of the biomolecule; however, these results proved to be inconclusive due to the low sample size of the peptide. Second, a fluorescence based analysis was studied to identify changes in the indole ring of the tryptophan.15 Using known excitation wavelengths for tryptophan, kynurenine, and ditryptophan,15 the fluorescence of the AuBP1 after the reaction with Au3+ was studied. Unfortunately, the overlaps of both the excitation wavelengths and the emission spectra of the three-species proved to be difficult to discern the identity of the residue in the peptide after the reaction. Additionally, Dynamic Light Scattering studies were performed to support the TEM results, however the polydispersity of the nonuniform samples did not allow for clear identification of the colloidal sizes. While there are clear differences in the reduction as a function of solvent pH, peptide protonation state, and AuCl4- structure, aromatic amino acids such as tryptophan are known to have varying degrees of interactions with cations in solution.2021 As such, the reactivity may be affected by the cation of the hydroxide salt used as the base. Thus, the reaction process under basic conditions was analyzed where the base was varied between KOH, NaOH, and LiOH in the pH adjusted aqueous solvent. Identical reaction procedures were employed using the new solvent systems for the AuBP1-driven reduction of Au3+ at a peptide:Au3+ ratio of 3. Figure 6a shows the pseudo-first order kinetic plot of Au3+ reduction in the KOH system at solvent pH values of 7, 9, and 10. Note that this is the same base discussed above where an increase in pH of the solvent increased the rate of metal ion reduction.

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When the base used in the solvent was switched to NaOH at solvent pH 7, 9, and 10 (Figure 6b), it was evident that different degrees of metal ion reduction and particle growth occurred. This was indicated by negligible plasmon formation at solvent pH 7 in the NaOH system (k = (0.2 ± 0.02) × 10-3 min-1), where production was substantially delayed to almost 1100 min after the reaction was initiated. When the solvent pH was increased to 9 and 10, more rapid plasmon formation was evident, but it was slower than the reaction processed using KOH at the same pH values. These systems gave rise to rate constants of (1.7 ± 0.05) × 10-3 and (2.1 ± 0.1) × 10-3 min-1 at solvent pH 9 and 10, respectively. Interestingly, reactions performed at solvent pH 11 and 13 were also tested for the NaOH solvent system; however, no plasmon formation was noted. While these results are consistent with the observations that no reaction occurred at these solvent pH values using KOH as the base, they contrast with those for a tryptophan containing trimer peptide with the ability to reduce Au3+ to Au0, as observed by Si and Mandal,15 where reactivity at a solvent pH 11 using NaOH and methanol was observed. This supports the indication that the peptide sequence and protonation state play a critical role in modulating the overall reduction process, likely through the effects of neighboring amino acids and their electronic interactions with the tryptophan side chain. When LiOH was employed to adjust the pH of the solvent to 7, 9 and 10 (Figure 6e and f), a different trend was observed as compared to both the KOH and NaOH systems. Here a dramatic increase in the k value was noted from solvent pH 7 ((0.2 ± 0.01) × 10-3 min-1) to 9 ((4.4 ± 0.1) × 10-3 min-1). This event was similar to what was observed with NaOH where negligible reactivity was noted at solvent pH 7; however, the rate of reduction at solvent pH 9 was substantially enhanced and, in the case of the LiOH system at solvent pH 9, provided the fastest reduction rate of all pH modified systems. Surprisingly a marked decrease in the reaction rate was observed using the LiOH system at solvent pH 10 ((1.3 ± 0.3) × 10-3 min-1). While the formation of a plasmon was evident, indicative of Au3+ reduction to Au0, it was significantly slower than LiOH at solvent pH 9. Such a dip at solvent pH 10 was only observed using LiOH. In comparing the rate constants of the reactions studied in the three different basic solvents (KOH, NaOH, and LiOH), it is evident that the process is dependent upon the cation of the basic system. It is anticipated that the reaction pH would be similar to those observed with KOH, regardless of the cation used for solvent pH selection, thus the peptide protonation state and the AuCl4- structure should be similar for each system at the indicated solvent pH value. Previous experimental and computational studies have suggested that these cations have varying degrees of interactions with the indole side chain of the tryptophan residue.20-21 For instance, cation-p interactions with tryptophan and other aromatic residues have been noted in the literature and have been identified as a crucial component of ion selectivity in biological systems.20-21 Ruan and Rodgers have examined such interactions between Na+ and K+ with phenylalanine, tyrosine, and tryptophan where they have shown that stabilization of the cation is gained through a tridentate binding motif to the carbonyl O atom, the amino N atom, and the p cloud of the aromatic ring for all of the aromatic amino acids, except for tryptophan interacting with K+. In this case, the preferred complex shows bidentate binding to the carbonyl O atom and

