NanodiamondâGold Nanocomposites with the Peroxidase-Like

Research Article www.acsami.org

Nanodiamond−Gold Nanocomposites with the Peroxidase-Like Oxidative Catalytic Activity Min-Chul Kim,†,∥ Dukhee Lee,‡,∥ Seong Hoon Jeong,§ Sang-Yup Lee,*,† and Eunah Kang*,‡ †

Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, Korea 03722 School of Chemical Engineering and Material Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, Korea 06980 § College of Pharmacy, Dongguk University, Goyang, Gyeonggi 10326 Korea ‡

S Supporting Information *

ABSTRACT: Novel nanodiamond−gold nanocomposites (NDAus) are prepared, and their oxidative catalytic activity is examined. Gold nanoparticles are deposited on carboxylated nanodiamonds (NDs) by in situ chemical reduction of gold precursor ions to produce NDAus, which exhibit catalytic activity for the oxidation of o-phenylenediamine in the presence of hydrogen peroxide similarly to a peroxidase. This remarkable catalytic activity is exhibited only by the gold nanoparticledecorated NDs and is not observed for either Au nanoparticles or NDs separately. Kinetic oxidative catalysis studies show that NDAus exhibit a ping-pong mechanism with an activation energy of 93.3 kJ mol−1, with the oxidation reaction rate being proportional to the substrate concentration. NDAus retain considerable activity even after several instances of reuse and are compatible with a natural enzyme, allowing the detection of xanthine using cascade catalysis. Association with gold nanoparticles makes NDs a good carbonic catalyst due to charge transfer at the metal−carbon interface and facilitated substrate adsorption. The results of this study suggest that diverse carbonic catalysts can be obtained by interfacial incorporation of various metal/inorganic substances. KEYWORDS: nanodiamonds, gold, hybrids, catalysts, oxidation

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to the Lewis basic surface carbonyl groups.16 Later, a composite of ND and carbon nanotube/SiC showed highly selective catalytic activity for the same dehydrogenation reaction under oxygen-lean conditions.17 The large surface-to-volume ratio of ND and the active functional groups on the surface of the sp2 carbon layer enhance the catalytic activity, producing metal-free carbonic catalysts. Implantation of ions or deposition of metallic compounds on ND induces a synergetic interaction between the metal and carbonic compounds, leading to diverse catalytic activities. Doping ND with nitrogen and boron results in catalytic oxygen reduction activity, applicable in the fabrication of battery cathodes. The above doping creates defects on the ND surface, resulting in numerous catalytically active sites.18 Besides, deposition of noble metal nanoparticles on ND, such as palladium and platinum nanoparticles, achieves outstanding catalytic activity for CO and methanol oxidation, respectively. Similarly to the effect of doping, metal-sp2-carbon bonds possibly function as active sites, changing the chemical bonds of sp2 carbons as well as the binding affinity of substrates.19,20 ND with deposited Ni clusters also displays

INTRODUCTION Nanodiamond (ND) is a carbonic nanomaterial with a particle diameter of 4−5 nm, having an sp3-carbon diamond core and a reconstructed sp2-carbon surface layer.1 ND exhibits fluorescence, surface functional groups, and biocompatibility distinguishable from those of other carbonic compounds, e.g., carbon nanotubes, graphene, and graphites.2−9 These appealing properties and dimensions of several nanometers made ND promising for the development of novel materials with designer functionalities. For example, ND-based lubricating additives,10,11 photoacoustic imaging agents,12,13 and photoenergy absorbers14 have been developed. In particular, the presence of crystalline carbon with hybrid orbitals allows the ND prepared by detonation to display unforeseen surface activities. The surface sp2 carbons are known to create a graphitic shell or amorphous carbon, and chemical modification of these sp2 carbons can introduce reactive functional groups.15 The reconstructed sp2 carbon surface and dangling surface functional groups exhibit unusual activity, leaving plenty of room to exploit multiple functions. Besides the above-mentioned applications, ND has been explored as a catalyst, exploiting the surface activity of sp2 carbons. ND surface-modified with an acidic solution exhibited catalytic activity for the dehydrogenation of ethylbenzene due © 2016 American Chemical Society

Received: August 20, 2016 Accepted: November 29, 2016 Published: November 29, 2016 34317

