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Bioinorganic Nanohybrid Catalyst for Multistep Synthesis of Acetaminophen, an Analgesic Boi Hoa San,†,‡,§ Subramaniyam Ravichandran,‡,§ Kwang-su Park,‡ Vinod Kumar Subramani,‡ and Kyeong Kyu Kim*,†,‡ †
Sungkyunkwan Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, Korea Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea
‡
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S Supporting Information *
ABSTRACT: A bioinorganic nanohybrid catalyst was synthesized by combining esterase with a platinum nanoparticle (PtNP). The combination of two catalysts resulted in enhanced catalytic activities, esterase hydrolysis, and hydrogenation in PtNPs, as compared to each catalyst alone. This hybrid catalyst can be successfully used in the multistep synthesis of acetaminophen (paracetamol), an analgesic and antipyretic drug, in a one-pot reaction with high yield and efficacy within a short time, demonstrating that the nanobiohybrid catalyst offers advantages in the synthesis of fine chemicals in industrial applications.
KEYWORDS: bioinorganic nanohybrid, platinum nanoparticle, catalysis, esterase, hydrolysis, hydrogenation, acetaminophen tions1−4,13,15 in synthetic chemistry, pharmaceuticals, environmental treatment, and food technology. Oligomeric enzymes with a hollow interior have been used as a template to construct nanobiohybrid catalysts using NPs because of their advantage in synthesizing homogeneous NPs with high stability, biocompatibility, and functional diversities.10,16−24 In the case of nanoparticles encapsulated within enzymes, it was observed that the enzyme activity was reduced due to the presence of nanoparticles close to the active site of enzymes.11 Therefore, it is required to overcome this limitation by developing a nanobiohybrid with different structural architecture. In this study, we synthesized a novel nanobiohybrid catalyst by combining oligomeric esterases (EST) with PtNPs that are grown outside of proteins. By this approach, catalytic sites of both platinum and enzymes are intact and fully accessible to the substrates. We demonstrated its capability of catalyzing multiple-step reactions in a novel one-pot reaction for the production of acetaminophen (paracetamol), a widely used antipyretic (Scheme 1), with enhanced catalytic activities. Onepot reactions are the most efficient strategy for organic synthesis and are highly desirable in the manufacture of pharmaceuticals. The combination of a one-pot synthesis reaction with green chemistry can result in significant benefits,
1. INTRODUCTION The synergistic integration of nanotechnology with biotechnology enables improved functionality and extended applications to be achieved in nanobiohybrid materials.1−4 Among various platforms for constructing nanobiohybrids, the combination of an enzyme and a highly active metallic nanoparticle is particularly interesting because of their enhanced biocatalytic functionality and capability for performing multiple reactions. Enzymes are extremely efficient at catalyzing a wide range of reactions with high yields and possess accurate substrate or product specificity under mild reaction conditions.5 Accordingly, there is a significant demand for their utility in food, energy, and various industrial processes as well as in the synthesis of fine chemicals for pharmaceutical, agricultural, and electronic purposes.1,5 However, metallic nanoparticles (NPs) made from highly active noble metals such as platinum and palladium have been extensively investigated for their applications in various fields such as chemical synthesis,6 hydrogen production,7,8 and biomedicine.9,10 The combination of an enzyme and a metallic NP with complementary activities can significantly enhance the biocatalytic activity, stability, capability, and engineering performances of both components in bioprocessing.11,12 In addition, it can contribute to creating new functionality. The versatility and reaction specificity of nanobiohybrid catalysts have been demonstrated with the generation of multifunctional hybrid materials with novel properties and enhanced functionalities,1,2,11,13,14 which make them promising biocatalysts for numerous fascinating applica© 2016 American Chemical Society
Received: Revised: Accepted: Published: 30058
October October October October
10, 19, 20, 31,
2016 2016 2016 2016 DOI: 10.1021/acsami.6b12875 ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Schematic of Multistep Reaction Synthesis of Acetaminophen Catalyzed by EST−PtNP Complexesa
a (1) Hydrolysis of 4-nitrophenyl acetate to produce 4-nitrophenol by enzymatic activity of EST−PtNPs. (2) Hydrogenation of 4-nitrophenol to 4aminophenol by hydrogenated activity of EST−PtNPs. (3) The formation of N-(4-hydroxyphenyl) acetamide (acetaminophen) in the presence of acetic anhydride. (4) Direct synthesis of acetaminophen from 4-nitrophenol acetate in the presence of EST−PtNPs and acetic anhydride.