Figure 6. Pseudo first order kinetic analysis of peptide-driven Au3+ reduction using (a and b) KOH, (c and d) NaOH, and (e and f) LiOH. For each reaction, a peptide:Au3+ ratio of 3 was employed. Parts (a, c, and e) present the kinetic analysis as a function of time, while parts (b, d, and f) present the k values determined at the indicated pH.

the aromatic ring. Using DFT ground state calculations, the interaction of the alkali metal cation with the aromatic side chain in the K+(Trp) complex is more favorable than the Na+(Trp) system. This is likely due to the conformations of the K+(Trp) where there is a more favorable K+-aromatic interaction, possibly due to the cation size. This effect likely plays a role in the modulation of the reductive reactivity of the peptide, where the effects of the K+ interaction at the reactive tryptophan residue may stabilize the side chain to facilitate the reaction. To the best of our knowledge, similar studies have not been conducted using Li+ as the cation; however, it is likely that altered interactions between the smaller cation and the aromatic side chain lead to the differing degrees of reactivity. From the changes in base identity, the cation plays a notable role in the reduction capacity of the peptide. This is likely due to interactions between the cation and the indole ring of the tryptophan side chain, which is the residue of the peptide identified to drive the reduction process. Such effects were further explored using reactions employing solvents containing various concentrations of LiCl, NaCl, KCl, and MgCl2•6H2O. These specific salts were chosen as complements of the bases used wherein the cation was the same, while the Mg2+ salt was selected to probe the cationic charge of the ion on the reaction process. For these analyses, the peptide-driven reduction reaction was processed using water as the solvent with 0.01, 0.1, and 1.0 mM concentrations of the indicated salt using the pep-

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tide:Au3+ ratio of 3. As a control, the reaction was also processed in water without any dissolved salt. Note that the selected salts are not anticipated to affect the reaction pH.

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0.2) × 10-3, (4.3 ± 0.3) × 10-3, and (1.9 ± 0.1) × 10-3 min-1 at MgCl2•6H2O concentrations of 0.01, 0.1, and 1.0 mM, respectively. It is worth noting that for all the salts selected, a 1.0 mM salt concentration showed a substantial decrease in reduction capacity of the AuBP1 peptide. Comparing all the k values of the peptide induced reduction of Au3+ where LiCl, NaCl, KCl, and MgCl2•6H2O were employed, there appears to be a notable difference in reactivity based upon the cation. There is evidence in the literature that identifies favored interactions between certain cations and aromatic amino acids,20-21 which may stabilize the tryptophan residue through its oxidation process. At present, no clear trends based upon cation size, charge, or charge density are observable; however, for each of the salts selected at the 1.0 mM concentration level, diminished reactivity was noted. This suggests that at sufficiently high concentrations, the ionic strength of the solution results in diminished reactivity. Scheme 1. Proposed approach for the peptide-driven reduction of Au3+ ions to Au0 for the fabrication of Au nanoparticles.

Figure 7. Comparison of the pseudo first order rate constants determined for the peptide-driven Au3+ reduction reaction processed using a Au3+:peptide ratio of 3 as a function of salt identity and concentration.