DOI: 10.1021/acsami.6b10471 ACS Appl. Mater. Interfaces 2016, 8, 34317−34326

Research Article

ACS Applied Materials & Interfaces Scheme 1. Preparation of the Nanodiamond−Gold Composite Exhibiting Peroxidase-Like Catalytic Activity

catalytic activity for acetylene hydrogenation.21 These studies indicate that ND catalytic activity is imparted by implantation of heterogeneous substances that can introduce a substantial number of surface functional groups and defects on the ND surface. Thus, it is possible to prepare a catalyst with excellent activity by depositing other metal nanoparticles on the ND surface. Graphene-based peroxidase mimetics have been widely explored for applications in biosensing, immunoassays, and theragnostic agents22,23 detectable by various colorimetric, voltammetric, fluorescence, and electrochemical methods. For a decade, ND has been focused on as a new biocompatible exogenous material source.24,25 Nanodiamond-based peroxidase mimics, which can act as various theragnostic agents in vivo, detect enzymatic activity ex vivo, as well as be a substitute for enzymes have not been investigated. To investigate the potential of ND as a catalytic platform, we herein present nanodiamond−gold nanocomposites (NDAus) displaying peroxidase-like catalytic activity. Gold nanoparticles were deposited on the surface of ND aggregates by simple reduction of gold ions, as shown in Scheme 1. The synergistic effect of ND and gold nanoparticles was exhibited as the eccentric oxidative catalytic activity of NDAus with lowered activation energy. The catalytic properties of NDAus were extensively examined using H2O2 and o-phenylenediamine (OPD) as substrates to determine the kinetic parameters and reaction mechanism. Finally, the reusability and compatibility of NDAus with the natural enzyme were verified for the future use of the former as a versatile catalyst in various fields.

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times. After washing, the color of the NDAu solution changed from dark purple to black, indicating the removal of unbound gold nanoparticles. The collected NDAus were dispersed in DI water to prepare the final stock solution (2.5 mg mL−1). The prepared aqueous NDAu suspension remained stable for weeks in a refrigerator. This suspension was used for characterization and was diluted for the catalytic activity test. For solid state characterization, water was evaporated in a drying oven for 2 h at 50 °C, followed by vacuumdrying for 24 h, furnishing NDAus as a soft powder. Characterization. Crystalline structures of ND and NDAus were investigated by XRD (D8-Advance X-ray diffractometer, Bruker Corp.) equipped with a Cu Kα radiation source (λ = 0.154 nm, 40 kV, 40 mA) and a high-speed LynxEye detector. XRD spectra of ND and NDAus were obtained for 2θ ranges of 10° to 80° with a step size of 0.02° at a rate of 6° min−1. Each spectrum was analyzed using the DIFFRAC plus Eva software package (Bruker AXS Inc., Germany). The morphology and elemental analyses of ND and NDAus were carried out using high-resolution transmission electron microscopy (HRTEM, JEM-3010, JEOL, 200 kV) coupled with EDX (Inca, Oxford, U.K.). Thermogravimetric analyses (TGA, TGA N-1000, Scinco, Korea) of ND and NDAus were used to monitor their degradation profile between 25−800 °C at a heating rate of 10 °C min−1 under a nitrogen atmosphere. Dynamic light scattering (DLS) and zeta potential measurements were performed using a particle analyzer (SZ-100, Horiba Ltd., Japan) at 25 °C. The light scattering detector angle was fixed at 90°. Suspensions of ND and NDAus at a concentration of 0.1 mg mL−1 were used for the size and zeta potential measurements. A disposable polystyrene cuvette (Sarstedt, Germany) and a disposable capillary carbon electrode zeta cell (Horiba Ltd., Japan) were used for the hydrodynamic radius and zeta potential measurements, respectively. These measurements were repeated five times for each sample. Oxidative Catalytic Activity of NDAus. The catalytic activity of NDAus was evaluated by monitoring the oxidation of OPD (98%, Sigma-Aldrich) in the presence of H2O2. Oxidation of OPD produces 2,3-diaminophenazine, which has a yellow-orange color. The corresponding progress of OPD oxidation (color change) was monitored by UV−vis spectroscopy (S-3100, Scinco, λabs = 450 nm).26 Catalytic activity measurements were performed using a 0.04 mg mL−1 NDAu suspension mixed with OPD solutions in various concentrations, with a drop of the H2O2 solution subsequently used to initiate oxidative catalysis at room temperature. The NDAu−substrate mixture solutions were maintained at pH 7.2 ± 0.2 during the catalytic reaction. The oxidation of OPD was monitored over time, and the oxidation rate was determined from the slope of the plot of 2,3diaminophenazine concentration vs time. For the kinetic analysis, the reaction rates were measured while changing both substrate concentrations. The kinetic parameters were determined from the double-reciprocal plot according to literature to infer the catalysis mechanism. Xanthine Detection using XOD-Coupled Catalysis. Coupled catalysis using XOD and NDAus was performed to demonstrate the possibility of xanthine detection. The catalyst mixture solution of XOD (2.56 units) and NDAus (0.04 mg mL−1) was prepared in the presence of the OPD substrate (10 mM). This catalyst mixture was treated with