component listed above were their final concentrations in the reaction mixture. The NaBH4 solution was added drop-wise to the reaction mixture with constant stirring for 1 h. The final product was kept at 4 °C until further use. The concentration of citrate-capped PtNPs was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). 2.3. Sample Characterization. The UV−visible absorption spectra at 200−500 nm were measured using a UV-550 spectrophotometer (Jasco, Tokyo, Japan) with samples in a 10 mm light path quartz cuvette. Transmission electron microscopy (TEM) images were taken using JEOL JEM-3010 microscopes (JEOL, Tokyo, Japan) operated at 300 kV. TEM samples were prepared by applying the EST−PtNP solution to a copper grid covered with a thin carbon film (JEOL, Tokyo, Japan) and dehydrating it overnight at room temperature. To prepare negatively stained PS−PtNP samples, the protein samples were stained on a copper grid with 2% uranyl acetate for 30 s. The size of the PtNPs was estimated by averaging 200 individual particles using the Gatan Digital Micrograph software (Gatan, Pleasanton, CA). Energy dispersive X-ray spectroscopy data were obtained from the samples prepared for TEM. Circular dichroism (CD) spectra of EST only and EST−PtNPs were measured with a Jasco J-810 CD spectrometer at 25 °C in a 1 mm quartz cell. High-performance liquid chromatography (HPLC) analysis was performed using a C18 reverse-phase column (Agilent Technologies, Santa Clara, CA) in an Agilent 1100 HPLC system (Agilent Technologies). X-ray photoelectron spectroscopy (XPS) was performed on a Thermo Scientific ESCALAB 250 Xi system. 2.6 nm EST−PtNPs were drop-coated on a 1 cm2 silicon wafer that was previously cleaned by ultrasonication in acetone. The samples were dried overnight before XPS analysis. The binding energy was calibrated using the C 1s peak intensity at 284.8 eV. The data was analyzed using CasaXPS and peak fittings carried out using Gaussian−Lorentzian functions. Liquid chromatography−mass spectrometry (LC−MS) was carried out on an Agilent Model 1100 HPLC system fitted with an ion trap mass analyzer. LC was carried out on a C18 reverse-phase column and the mass analyzed using the ESI system in both positive and negative ion modes. The multistep synthesis reaction mixture was filtered using a 0.22 μm filter before analysis. A 1 g/mL solution of Acetaminophen (Sigma) in DMSO was used as standard. 2.4. Hydrolytic Activity of EST−PtNPs. The hydrolytic activities of the EST−PtNPs were determined using 4-nitrophenyl acetate as a substrate. In a typical experiment, 200 μL of reaction mixture containing 1 mM substrate and 1 μM soluble EST−PtNPs in 10 mM Tris−HCl buffer at pH 7.0 was incubated at 37 °C in a 96 well plate. The release of 4-nitrophenol was then monitored using a SpectraMax Plus384 (Molecular Devices, Sunnyvale, CA) at a wavelength of 405
and this study comprehensively demonstrates the advantage of using nanobiohybrid catalysts for industrial applications.
