Hybrid Enzyme-Bimetallic Nanoparticle System for Tandem Oxidation Catalysis

Metallic nanoparticles are commonly used as catalysts in industrial and academic settings. Recent observations that bimetallic nanoparticles can have enhanced activity over their monometallic counterparts has increased the interest in their synthesis and application to new catalytic systems. Combining enzymes and metallic nanoparticle active sites in hybrid materials shows promise in performing sequential, tandem reactions. A goal of this research was to investigate new synthesis methods for synthesizing these tandem catalytic materials. A direct synthesis method for using glucose oxidase as both a reducing agent and a stabilizing agent for synthesis of glucose oxidase-bound Au nanoparticles was performed. It was shown that the glucose oxidase is deactivated during this synthesis and did not retain its catalytic activity. Additionally, a method for synthesizing bimetallic, gold-palladium nanoparticles was investigated with promising results. Future work will post-synthetically bind enzymes to the surface of these AuPd nanoparticles. Additionally, in a separate reaction system, the use of a Fe2+ catalyst to degrade p-nitrophenol was explored as preliminary data for future work, but more experimentation is needed to draw a precise conclusion.

HYBRID ENZYME-BIMETALLIC NANOPARTICLE SYSTEM FOR TANDEM OXIDATION CATALYSIS by Kathryn K. Jones A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Chemistry Approved: ______________________________ Michael Nigra, PhD Thesis Faculty Supervisor _____________________________ Matthew Sigman, PhD Chair, Department of Chemistry _______________________________ Thomas Richmond, PhD Honors Faculty Advisor _____________________________ Sylvia D. Torti, PhD Dean, Honors College May 2020 Copyright © 2020 All Rights Reserved ABSTRACT Metallic nanoparticles are commonly used as catalysts in industrial and academic settings. Recent observations that bimetallic nanoparticles can have enhanced activity over their monometallic counterparts has increased the interest in their synthesis and application to new catalytic systems. Combining enzymes and metallic nanoparticle active sites in hybrid materials shows promise in performing sequential, tandem reactions. A goal of this research was to investigate new synthesis methods for synthesizing these tandem catalytic materials. A direct synthesis method for using glucose oxidase as both a reducing agent and a stabilizing agent for synthesis of glucose oxidase-bound Au nanoparticles was performed. It was shown that the glucose oxidase is deactivated during this synthesis and did not retain its catalytic activity. Additionally, a method for synthesizing bimetallic, gold-palladium nanoparticles was investigated with promising results. Future work will post-synthetically bind enzymes to the surface of these AuPd nanoparticles. Additionally, in a separate reaction system, the use of a Fe2+ catalyst to degrade p-nitrophenol was explored as preliminary data for future work, but more experimentation is needed to draw a precise conclusion. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 5 RESULTS AND DISCUSSION 8 CONCLUSIONS 15 REFERENCES 17 iii INTRODUCTION Metallic nanoparticles have long provided active sites in heterogeneous catalytic materials. The use of metallic nanoparticle active sites has allowed for the improvement of processes across industries such as chemical manufacturing and environmental technologies.1 Gold, specifically, was initially thought to be inert in all forms until it was found that gold nanoparticles (AuNPs) are highly redox active when attached to an oxide support.2 This was discovered in 1982 when Masatake Haruta found that AuNPs are highly active for carbon monoxide oxidation.3 Additional work has found that gold nanoparticles are active in solution for the reduction of p-nitrophenol, the