EXPERIMENTAL SECTION

Materials. Acid-washed and highly purified nanodiamond with numerous surface carboxyl groups was gifted by Nanoresource, Inc. (Seoul, Korea). Gold nanoparticles were deposited on nanodiamond using ethylene glycol (99.5%, Sigma-Aldrich), sodium borohydride (NaBH4 , 98+%, Acros Organics), and AuCl3 (99%, Stream Chemicals). In catalysis experiments, o-phenylenediamine (98%, Sigma-Aldrich) and xanthine oxidase (from bovine milk, 0.1−0.4 unit mg−1, Sigma) were used as the substrate and enzyme, respectively. Preparation of NDAus. Catalytically active NDAus were synthesized via in situ reduction of gold ions by NaBH4. The ND powder was dispersed in 10 mL of 1 wt % ethylene glycol solution in water to a concentration of 1 mg mL−1 under sonication for 10 min. AuCl3 (20 mg) was added to the homogeneous ND suspension, and the ND−AuCl3 mixture was incubated at 70 °C for 10 min. A 0.1 mL aliquot of NaBH4 solution (100 mg mL−1) was slowly added to the incubated mixture to produce gold nanoparticles. Immediately after the addition of NaBH4, the ND−AuCl3 mixture changed color to dark purple. After 10 min vigorous stirring, the reactor was cooled to room temperature and stirred for another 24 h. The produced solid NDAus were centrifuged (10 000 rpm, 20 min) and washed with DI water five 34318


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ACS Applied Materials & Interfaces

Figure 1. Characterization of NDAus. (a, b) Field emission transmission electron microscopy (FE-TEM) images of nanodiamonds (scale bars 20 and 5 nm, respectively), (c, d) nanodiamond-gold nanocomposites (scale bars: 20 and 2 nm, respectively), (e) comparative XRD patterns of intact ND and NDAus, and (f) thermogravimetric analysis (TGA) curves of ND and NDAus.

deposited via in situ chemical reduction of Au3+ ions to produce NDAus (Figure 1c). Discrete gold nanoparticles (AuNPs) were generated, suggesting that most gold nanoparticles were grown on the ND surface, and the gold nanoparticles created in bulk solution were removed during washing and separation. The carboxyl groups on the ND surface were suggested to function as binding sites for Au3+ ions, leading to subsequent nucleation and growth. Gold nanoparticles exhibited a broad size distribution with an average diameter of 10.1 ± 6.8 nm (see Supporting Information (SI), Figure S1). This broad distribution implies that the growth of gold nanoparticles after nucleation on the ND surface proceeded without size regulation. The size and uniformity of gold nanoparticles could not be controlled here because of the absence of size-regulating capping agent and nonuniformity of the ND surface where nucleation−growth of gold nanoparticle occurs. Energydispersive X-ray spectroscopy (EDX) analysis confirmed that NDAus were composed of only carbon and gold, without other

the xanthine target substrate to achieve a concentration of 1 mM. As soon as the xanthine solution was added, the cascade catalysis of xanthine oxidation and subsequent OPD oxidation occurred. The progress of OPD oxidation was monitored using UV−vis spectroscopy (λabs = 450 nm) for 300 s at pH 7.8. Control experiments were performed in the presence/absence of XOD and NDAus.