2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received without further purification. 2.2. Sample Preparation. The preparation of EST and the synthesis of the platinum nanoparticles (PtNPs) using EST were described previously.11,25 In brief, the EST enzyme with hydrolytic activity was prepared by cloning the gene encoding EST from Mycobacterium smegmatis (GeneID: 4532379) into a pET-based vector with a tobacco etch virus (TEV) protease-cleavable N-terminal hexahistidine tag. The recombinant plasmid, named pVFT1S-EST, was transformed into Escherichia coli BL21 (DE3) cells (Novagen, Billerica, MA). Cells were cultured in Luria−Bertani medium at 37 °C to an OD600 of 0.5 before being induced with 0.5 mM isopropyl-b-Dthiogalactopyranoside. The protein was first purified by metal affinity chromatography on a HiTrap chelating column (GE Healthcare, Pittsburgh, PA). The N-terminal His-tag was removed using TEV protease in buffer A (20 mM Tris−HCl, pH 7.5, and 10 mM NaCl). The cleaved protein was further purified by anion exchange chromatography on a HiTrap Q column (GE Healthcare). The purified protein was dialyzed against buffer B (20 mM Tris−HCl, pH 7.5) and concentrated using YM-3 ultrafiltration and Centricon (Millipore, Billerica, MA). The purity of EST was determined using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). In situ synthesis of PtNPs was performed in solution by reducing PtII in the presence of EST at 22 °C. To synthesize EST−PtNPs, purified recombinant EST protein was mixed with K2PtCl4 in 10 mL of buffer (50 mM HEPES, pH 8.0) followed by 1 h incubation with constant stirring. In the reaction mixture, the final concentration of EST was 1 μM, and the final concentration of K2PtCl4 was 0.5, 1.0, and 3.0 mM for the synthesis of 1.8, 2.6, and 3.8 nm EST−PtNPs, respectively. After 1 h of incubation of protein and platinum precursor, 0.5 mL of 100 mM ice-cold NaBH4 solution was added into the reaction mixture, followed by 2 h of incubation with constant stirring. The reaction mixture containing the EST−PtNPs was concentrated using Centricon centrifuge (30 kDa cutoff, Sartorius, Gottingen, Germany) at 16 100 rcf for 30 min at 4 °C to remove aggregates from the supernatant that is used for further analyses as the EST−PtNPs. The concentration of EST was determined by the Bradford assay26 using bovine serum albumin to generate the standard curve. Citrate-capped PtNPs were synthesized according to reported procedures with some modifications.27 Briefly, PtNPs were obtained by reducing 1 mM H2PtCl6 with 10 mM NaBH4 in the presence of 3 mM sodium citrate in 50 mL of reaction mixture. The amounts of each 30059
DOI: 10.1021/acsami.6b12875 ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
Research Article
ACS Applied Materials & Interfaces
Figure 1. Crystal structure (PDB ID: 2Q0Q) of EST. (A) Ribbon diagram of an EST octamer. The EST subunits are shown in different colors. (B) Charge distribution on the exterior surface of EST; negative charges are red, and positive charges are blue. (C) Charge distribution on the internal surface of EST. The dimensions of the inside and outside of EST are indicated.
Figure 2. TEM images of EST−PtNPs. (A) EST−PtNP complexes were confirmed using electron microscopy with 2% uranyl acetate staining. The insets are representative images of EST−PtNP complexes at a 2:1 (left) and 3:1 (right) ratio between EST and PtNP. (B) EDX analysis of EST− PtNP complex. (C−E) Ratio-controlled synthesis of EST−PtNPs. Platinum precursors were incubated with EST at varying molar ratios of 500:1, 1000:1, and 3000:1 for 2 h, resulting in complexes with average sizes of 1.8, 2.6, and 3.8 nm, respectively. The inset in (D) shows the magnified image of an unstained 2.6 nm EST−PtNP. The periodicity of the lattice fringes was found to be 0.226 nm in the [111] direction. The bottom panels in (C−E) are the corresponding size distribution histograms, with the numbers indicating the average size of the NPs. hydrogenation reaction. The hydrogenation of 4-nitrophenol to 4aminophenol was also monitored at 405 nm via UV−vis scanning for 5 min at 1 min intervals. Finally, 0.12 g of acetic anhydride was added to the reaction mixture to yield the final product of acetaminophen. The formation of the final product was monitored at 5 min intervals for 30 min. All reactions were performed at 22 °C with vigorous stirring. For all UV measurements, 50 μL aliquots of the sample were diluted in 450 μL of distilled water. 2.7. Calculation of the Yield of Final Product (Acetaminophen). Commercially available acetaminophen in powder form was dissolved in dimethyl sulfoxide (DMSO) as a standard and injected into a C18 reverse-phase column (Agilent Technologies, Santa Clara, CA) in an Agilent 1100 HPLC system (Agilent Technologies). The elution profile of acetaminophen was monitored at 250 nm. A standard curve of acetaminophen was plotted using the peak area of the elution peak on the y-axis and the number of moles on x-axis. The amount of acetaminophen produced was calculated by extrapolating the values through a calibration curve (R2 = 0.9999) (Figure S7). The yield of acetaminophen production was obtained using the following equation:
nm for 30 min, and all experiments were repeated three times to obtain a mean value. 2.5. Hydrogenation Activity of EST−PtNPs. The catalytic activities of the EST−PtNPs were measured using 4-nitrophenol as a substrate in the liquid phase.14 Briefly, a 9 mL reaction mixture containing EST−PtNPs (20 ppm PtNP) and 0.15 M NaBH4 was stirred for 15 min at RT. A total of 1 mL of 4-nitrophenol was added to the mixture to yield a final concentration of 0.5 mM, and this mixture was stirred until the reaction was completed. The reaction progress was spectrophotometrically monitored at 405 nm using a UV−vis spectrometer (UV550, JASCO, Tokyo, Japan) for 6 min. Each 50 μL aliquot of the sample was diluted in 450 μL of distilled water. The hydrogenation activity was determined as a first-order reaction. The hydrogenation activity of the sodium citrate-capped PtNPs at 20 ppm was assessed in a similar way. 2.6. Multistep Catalytic Activities. The synthesis of acetaminophen was carried out in a one pot reaction by mixing 1 mM of 4nitrophenyl acetate substrate with EST−PtNP complexes for a concentration of 20 ppm Pt in a final reaction volume of 5 mL. To measure the release of 4-nitrophenol from 4-nitrophenyl acetate, UV− vis spectra with a wavelength ranging from 250 to 500 nm were scanned for 5 min at 1 min intervals (UV550, JASCO, Tokyo, Japan). Subsequently, 28.5 mg of NaBH4 was added to initiate the
yield of acetaminophen (%) number of molecules of acetaminophen = × 100 number of moles of 4‐nitrophenylacetate 30060
DOI: 10.1021/acsami.6b12875 ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
Research Article
ACS Applied Materials & Interfaces 2.8. Calculation of the Turnover Frequency of the Multistep Reaction. The turnover frequency (TOF) value of the EST−PtNP complexes in multistep reaction was determined using the following formula:13 TOF =
number of moles of acetaminophen 1 × number of moles of Pt atom on the PtNP surface time
3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of EST−PtNPs. Esterase (EST) from M. smegmatis, which self-assembles into a well-defined cube-like octameric complex28 with overall exterior dimensions of 8.0 nm × 8.0 nm × 6.0 nm, has been introduced as a PS for the synthesis of PtNPs. The cube-like architecture of EST reveals a cavity with a diameter of approximately 2.5 nm located in the center (Figure 1). EST, an SGNH hydrolase enzyme, efficiently catalyzes acyl group transfer to alcohols (alcoholysis) in aqueous conditions. It also preferably catalyzes perhydrolysis rather than the hydrolysis reaction when peroxide acts as the acceptor.28 A total of six channels, two large and four small with diameters of 2 and 1 nm, respectively, presented on each face of the cube are likely to serve as entries for the substrate and exits for the product during catalysis. The active sites of EST face inward in the cube-like cavity. EST−PtNPs were synthesized using EST protein as a template by slowly reducing K2PtCl4 with NaBH4 as a reducing agent in an aqueous solution at 25 °C. The formation of PtNPs was visibly observed by a color change from light yellow to dark brown, which was also confirmed by UV−vis spectra measurements (Figure S1). Consistent with previous reports,9−11 the EST−PtNP solution showed a broad UV−vis absorption spectrum with a maximum peak at 280 nm, a representative fingerprint of the EST protein (Figure S1). On the contrary, the two major peaks from the K2PtCl4 solution at 320 and 375 nm completely disappeared (Figure S1), indicating the formation of PtNPs. EST−PtNPs were purified using highspeed centrifugation (16 100 rcf) and were characterized using TEM (Figure 2). As a control, PtNPs with a size of 2.4 nm were chemically synthesized and stabilized by sodium citrate (Figure S2). TEM images of EST−PtNPs, stained with 2% uranyl acetate, revealed PtNPs stabilized by several EST proteins (Figure 2A). In some instances, one PtNP can be stabilized by either two or three EST proteins (Figure 2A, inset). However, overall, the distribution of EST around the PtNP remained heterogeneous. We observed that the final EST−PtNPs were quite welldispersed; very little clumping was seen, which was the result of stabilization of nanoparticles by the proteins. The molar ratio between the platinum precursors and EST in the initial reaction mixture largely contributes to the size of the PtNPs. When 1 μM of octameric esterase was incubated with platinum precursor at the concentration of 500 μM, 1 mM, and 3 mM, the size of resulting PtNPs in the EST−PtNP nanohybrids were 1.8 ± 0.3, 2.6 ± 0.4, and 3.8 ± 0.9 nm, respectively (Figures 2C−E). The high-resolution image revealed that the lattice spacing was 0.226 nm, which corresponds to the (111) plane of platinum exhibiting fcc crystal structure (Figure 2D, inset). Energy-dispersive X-ray (EDX) analysis confirmed the presence of platinum ion in the protein−NP complexes (Figure 2B). X-ray Photoelectron Spectroscopy (XPS) was used to analyze the surface characteristics of the EST−PtNP complex (Figures 3 and S3). Region-specific XPS spectra were acquired and fitted for the analysis of Pt, C, O, and N (Figure 3) using
Figure 3. XPS region spectrum of EST−PtNP complex. (A) O 1s, (B) Pt 4f7/2 and Pt 4f5/2, (C) C 1s, and (D) N 1s. The red and dotted lines represent the fitted and experimental curves, respectively. Black, green, blue, and brown lines represent the binding energy peaks.