synthesis of ammonia, and in other reactions.4 Increasingly, research is trending more into bimetallic nanoparticles because of the high activity seen in the alloyed materials due to synergetic effects between the metallic components.5 As an example, AuPd nanoparticles have demonstrated activity in the direct partial oxidation of methane to methanol, which is an important reaction for the valorization of methane.6,7 The combination of enzymatic catalysts and metallic nanoparticle catalysts in new hybrid catalytic materials has been hypothesized as a means to create new or more efficient catalytic pathways. The Nigra research group has investigated using Au nanoparticles bound to glucose oxidase (GOx) to demonstrate tandem reactivity.8 The system used glucose oxidase to oxidize glucose to gluconic acid and hydrogen peroxide. The gluconic acid and hydrogen peroxide reacted on the Au surface to produce glucaric acid. Catalytic cooperativity was demonstrated between the GOx and the Au nanoparticle catalysts to produce the glucaric acid. The initial scope of the work described in this thesis was an extension of the previous work where we investigate a two-step catalyzed mechanism consisting of bimetallic nanoparticles bound to an enzyme, also GOx. In this system, the GOx would act as the reducing agent for the formation of the nanoparticle, as outlined by Sharma et al. The glucose oxidase would also act as a stabilizing agent to protect the metallic nanoparticles against aggregation. The system by Brindle, et al. synthesized Au nanoparticles first, and then bound GOx to their surfaces. This synthesis is a basic “dump and stir” methodology, where the enzyme and metal salts are added to a buffered solution and left to stir on heat for approximately 36 hours. In the procedure, chloroauric acid (HAuCl4) is added to a 0.7 𝑚𝑚𝑚𝑚 𝐿𝐿 GOx solution such that the final gold concentration is 6 × 10−4 𝑀𝑀. After heating, the solution changes colors from light yellow to dark pink, indicating the success of nanoparticle formation through the reduction of Au3+ to Au0.9 This project has GOx acting as the reducing agent to form bimetallic nanoparticles consisting of gold and palladium. This material is used in a tandem oxidation reaction where, first, glucose is oxidized to gluconic acid on the GOx, producing H2O2. The peroxide is used as the oxidizing reagent for the nanoparticle catalyzed conversion of CH2OH O OH HO O H2O2 + OH OH CH2OH O OH OH HO OH NP GOx O H Figure 1: Tandem Oxidation of Glucose and Benzyl Alcohol on Hybrid Enzyme/Nanoparticle Material 2 benzyl alcohol to benzaldehyde. This would allow for a two-step tandem oxidation that produces benzaldehyde from benzyl alcohol with only the addition of the catalyst and glucose (Figure 1). The investigation into the oxidation of benzyl alcohol to benzaldehyde is valuable because of the widespread usefulness of aldehydes as an reagent in industries like pharmaceuticals and perfumes.10,11 This reaction uses oxygen or some peroxides as an oxidizer to form benzaldehyde, benzoic acid, and benzyl benzoate. Using gold as the catalyst for this reaction, has both high conversion of benzyl alcohol and high selectivity for the benzaldehyde, compared to the more highly oxidized products.11 Other catalysts can perform this conversion, but they have reduced selectivities. The investigation 𝐹𝐹𝐹𝐹 2+ + 𝐻𝐻2 𝑂𝑂2 → 𝐹𝐹𝐹𝐹 3+ + 𝐻𝐻𝑂𝑂∙ + 𝑂𝑂𝐻𝐻 − (1) 𝑅𝑅𝑅𝑅 + 𝐻𝐻𝑂𝑂∙ → 𝐹𝐹𝐹𝐹 3+ + 𝐻𝐻𝑂𝑂2 + 𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 (4) 𝐹𝐹𝐹𝐹 3+ + 𝐻𝐻2 𝑂𝑂2 → 𝐹𝐹𝐹𝐹 2+ ⋅ + 𝐻𝐻𝑂𝑂2 + 𝐻𝐻 𝐹𝐹𝐹𝐹 3+ + 𝐻𝐻𝑂𝑂2 ⋅ → 𝐹𝐹𝐹𝐹 2+ + 𝑂𝑂2 + 𝐻𝐻 + (2) + (3) 𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 + 𝐻𝐻𝑂𝑂∙ → 𝐶𝐶𝐶𝐶2 + 𝐻𝐻𝑂𝑂2 (5) Scheme 1: Ferrous Iron Catalyzed Mechanism for the Degradation of Organic Compounds.14 into the degradation of PNP by using a mixture of ferrous iron (Fe2+) and H2O2, known as Fenton’s reagent, is valuable because PNP is considered a priority