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RESULTS AND DISCUSSION Characterization of NDAus. The prepared NDAus exhibit a morphology of random ND aggregates with deposited gold nanoparticles on the surface. Figure 1 presents the TEM images of intact ND and NDAus. The former exists as small aggregates (50−80 nm), while every single ND is 4−5 nm in size (Figure 1a). Although the ND surface was modified to include carboxyl groups enhancing dispersion in water, ND particles coagulated to form small aggregates of tens of nanometers, presumably due to the residual hydrophobic carbon on their surface. The ND aggregates were very stable and did not dissociate after 30 min ultrasonication (200 W). A number of gold nanoparticles were 34319


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ACS Applied Materials & Interfaces artifacts (see Figure S2). The prepared nanocomposites formed aqueous dispersions stable (without aggregation) for over two months, allowing their use in combination with natural biomolecules. This colloidal stability presumably originates from the negative surface potential of the ND carboxyl groups and hydrated gold nanoparticles (see Figure S3). Magnified ND and NDAu TEM images showed that the former exhibited a crystalline structure with a lattice distance of 0.2 nm (Figure 1b). Although the ND surface was reported to have a graphene-like structure with difference lattice distances, we could not observe different lattice layers at the ND boundary. The produced gold nanoparticles also exhibited a polycrystalline structure, as shown in Figure 1d. Considering that many crystalline noble metal nanoparticles exhibit catalytic activity, this crystalline gold structure was also expected to contribute to the catalytic activity of NDAus. Details of the crystalline structures of ND and gold were obtained by X-ray diffraction (XRD) analysis. X-ray diffraction peaks at 2θ = 44.0° and 76.0° correspond to the (111) and (220) lattice planes of the ND core, respectively (Figure 1e), and are indicative of the cubic structure of the ND core crystal. The peak at 2θ = 19.8° corresponds to the (002) graphite crystal plane, implying that the core ND cubic structure might be wrapped by a graphite shell.27 The noisy background between 20° and 30° and the string background below 15° also indicate the presence of amorphous carbon in the ND powder. However, NDAus showed four additional diffraction peaks at 2θ = 38.1°, 44.4°, 64.7°, and 77.7°, which were indexed as the (111), (200), (220), and (311) planes of gold crystals, respectively.28 These peaks showed that the gold nanoparticles possess face-centered cubic phases. The Au (111) peak had the highest intensity compared to Au (200) and Au (220) peaks; thus, most of the Au structure was attributed to the Au (111) facet. It is plausible that the crystalline gold nanoparticles were created by the fast nucleation growth of the Au3+ ions reduced by a strong reducing agent (NaBH4) on the carboxylated ND substrate surface. TGA thermograms of intact ND and NDAus showed that the thermal decomposition of intact ND began at 548 °C, whereas NDAus showed only 11% weight loss even at 800 °C (Figure 1f). The thermal oxidation of nanodiamond at 550 °C was reported to be due to the nonintegrity of its crystal structure, implying the presence of a graphitic shell on the ND surface.27 The negligible thermal degradation of NDAus might be due to the heat of fusion of gold nanoparticles. Results of XRD and TGA analyses imply that crystalline gold nanoparticles were generated on the graphitic shell of the ND. Crystalline gold and the graphitic shell may interact and potentially give rise to catalytic activity, working as an active site. NDAus as Oxidative Catalysts. NDAus displayed peroxidase-like oxidative catalytic activity, which was not observed neither for the intact ND nor for 50 nm gold nanoparticles. Citrate-capped gold nanoparticle in 10 nm showed weak oxidative catalytic activity; however, its activity was approximately one-fifth of that of NDAu. Figure 2 shows the progress of OPD oxidation by H2O2 in the presence of NDAus and the results of control experiments using intact ND and gold nanoparticles. The progress of OPD oxidation was clearly observable from the solution color change (Figure 2, inset); the solution containing NDAus became yellow-orange in color, while the others remained transparent. Production of 2,3-diaminophenazine by OPD oxidation was verified through HPLC-mass spectrometry (see Figure S4). The catalytic activity