Gaussian−Lorentzian curves (Table S1). The XPS peaks at 69.45 and 72.88 eV can be attributed to the Pt 4f7/2 and Pt 4f5/2 core levels, respectively. The separation between the 4f7/2 and 4f5/2 peaks was 3.4 eV, which is consistent with the previous report.29 However, an additional peak in each core level was observed, possibly due to the energy shift caused by protein binding. The C 1s peak was deconvoluted into three peaks at 284.70, 285.58, and 287.15 eV, which can be assigned to C−C and C− H, CN, and CO bonds, respectively.30 The O 1s peaks were fitted to three peaks at 530.56 and 531.54 eV and a weak peak at 535.51 eV; these positions are consistent with earlier literature.31 The binding energy at 531.54 eV is likely corresponding to the oxygen atom in the COO− group bound to the Pt surface. The region spectrum for N 1s was fitted to peaks at 398.51 and 400.54 eV that can be assigned to the chemical states of nitrogen NH2 and NH3+ connected to carbon atoms, respectively.30,31 The NH3+ binding energy was shifted relative to NH2, possibly due to the binding of NH3+ group to the PtNP surface.30 The circular dichroism (CD) spectra of EST−PtNPs and EST revealed that the compositions of their secondary structures are almost identical, indicating that the overall conformation is well maintained during PtNP synthesis. However, local conformational changes are expected from the minor deviations in the two spectra (Figure S4). Together with TEM analysis, these results suggest that, unlike other known NPs deposited inside PSs,10,18,32 PtNPs are preferably synthesized outside of EST, possibly because of the difference between the charge distribution of EST and that of other known PSs such as PepA, ferritin, and ClpP, which have negatively charged residues inside.10,18,32 However, EST shows a highly negatively charged surface, whereas its interior is positively charged (Figure 1B,C). Considering that negatively charged residues in PSs play a role in metal deposition in the initial stage of NP synthesis, NPs are likely to grow from the exterior of EST. Considering all of the physicochemical properties of EST− PtNPs, we propose the following mechanism for PtNP synthesis in EST: (1) binding of platinum ions, incubation of 30061
DOI: 10.1021/acsami.6b12875 ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
Research Article
ACS Applied Materials & Interfaces Pt2+ ions with EST molecules causes Pt2+ ions to bind to the EST surface via electrostatic interactions with the negatively charged surface of EST, which serves as nucleation sites; (2) nucleation of platinum crystal, addition of an excess amount of strong reducing agent (NaBH4) subsequently reduces Pt2+ to Pt0, after which Pt0 nuclei are formed on the surface of EST; (3) growth of PtNPs, in which from the Pt0 nuclei, PtNPs continue to grow until Pt2+ are depleted in the solution. 3.2. Evaluation of the Catalytic Ability of EST−PtNPs. We first investigated hydrolytic and hydrogenation activities, separately, in the EST−PtNP hybrid catalyst. 4-nitrophenyl acetate was used as a substrate for the measurement of the hydrolytic activity of EST−PtNPs. The release of 4-nitrophenol from 4-nitrophenyl acetate was observed for 30 min (Figure 4).