Figure 7 presents a comprehensive comparison of the calculated k values for the peptide-driven Au3+ reduction reaction at each salt concentration. For comparison, the rate constant for the reaction processed in the absence of any added salt is also presented, which demonstrated a k value of (6.4 ± 0.2) × 10-3 min-1. For reactions in 0.01 mM and 0.1 mM of KCl, negligible differences in reactivity were observed, as compared to the salt free control, with k values of (6.2 ± 0.5) × 10-3 and (6.6 ± 0.2) × 10-3 min-1, respectively. At a higher KCl concentration of 1.0 mM, a substantially slower reduction reaction was observed (k = (2.1 ± 0.2) × 10-3 min-1). Interestingly, the reactions performed in the KCl solvent at 0.01 and 0.1 mM salt concentrations exhibited higher k values than those of KOH at pH 7, 9, or 10. For LiCl, an initial increase in reactivity was observed for 0.01 mM LiCl reactions as compared to the control. Here at 0.01 mM LiCl, a k value of (10.6 ± 0.2) × 10-3 was observed, which is 1.7 times faster than its salt free parent and 2.5 times faster than the LiOH system at pH 9. Dramatically decreased reactivity was noted with each increase in LiCl concentration, where 0.1 and 1.0 mM LiCl resulted in k values of (3.9 ± 0.3) × 10-3 and (2.1 ± 0.1) × 10-3 min-1, respectively. Unlike K+ and Li+, Na+ and Mg2+ show diminished reactivity as compared to the salt-free control, regardless of the salt concentration in the reaction. For NaCl, a diminished reaction rate of (4.9 ± 0.1) × 10-3 min-1 was observed at 0.01 mM salt that further decreased to (4.3 ± 0.1) × 10-3 min-1 at 0.1 mM NaCl. At the highest NaCl concentration (1.0 mM), a k value of (2.8 ± 0.06) × 10-3 min-1 was observed, displaying substantially diminished reactivity. Finally, MgCl2•6(H2O) was chosen to elucidate the effects of cationic charge on the reaction process. Like NaCl, a diminished k value trend was observed with reaction rates of (4.5 ±

From these collected results, further insights into the peptidedriven reduction of Au3+ ions for nanoparticle production can be considered. As shown in Scheme 1, the peptides and metal ions are commixed in solution. Over time, Au3+ reduction to Au0 is observed, arising from the oxidation of the tryptophan residue in the sequence. Initially larger Au dendritic structures are observed in the product mixture that disaggregate over time to form a polydisperse set of final individual nanoparticles. This study has demonstrated that solution conditions, including solution pH, ionic strength, and cation identity play substantial roles in controlling the reduction, nucleation and growth process. More specifically, the solution pH greatly affects two factors that substantially contribute to the reaction rate: the peptide protonation state and the structure of the Au3+ complex. Furthermore, the identity of the cation in the reaction played a role, most likely in complexation to the reactive tryptophan residue that modulated the reductive capability. In general, the greatest reduction rates were observed when basic solvents were employed or with solutions of low ionic strength. It is possible that different reaction steps may be occurring based upon the peptide protonation state and Au3+ structure, which would be consistent with similar previous results for both Au and Pt nanomaterials. Advanced computational and experimental understanding of the system, including exact identification of the peptide protonation state and the complexation environment of the peptide are required; however, such analysis remain exceedingly challenging.

CONCLUSIONS

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In summary, we have demonstrated the effects and importance of solution conditions on the metal ion reductive capacity of a well-studied Au binding peptide, AuBP1. These results indicate that a combination of multiple solution conditions, including pH, ionic strength, and cationic species, work in concert to control the overall reaction process. These solution conditions likely greatly alter the peptide protonation state and Au3+ chemical structure, which leads to variations in the reductive capability of the biomolecule for the metal ions for nanoparticle production. Minor differences in the overall reaction mechanism may occur because of the protonation state and structural differences; however, the overall material production process remains similar in that large aggregated Au structures are initially observed that break apart into individual nanoparticles over time. Furthermore, cations in the reaction mixture are also likely to affect the process through tryptophan stabilization to facilitate the redox reaction. Together, these solution components may work in concert where the cations of the base stabilize the indole ring of the tryptophan side chain throughout the reduction process that is modulated by the solution ionic strength and pH that modify the peptide protonation state and the Au3+ structure. Additional molecular level understanding is required to elucidate the reaction pathway under various conditions, where such studies are presently hampered by the complex mixtures of reduced and oxidized biomolecules, Au3+ ions, Au0 atoms, and growing Au nanoparticles in solution.

ASSOCIATED CONTENT Supporting Information Complete UV-vis and TEM analysis of conducted experiments. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *MRK – [email protected]

ACKNOWLEDGMENT This material is based upon work supported by the Air Force Office of Scientific Research, grant number FA9550-12-1-0226. C.J.M. would also like to thank the University of Miami Dean’s Summer Fellowship for support.

ABBREVIATIONS TFA, trifluoroacetic acid; TIS, tri-isopropyl silane; DMF, N,N-dimethylformamide; FMOC, Fluorenylmethyloxycarbonyl; HPLC, High Performance Liquid Chromatography; ESI, Electrospray ionization.

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