Figure 2. Progress of the catalytic oxidation reaction with time. The intensity of the characteristic UV absorbance peak of 2,3diaminophenazine at 430 nm was monitored. The catalytic reaction was performed at pH 7.2 in the presence of NDAus (0.02 mg mL−1), OPD (10 mM), and H2O2 (20 mM). Inset is a photo of the NDAu suspension showing the OPD oxidation. Each vial contains (i) 10 nm gold nanoparticles, (ii) 20 nm gold naniparticles, (iii) intact NDs, and (iv) NDAus. In the absence of NDAus, the solution remained transparent with little OPD oxidation. Weak violet color of (i) and (ii) originates from the gold nanoparticles. The vial containing NDAus exhibits yellow color because of the catalytic oxidation of OPD.

of 10 nm gold nanoparticles were originated from the electron exchange interaction at the surface,29 while the citrate retarded the catalytic activity due to its redox property.30 The uncapped gold nanoparticle produced on the ND surface would have higher catalytic activity than citrate-capped ones. Furthermore, the high catalytic activity displayed by NDAus implies a syncretization of ND and gold nanoparticles to express oxidative activity. This synergistic effect was observed for similar protein-stabilized gold nanoparticles and graphene-gold composite systems showing enhanced peroxidase-like activities.31−33 The oxidative catalytic activity is thought to originate from the promoted charge transfer from gold nanoparticles to ND substrate due to the hybridization of metal nanoparticles with sp2 dangling bonds of surface carbons.34,35 In particular, the gold nanoparticles were supposed to affect the surface graphitic layer of ND similarly to doping, presumably causing the catalytic activity to be expressed at the interfaces between these metal and carbonic compounds.36 To rationalize the synergistic interaction of gold nanoparticles and ND, the surfaces of intact ND and NDAus were investigated using X-ray photoelectron spectroscopy (XPS). The binding energy survey of NDAu was characterized by the doublet of two spin−orbit components. The peaks of Au 4f7/2 and 4f5/2 serve as evidence of NDAu formation (Figure 3f).32 The peaks at 83.5 and 86.8 eV were attributed to the Au0 metallic state of AuNPs and peaks at 84.5 and 88.1 eV were attributed to Au+ ionic oxidative state. It was reported that oxidation of a substrate using gold catalyst was affected by the presence of partially oxidative gold species or phase ratio of metallic Au0 to oxidative Au+ ion.37 The Au+ ion is postulated to facilitate the generation of hydroxyl groups from H2O2, which may elevate initial activity for OPD oxidation.38,39 NDAu composite includes both gold metallic atoms and ion in oxidative state, leading to highly active catalytic activity. The deconvoluted C 1s spectra describe the association of gold nanoparticles with the oxygenated carbonic components on the 34320


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Figure 3. XPS spectra of intact ND and NDAus, carbon (C 1s) in (a) and (d), O 1s in (b) and (e), Au in (f), XPS survey (c) of ND (black), and NDAus (red). Each spectrum was deconvoluted using Gaussian-shaped peaks. The COOH peak intensity at 289.0 eV notably decreased after deposition of gold nanoparticles.

ND surface (Figure 3a, c). The COOH peak at 289.0 eV significantly decreased after deposition of gold nanoparticles, compared to other (CC and CO) carbonic peaks.36 This decay evidence that carboxyl groups on the ND surface are mostly involved in the reduction of Au3+ to form AuNPs on ND. The associated Au3+−carboxylate complexes on the graphitic layer presumably function as nucleation sites for gold nanoparticles. Aside from the decay of the COOH peak, the shift of the sp2 CC peak from 283.1 eV to a higher binding energy of 283.4 eV implies that the gold nanoparticles exhibit an n-type doping effect on the ND graphitic layer. The presence of electron-rich gold nanoparticles at the ND surface enabled electron transfer to the graphitic layer of ND, leading to increased electron mobility at the ND−metal interface.33−36 This enhanced electron mobility is supposed to promote the catalytic reaction, so that the catalytic oxidation of OPD is observed only in the presence of NDAus. The conventional binding energies of the O 1s peak were deconvoluted at 531.5 (CO) and 532.9 eV (COC/C OH) (Figure 3b, d). The ND O 1s peak was also deconvoluted at 531.5 eV, showing a high density of carboxyl groups on the ND surface. The NDAu peak at 531.5 eV was weak compared to that of the carboxyl-rich ND, indicating that the carboxyl groups were reduced concomitantly with gold deposition. Supporting the formation of NDAus, the binding energy of the latter increased at 532.9 eV, attributed to COH functionalities. Activation Energy of the NDAu Catalyst. In the catalytic activity analysis, the activation energy of NDAus was evaluated based on reaction rates measured at various temperatures. The reaction rates were plotted according to the Arrhenius equation, and the activation energy was determined from the slope of the linear regression (Figure 4). The activation energy of NDAus was 93.3 kJ mol−1, which was higher than that of a different carbon-based peroxidase-mimetic catalyst (59.3 kJ