toxic pollutant by the EPA.12 Additionally, PNP is a difficult to remove pollutant because of the stabilizing nitrite substituent on the aromatic ring.13 Rodrigues et al. were able to completely degrade PNP within a bubble column over the course of 120 minutes using a Fe2+ catalyzed reaction.14 The reaction was run under acidic conditions (𝑝𝑝𝑝𝑝 = 3), with ambient temperatures (22-24°C) and a 1 𝑚𝑚𝑚𝑚/𝑚𝑚𝑚𝑚𝑚𝑚 flow rate of air through the reactor for mixing purposes. The mechanism for the oxidation of any organic (RH) via a ferrous iron 3 catalyzed hydroxyl radical oxidation is summarized in Scheme 1. The reaction proceeded with a high 𝐹𝐹𝐹𝐹 2+ + 𝐻𝐻𝑂𝑂∙ → 𝐹𝐹𝐹𝐹 3+ + 𝐻𝐻𝐻𝐻− 𝐻𝐻2 𝑂𝑂2 + 𝐻𝐻𝑂𝑂2 ⋅ → 𝐻𝐻𝐻𝐻2 + 𝐻𝐻𝑂𝑂2 ∙ (6) (7) Scheme 2: Terminating Reaction Steps from Hydroxyl Radical Oxidation of Organic Compounds.14 amount of Fe2+, relative to what is expected for a catalyst. The high initial concentration of Fe2+ salt was necessary because of undesirable competing reactions that both consume the hydroxyl radicals and the Fe2+ ions.14 Some of these reactions are described in Scheme 2. 4 METHODS Preparation of Stock Solutions The buffer was prepared by mixing a 50:50 ratio of 10 𝑚𝑚𝑚𝑚 monobasic phosphate and 10 𝑚𝑚𝑚𝑚 dibasic phosphate. It was then brought to the appropriate pH using concentrated HCl and NaOH. The gold stock solution was created by dissolving approximately 20 𝑚𝑚𝑚𝑚 of HAuCl4 in 20 𝑚𝑚𝑚𝑚 of purified water, creating a stock concentration of approximately 2.54 𝑚𝑚𝑚𝑚. The palladium stock was created by dissolving 17.5 𝑚𝑚𝑚𝑚 of PdCl2 into 10 𝑚𝑚𝑚𝑚 HCl, creating a stock of approximately 4.91 𝑚𝑚𝑚𝑚. The HCl was included to increases the solubility of the PdCl2. The solution of 0.06 𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 horseradish peroxidase and 0.12 glucose oxidase kinetic measurements. 𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 ABTS was created for Formation of Monometallic and Bimetallic Nanoparticles To synthesize the metallic nanoparticles, a 3.3 × 10−4 𝑀𝑀 metal solution was added to 3 equivalents of NaBH4 in a pH 7, 10 𝑚𝑚𝑀𝑀 phosphate buffer. Monometallic nanoparticles were formed using Au3+ and Pd2+ (AuNP and PdNP, respectively). Bimetallic nanoparticles were formed at a gold to palladium (AuPdNP) ratio of 9:1, 19:1, and 99:1. Initially, samples were gently stirred after the addition of the NaBH4 for 90 minutes to facilitate the formation of the NPs. It was found that the PdNP and AuPdNP tended to aggregate when stirred for 90 minutes. Because of this, 90 minutes of stirring was replaced by a single shake of the scintillation vial. All NP solutions underwent a UV-Vis spectroscopy scan from 300-650 nm. This was done to identify characteristic peaks unique to the NPs formed. 5 Synthesis of Hybrid Nanoparticle/Enzyme Materials To synthesize the metallic nanoparticle/enzyme hybrid material (EnNP), the protocol outlined in the Sharma et al. paper was followed, with slight modifications to investigate the addition of metals other than gold.5 The synthesis was completed in a 10 𝑚𝑚𝑚𝑚 phosphate buffer at a pH of 7. Metal concentration was held constant at 6 × 10−4 𝑀𝑀. The synthesis was completed using Au, in the form of HAuCl4, and Pd, in the form of PdCl2. The metal salts were added to a mixture of 0.7 𝑚𝑚𝑚𝑚 𝐿𝐿 GOx in 3 𝑚𝑚𝑚𝑚 of buffer in one the following ratios: 100% Au, 100% Pd, 9:1 Au:Pd. The solutions were placed in a 37℃ paraffin oil bath with gentle stirring for 36-48 hours. For some experiments, 1 equivalent of NaBH4 was added to the solution, to assist in reducing the metals to explore the addition of an external reduction agents to assist in NP formation. Characterization and Kinetic Studies of EnNPs A UV-Vis spectrum of the EnNPs was taken from 300 to 600 nm, to ensure the formation of the nanoparticles. To test the first step of the tandem oxidation mechanism, a kinetic experiment was devised. This experiment used a proxy reaction to measure