protein, we compared the activity of EST−PtNP complex with a mixture containing EST and citrate-stabilized PtNPs (Figure 4). The average sizes of PtNPs in hybrid and citrate-stabilized were 2.6 and 2.4 nm, respectively. The concentrations of protein and Pt in the EST−PtNP complex were analyzed using the Bradford assay and ICP-OES, respectively. When the mixture of EST and citrate-stabilized PtNP were used for the activity assay, their concentrations were adjusted to those in the EST−PtNPs. The hydrolysis activity of the EST−PtNP complex was significantly higher than that of the mixture containing EST and citrate-stabilized PtNPs, indicating that PtNPs in the EST−PtNP complex contributed to the enhanced catalytic activity of EST. Similarly, the hydrogenation activity of EST−PtNPs was compared to that of the PtNPs in the mixture of EST by monitoring the reaction rate of hydrogenation from pnitrophenol to p-aminophenol for 6 min (Figure 5A,C). The
Figure 4. Enzymatic activity comparison of 1.8, 2.6, and 3.8 nm EST− PtNPs with EST only and a mixture of EST with citrate-stabilized 2.6 nm PtNPs. The hydrolysis of 4-nitrophenyl acetate to 4-nitrophenol was monitored by taking the absorbance at 405 nm. The absorbance of EST only was set as 100%, and the relative activity percent of other conditions was calculated relative to EST only. All experiments were done in duplicate; error bars represent standard deviation of three individual trials (n = 3).
Figure 5. Hydrogenation activities of (A) mixture of EST and 2.4 nm citrate-stabilized PtNPs and different-sized EST−PtNPs (B−D). Inset shows the rate constant.
We compared the hydrolytic activities of EST−PtNPs of different sizes to ascertain whether the size had any effect on activity, which was one of the limitations of earlier nanobiohybrid catalysts.11 The results showed that the hydrolytic activity was independent of the size of the nanoparticle in the EST−PtNP complex. Because the nanoparticles are stabilized externally, the interior active site of EST cannot be affected by the binding of PtNPs on the exterior surface. Surprisingly, the hydrolytic activity of EST−PtNPs was significantly higher than that of EST alone, suggesting that the substrate binding site of EST in the hybrid complex is more exposed to the substrate than that of EST alone possibly by conformation alteration accompanied by PtNP binding, which was evidenced by CD spectra (Figure S4). Consistently, it has been also reported that gold nanoparticles can positively affect the catalytic activity of lysozymes when they form an enzyme−NP conjugate by inducing conformation changes in the lysozymes.33 Additionally, the conjugation of protein to metal nanoparticles can also increase enzyme activity by increasing collisions of the substrate with the enzyme aided by the Brownian movements of nanoparticles.12 In addition, to check whether the enhanced catalytic activity was a result of true synergy between the nanoparticle and EST
hydrogenation activity of EST−PtNPs was higher than that of citrate-stabilized PtNPs physically mixed with EST, indicating that the PtNP stabilized by EST proteins is more active. This is consistent with previous reports in which the catalytic activity of metallic nanoparticles was enhanced when they were bound to proteins.10,11 Taking these results together, it can be concluded that EST−PtNPs possess higher hydrolytic and hydrogenation activities as compared to the catalytic activities of EST and PtNP in a simple mixture. In addition, we also examined whether the size of PtNPs in the nanobiohybrid catalyst affects the hydrogenation activity (Figure 5). We observed that the rate constant of hydrogenation was inversely proportional to the size of the nanoparticles in the EST−PtNP complex (Figures 5B−D). This result is consistent with the results of the size-dependent activity change of Pt nanoparticles reported in other literature.34 We also confirmed that the nanobiohybrid catalyst was very stable over multiple cycles of reactions by verifying that the hydrogenation activity did not decrease (Figure S5). This result indicates that there had not been any catalyst deactivation.35,36 It is well-known that leaching and catalyst deactivation have a cause−effect relationship.37 Therefore, we believe that there was no leaching. To verify this analysis, we 30062
DOI: 10.1021/acsami.6b12875 ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
Research Article
ACS Applied Materials & Interfaces filtered the reaction mixture through a membrane with 10 kDa molecular weight cut off and analyzed the filtrate using ICPOES. However, we could not detect Pt in the filtrate, which suggested that the amount of leached Pt was lower than the detection limit of our machine. Therefore, we can conclude that there was no leaching in current setup of experiment. It has been well documented that the size of PtNPs (especially