Figure 4. Arrhenius plot used to determine the activation energy of OPD oxidation catalyzed by NDAus. Assuming a pseudo-first-order reaction, the reaction rate constant (k) was determined from the initial reaction rate at each temperature.

mol−1),40 hemin−graphene composite catalyst (20.37 kJ mol−1),41 and few-layered graphene (28 kJ mol−1).42 This rather high NDAu activation energy implies that the ND surface layer is not as active as graphene or other reported carbonic supports. Since the gold nanoparticle-induced catalytic oxidative dehydrogenation of the amine substrate (OPD) was highly influenced by properties of the catalyst support,43,44 specifically by the interaction between gold nanoparticles and ND in this study, the inertness of the ND surface might diminish the catalytic activity. Although sp2 carbon layers (like graphene with dangling functional groups) are expected to be present on the ND surface, the density of such dangling functional groups might not be as high as that of graphene, since the surface oxygenated functionalities (mainly carboxyl groups) were reduced during the formation of gold nano34321


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Figure 5. Steady-state reaction kinetic assay of NDAus. Apparent reaction rate profiles and double reciprocal plots were drawn based on the Michaelis−Menten kinetic model. The apparent reaction rate (v0) and [E0]/v0 were plotted (a, b) for variable H2O2 concentrations at fixed concentrations of OPD (2.5, 5, and 10 mM), and (c, d) for variable OPD concentrations at fixed concentrations of H2O2 (20, 40, and 80 mM), respectively. All reactant solutions were at pH 7.2 ± 0.2 with 0.04 mg mL−1 of NDAus.

assumption of a pseudo-first-order reaction (Table 1). At the same OPD concentration, the catalytic efficiency increased with

particles on ND. Alternatively, active functional groups can be screened by the relatively large gold nanoparticles. Considering that carbonyl or carboxyl groups of the carbonic compounds are required to facilitate the catalytic reaction,33 the synergetic catalytic effects were suppressed, leading to a higher activation energy of NDAus for the oxidation reaction. Catalytic Reaction Kinetics and Suggested Mechanism. To analyze the catalytic activity and reaction mechanism in detail, comprehensive kinetic parameters of NDAus were obtained using the enzyme kinetic model. The peroxidase-like catalyst followed the bisubstrate Michaelis−Menten (MM) kinetic model, with OPD and H2O2 as substrates. The latter model can be expressed as the following eq 1, where KM and kcat represent the Michaelis constant and the apparent turnover number, respectively.45 v0 =

Table 1. Apparent Kinetic Parameters for OPD Oxidation by H2O2 Catalyzed by NDAus H2O2 conc. [mM]

kcat [mM s−1 mg−2]

KMH2O2 [mM]

OPD conc. [mM]

377.6 ± 22.9 474.3 ± 19.6 565.6 ± 18.8 kcat [mM s−1 mg−2]

KMOPD [mM]

1.8 ± 0.2 2.3 ± 0.3 6.3 ± 0.8 kcat/KMOPD [s−1 mg−2]

2.5 5.0 10.0

290.4 ± 27.2 300.0 ± 25.1 319.3 ± 23.0

48.7 ± 0.2 33.0 ± 0.4 6.4 ± 1.0

5.9 ± 0.6 10.1 ± 1.1 50.5 ± 9.3

20 40 80

208.7 ± 14.7 188.4 ± 15.6 89.7 ± 9.6

kcat/KMH2O2 [s−1 mg−2]

kcat[E0][OPD][H 2O2 ] OPD KM [H 2O2 ]

H 2O2 + KM [OPD] + [OPD][H 2O2 ]

increasing H2O2 concentration, showing maximum efficiency for 80 mM H2O2. An increase of kcat and decrease of KM were observed with increasing H2O2 concentration, suggesting simultaneous promotion of substrate binding and reaction at the NDAu active site. Similar MM curves were obtained for different H2O2 concentrations at a constant concentration of OPD (Figure 5c,d). The kinetic parameters also displayed an increase of catalytic efficiency with substrate concentration. Michaelis constant of this NDAu is compared with other carbon−metal catalysts (see Table S1). NDAu has high KM value presumably due to the relatively small surface active area of ND than other carbonic compounds and due to the difference in the substrate used.