the concentration of H2O2 produced from the oxidation of glucose. By using the enzyme, horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), the rate of H2O2 production from the oxidation of glucose could be measured. This reaction was done in a 10 𝑚𝑚𝑚𝑚 phosphate buffer at pH 7. The HRP/ABTS concentrations was 0.48 𝑚𝑚𝑚𝑚 𝐿𝐿 and 0.98 𝑚𝑚𝑚𝑚 𝐿𝐿 respectively and 50 𝜇𝜇𝜇𝜇 of the EnNP solution was added. This was placed in the UV-Vis with sufficient stirring. Glucose was then added to a final concentration of 2 𝑚𝑚𝑚𝑚. 6 Analysis of PNP Catalyzed Degradation To measure the decomposition of PNP into p-nitrocatechol, p-benzoquinone, hydroquinone, and various carboxylic acids using Fenton’s reagent, a UV-Vis based kinetic experiment was run. For this experiment, 80 𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 of Fe2+ from FeSO4 was added to 10 𝑚𝑚𝑚𝑚 phosphate buffer at pH 4 along with varying amounts of 500 𝑚𝑚𝑚𝑚 𝐿𝐿 PNP. This was placed inside the UV-Vis spectrophotometer and stirred to ensure the mixture was well 𝑔𝑔 mixed. Then H2O2 was introduced to the cuvette at a final concentration of 1.6 and the reaction was monitored at 320 nm for 10 minutes. 7 𝐿𝐿 RESULTS AND DISCUSSION Monometallic and Bimetallic Nanoparticle Synthesis and Characterization Initially, bimetallic nanoparticles of varying ratios of gold and palladium were synthesized to determine the best synthesis method and metallic ratio to form gold and palladium bimetallic nanoparticles. In these experiments, total metal concentration was 2 × 104 𝑀𝑀. After stirring, there was a visual color difference in the solutions. The 99:1 Au:Pd NPs were very close to the characteristic dark pink seen when producing pure AuNPs. The 9:1 ratio was more of a muted pinkish, purplish, grey color, while the 19:1 was somewhere in between the two extremes (Figure 2). Visually, this indicated the most difference from the pure AuNP. 0.5 Absorbance 0.45 0.4 0.35 0.3 99:1 19:1 0.25 A B C Figure 2: Gold and Palladium Bimetallic Nanoparticle Synthesis Synthesis. Ratios of Au to Pd were 99:1 (A), 19:1 (B), and 9:1 (C). 0.2 9:1 450 500 550 600 Wavelength (nm) Figure 3: Spectrum of Each Bimetallic Nanoparticle Ratio. As the percentage of palladium increases, the location of the peak height decreases. As seen in Figure 3, increasing the fraction of palladium metal ions, broadens the peak in that region. Based on this result, it was decided to move forward with the 9:1 ratio, since 8 it gave the most spectral shift and visual color difference. The PdNPs and the AuPdNPs were very sensitive to aggregation, so agitation during the synthesis was reduced to one manual shake to ensure reagents were fully mixed. 1 0.9 0.8 Absorbance 0.7 0.6 0.5 PdNP 0.4 AuNP 0.3 9:1 AuPdNP 0.2 0.1 0 50:50 AuNP:PdNP 9:1 AuNP:PdNP 250 350 450 Wavelength (nm) 550 650 Figure 4: Spectrum Comparison of Monometallic, Bimetallic, and Mixture Nanoparticles. AuNP, PdNP, AuPdNP, 9:1 mixture of AuNP and PdNP, and a 1:1 mixture of AuNP were measured PdNP. 0.03 0.025 Absorbance 0.02 0.015 0.01 GOx 0.005 0 350 GOxAuNP 400 450 500 550 Wavelength (nm) 600 650 (a) (b) Figure 5: (a) UV-Vis spectrum of the GOx compared to the GOx bound AuNPs. (b) Color Comparison between unbound GOx and AuNP bound GOx. Vial on the left is unbound GOx, on the right is the EnAuNP. 9 As seen in Figure 4, the PdNP (blue line) exhibited a noticeable peak at 263 nm, the AuPdNP (green line) showed no measurable peak, and the AuNP (red line) showed its characteristic peak at 512 nm. More interestingly, the spectra for both mixtures of pure AuNP and PdNP did not match the profile of the AuPdNP, leading to the conclusion that bimetallic nanoparticles were formed rather than a mixture of monometallic nanoparticles. Bound Enzyme/Nanoparticle Material Synthesis and Characterization Based on the synthesis method presented by the Sharma et al. and outlined above, gold nanoparticles bound to glucose