(1)

Reaction rates at the onset of the reaction (v0) were determined from the concentrations of 2,3-diaminophenazine by changing the concentration of one substrate while keeping the other one fixed. The plots of reaction rates vs substrate concentration obeyed the MM kinetic model well. The steadystate MM kinetic and double reciprocal plots obtained under fixed OPD concentrations are shown in Figure 5a,b, respectively. The double-reciprocal plots were used to determine the apparent Michaelis constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) under the 34322


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ACS Applied Materials & Interfaces The decay of KM with increasing substrate concentration is noticeable, considering that many previous peroxidase-mimetic catalysts showed invariance of KM.45,46 This substrate-dependent KM change implies that both H2O2 and OPD substrates bind to the NDAu active sites. Quan et al. proposed that the attractive interaction between carbon 2p and Au 5d orbitals of graphene defects and gold nanoparticles is the driving force of H2O2 binding to the active site, and that this H2O2 adsorption promotes the production of OH• radicals during the catalytic oxidation.33 Therefore, it is very likely that a similar H2O2 binding and OH• radical generation mechanism operated at the active sites of NDAus, so that KMH2O2 increased with H2O2 concentration. The kcat values increasing concomitantly with H2O2 concentration also support H2O2 binding and subsequent radical formation during the catalytic reaction. The much lower KM values of OPD than those of H2O2 are indicative of the higher affinity of the former to NDAus. This high affinity is presumably due to couple of factors, one of them being the aromatic structure of OPD displaying a binding affinity to the ND surface via aromatic interactions.32 The other factor is that the amine groups of OPD can promote electrostatic attraction to the remnant carboxyls on the ND surface or interact with the local dipole moment of ND.47,48 These two factors enhance OPD binding to the ND surface and promote the catalytic reaction. Taken together, both H2O2 and OPD substrates bind to the active sites of NDAus via attractive intermolecular forces originating from the ND−Au interface, leading to a synergetic catalytic reaction. The parallel double reciprocal plots shown in Figure 5b,d are indicative of a ping-pong mechanism, where a transitory catalyst complex is proposed to form during the catalytic reaction.49 As observed from kinetic parameters, the substrates associated with the active sites of NDAus to create active intermediates for OPD oxidation. Gold species near the oxygenated graphitic surface facilitated the formation of such intermediates, leading to enhanced catalytic activity. This ping-pong mechanism is commonly observed for many peroxidase-like catalysts based on graphene as well as biological substances.45,46,50−52 Reusability of the NDAu Catalyst. Previous results proved that NDAus can be used as a heterogeneous catalyst. Therefore, the reusability of NDAus was examined as a practical requirement. The nanocomposites were collected by centrifugation after the reaction, washed with deionized (DI) water, and redispersed in aqueous solution for reuse. The catalytic activity decreased up to the third reuse cycle and then remained invariant (Figure 6). The decay of catalytic activity is presumably due to the growth of gold nanoparticles induced by Ostwald ripening (Figure 6, inset). Since the solubility of gold nanoparticles increases with decreasing size, the growth of larger gold nanoparticles might occur by the sacrificial dissolution of small ones during the washing step. This ripening of gold nanoparticles on the ND substrate reduces the number of active sites of the NDAu nanocomposite and leads to decreased catalytic activity. TEM images show the generation of many larger gold nanoparticles of ∼40 nm diameter after 5-fold cycling of NDAus, while such large particles were hardly observed in fresh NDAus (see Figure S5). These ripening changes of gold nanoparticles also support the synergistic effect of ND and gold nanocomposites to evolve catalytic activity. Xanthine Detection Using the NDAu Catalyst Coupled with Xanthine Oxidase. As an application of the NDAu catalyst, detection of xanthine was demonstrated using coupled

Figure 6. Reusability of NDAus. The concentration of 2,3diaminophenazine, the product of OPD oxidation, was read after 400 s of reaction. After every catalytic reaction, NDAus were collected, washed, and dispersed in a solution containing 5 mM OPD and 20 mM H2O2 at pH 7.2. The amount of NDAus was adjusted to 0.12 mg mL−1 for every reuse test. Inset is a TEM image of NDAus after five reuse cycles (scale bar: 20 nm).

catalysis with xanthine oxidase (XOD). Xanthine, a purine base found in human fluids, was used as a XOD substrate to produce uric acid and H2O2. Subsequently, the generated H2O2 oxidized OPD in the presence of the NDAu catalyst, resulting in a color change of the xanthine solution from transparent to orange. A schematic illustration of the XOD coupled catalysis is shown in Figure 7a. In control experiments, the color change of the xanthine solution was examined in absence of XOD or NDAus. In both cases, no notable color change occurred (see Figure S6). This verified the combined catalysis of XOD and NDAus for detecting xanthine in solution. Progress of the coupled catalysis was monitored using the intensity change of the UV absorbance peak at 450 nm (see Figure S7). The corresponding peak intensity increased with time, revealing the sequential catalytic reaction of OPD oxidation. This oxidation was promoted by increased concentrations of XOD that supplied more H2O2 for OPD oxidation catalyzed by NDAus (Figure 7b). These results indicate that NDAus are compatible with natural enzymes without degradation and/or denaturation.

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CONCLUSIONS Studies are underway to investigate the properties and versatile new applications of the nanodiamond as a relatively new carbonic material. This study offers a promising way of using ND by exploiting it as an active nanocomposite biomimetic catalyst. Although the sp2 graphene-like layer was likely to exist on the surface, ND itself did not show intrinsic catalytic properties, whereas graphene was intrinsically active as a peroxidase.53,54 We demonstrated that the nanocomposite of ND with gold nanoparticles displayed unforeseen chemical activity driven by the synergistic effect between gold and the sp2 carbon layer. Specifically, the ND−gold nanoparticle nanocomposite exhibited oxidative catalytic activity following a pingpong mechanism for multiple substrates. The extraordinary catalytic activity of ND−metal composite combined with higher mechanical stability and optoelectronic property from ND crystal may grant a new application area once we synergistically exploit the inherent strong multifaceted ND adsorption properties showing diverse intermolecular attractions like van 34323


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Figure 7. Coupled catalysis by xanthine oxidase (XOD) and NDAus used for the detection of xanthine. (a) Illustration of coupled catalysis, where XOD produces uric acid and H2O2 from xanthine and subsequent OPD oxidation is catalyzed by NDAus (XOD was drawn by using 3unc.pdb). Oxidation of OPD produces 2,3-diaminophenazine, resulting in a solution color change from transparent to yellow. (b) Concentration of 2,3diaminophenazine obtained for various XOD amounts. The concentration of XOD was adjusted to the desired activity (units) in a solution containing NDAus (0.04 mg mL−1), xanthine (1 mM), and OPD (10 mM). The concentration of 2,3-diaminophenazine was read after 300 s of the coupled catalytic reaction.

der Waals forces, electrostatic interactions, and dipole moments.48 We also demonstrate that this ND−gold composite could be utilized for analysis of biological samples in association with a natural enzyme. Considering that various combinations of ND with other heterogeneous compounds are available, many applications and functions are available by proper design of the ND composite. The new interface of NDAus is potentially useful for a hybrid system containing biomolecular substances, due to the repetitive catalytic activity and suspension stability in aqueous solutions.

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REFERENCES

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ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10471. Size distribution of gold nanoparticles, EDX analysis on NDAu, zeta potential of ND, and NDAu, LC-mass spectrometry of the product, comparison of Michaelis constant, TEM images of reused NDAus, and photo image of combinational catalysis (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-2-2123-5758; fax: +82-2-312-6401; e-mail: leessy@ yonsei.ac.kr (S.-Y.L.). *Tel:+82-2-820-6684; e-mail: [email protected] (E.K.). Author Contributions ∥

ACKNOWLEDGMENTS

This work was supported by the Human Resources Program in Energy Technology of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) funded by the Ministry of Trade, Industry & Energy, Republic of Korea (No. 20154010200810) and by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053307).

S Supporting Information *

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These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 34324


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