oxidase (EnAuNP) were successfully synthesized. When a spectrum of the enzyme stabilized AuNP, a peak at 540 𝑛𝑛𝑛𝑛, as well as the deep pink color (Figure 5), confirm the production on the EnAuNPs. The initial stage of the kinetic study for the two-step, tandem oxidation was to measure the activities of the bound and unbound GOx. This was done by measuring the production of H2O2 from the oxidation of glucose using HRP and ABTS. As the reaction proceeds, the ABTS formed a green product, the concentration of which is easily measured at 414 𝑛𝑛𝑛𝑛. In Figure 6, it can be seen that the GOx bound to the AuNPs is not showing any production of H2O2 that can be measured by the HRP/ABTS reaction. 10 that the GOx in the EnAuNP isn’t catalytically active, since the H2O2 could be consumed by other oxidation reactions occurring on the surface of the AuNP. To determine what actually is occurring, another experiment needed to 3.5 3 2.5 Absorbance This result does not necessarily indicate 2 1.5 GOx 1 0.5 0 0 100 200 GOxAuNP Time (sec) 300 Figure 6: Measuring the Activity of the GOx using through the production of H2O2. The concentration of H2O2 is monitored via the HRP/ABTS mechanism, be performed. To determine whether the lack of HRP/ABTS reaction stemmed from GOx being inhibited or whether the H2O2 was being consumed by another, faster reaction, the ratios GOx solution and HRP/ABTS were varied. This was done to favor the HRP/ABTS reaction mechanism for any H2O2 that was produced. The EnAuNPs were then placed in the same reaction conditions as before, but the molar ratios of GOx and HRP were varied between a 1:1 and a 1:100 ratio. If the H2O2 was being consumed by an unmeasurable reaction with the AuNPs, then increasing the amount of HRP/ABTS in solution would make the AuNP reaction statistically less favorable. This would cause the slope of the absorbance versus time curve to increase until the system was limited by the rate at which glucose is oxidized, producing H2O2. This behavior can be seen in Figure 7a, where unbound GOx was allowed to react with the glucose and HRP/ABTS solutions. Alternatively, in Figure 7b, the GOx is bound in the EnAuNP system. The slope of the lines is essentially zero for all ratios of GOx to HRP/ABTS. This indicates that using the GOx as a reductive and stabilizing element for the nanoparticle formation causes the 11 active site of the GOx to become inactive. This could be caused by the active site being the source of the reduction or the formation of the nanoparticles causes a confirmation change in the enzyme that does not allow substrate access. 5 Absorbance 4 3 2 1:1 1:10 1:15.2 1 (b) 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 Time (sec) 200 1:1 Absorbance (a) 0 1:10 1:15.2 1:100 0 50 100 150 Time (second) 200 250 300 Figure 7: (a) Free GOx with Various Ratios of HRP/ABTS (b) GOx Bound to AuNPs with Various Ratios of HRP/ABTS This project was originally designed to examine the catalytic activity for these enzymenanoparticle structures for the tandem oxidation mechanism. Unfortunately, the inactivation of the enzyme doesn’t allow this two-step system to work, but these enzyme nanoparticle clusters may still be useful for other applications in the future. Degradation of p-Nitrophenol via an Oxidative Mechanism using Fenton’s Reagent When tracking the degradation of PNP using Fe2+ as a catalyst and H2O2, also known as Fenton’s reagent, the absorbance of the solutions was expected to decrease over time as 12 the concentration of UV-Vis active PNP decreased. Unexpectedly, when monitoring the absorbance at 320 𝑛𝑛𝑛𝑛, the shape of the curve for all initial concentrations of PNP increased slightly and leveled out, as seen in Figure 8. This could be caused by a variety 4 5 mg/L 20 mg/L 40 mg/L 3.5 3 Absorbance 2.5 10 mg/L 30 mg/L 2 1.5 1 0.5 0 0 20 40 Time (sec) 60 80 100 Figure 8: Catalyzed Degradation of PNP using Fenton’s Reagent. Ferrous Iron catalyst is at initial concentration of 80 mg/L. of sources. One possibly source for an absorbance profile like this is the presence of reactive intermediates that absorb at a similar wavelength. Many of the initial intermediates (pnitrocatechol, p-benzoquinone, hydroquinone, etc.) are very similar in structure (Scheme 3). If the reactive intermediate products are absorbing in the same wavelength, then the consumption of PNP wouldn’t be seen during the reaction. To determine whether this is the cause, UV-Vis spectra for OH Fe2+, H2O2 each intermediate would need to be measured to determine whether they absorb at 320 𝑛𝑛𝑛𝑛. OH NO2 OH + + NO2 OH O O OH Scheme 3: Select Products from the Degradation of PNP using Fenton’s Reagent. Products are p-nitrocatechol, p-benzoquinone, and hydroquinone. 13 The shape of the graph also seems indicative of this possibility because the shape, increasing and then leveling off. This looks a lot like the concentration over time profile of a reactive intermediate during a multistep reaction mechanism, like this degradation. If the PNP is being consumed very quickly, then the profile could be very indicative of the production and consumption of one of the intermediates. Unfortunately, more testing is needed to explore the profile seen and, because of the COVID-19 pandemic, this was unable to collect before this thesis was written. 14 CONCLUSION The main focus of this project was to investigate bimetallic nanoparticles, their synthesis, and the use of them as a portion of a tandem oxidation catalyzed mechanism. With respect to synthesizing the nanoparticles, there were promising results. The lack of matching shape to the UV-Vis absorbance profiles was indicative that alloyed AuPdNPs were successfully formed rather than forming a mixture of AuNP and PdNP. More characterization of the AuPdNPs is needed to see their capabilities as a catalyst. Additionally, more exploration into stabilizing agents is needed, as the metal nanoparticles are sensitive to agitation and aggregated. When looking at the EnAuNP material, there was less promise. The discovery that using the GOx as the reducing agent to form the AuNPs in the material inactivates the GOx as an enzyme means that this material as synthesized in this manner would not be useful as a catalyst for the tandem oxidation of interest. This material is still capable of being used in mechanisms where AuNP is the catalyst.5 Other work in the Nigra research group has indicated that by synthesizing the AuNP first and then binding the GOx, the catalytic activity of the enzyme and the Au is preserved.8 Future work will investigate synthesizing the bimetallic AuPd nanoparticles first and then binding the enzyme to the metallic nanoparticle surface. Finally, the results of the Fe2+ catalyzed degradation of PNP were unexpected. The absorbance profile was not expected for a reactant being consumed. It instead looked more like the profile expected for a reactive intermediate, increasing concentration as the intermediate is produced then tapering off as subsequent steps consume it. 15 The procedure outlined needs to be repeated to confirm the shape of the absorbance profile over time. Future steps to this project will include using GOx as a source of H2O2 for the oxidation and exploring options to include this reaction as the second step in a tandem mechanism. 16 REFERENCES (1) Schauermann, S.; Nilius, N.; Shaikhutdinov, S.; Freund, H. J. Nanoparticles for Heterogeneous Catalysis: New Mechanistic Insights. Acc. Chem. Res. 2013, 46 (8), 1673–1681. https://doi.org/10.1021/ar300225s. (2) Sardar, R.; Funston, A. M.; Mulvaney, P.; Murray, R. W. Gold Nanoparticles: Past, Present, and Future. Langmuir 2009, 25 (24), 13840–13851. https://doi.org/10.1021/la9019475. (3) Okumura, M.; Fujitani, T.; Huang, J.; Ishida, T. A Career in Catalysis: Masatake Haruta. 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