Ir nanoparticles with multi-enzyme activities and its application in the selective oxidation of aromatic alcohols

Applied Catalysis B: Environmental 267 (2020) 118725

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Ir nanoparticles with multi-enzyme activities and its application in the selective oxidation of aromatic alcohols

T

Guangxia Jin, Jie Liu, Chan Wang, Wenxiu Gu, Guoxia Ran, Bing Liu, Qijun Song* Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu Province 214122, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Iridium nanoparticles Superoxide ion Oxidase-like activity Aerobic oxidation Alcohol transformation

An enzyme-like nanoplatform obtained from sodium citrate-modified iridium nanoparticles (Cit-IrNPs) was presented, which exhibits excellent peroxidase, catalase and oxidase like activities thanks to the large accessible surface area and high-index facets of the mono-dispersed nanoparticles. The morphology and structures of CitIrNPs were comprehensively characterized by TEM, HRTEM, XRD, IR and XPS. The surface chemistry of CitIrNPs reveals that the oxidase-like and peroxidase-like activities can be ascribed to the formation of O2¯ from the activation of dissolved oxygen on the high-index facets of IrNPs. The oxidase-like activity of Cit-IrNPs was further manifested by the oxidation of 33.3 mM aromatic alcohols in the presence of 3 mg/mL Cit-IrNPs in ambient conditions. Over 90 % conversion rates were readily obtainable in 10 h for all the tested alcohols with an initial reaction rate of ca. 20 μmol/h. More significantly, apart from the aldehydes no other byproducts were detected. The kinetic analysis of the enzyme mimic suggests the reaction follows a classical Michaelis-Menten model. The enzyme mimic also shows a good stability, as no obvious decrease in catalytic activity was observed after recycled use for 6 times. Furthermore, DFT calculation was employed to elucidate the reaction mechanism and it was found that the alcohol is initially bond to Ir(0) and subsequently forms Irδ+-alkoxide species and the carbonyl product. Meanwhile, the Irδ+-hydride species reductively eliminates O2¯ and returns back to Ir(0). To the best of our knowledge, this is the first report of metal nanomaterials which can effectively transform aromatic alcohols to corresponding aldehydes at ambient conditions without need of external energy input.

1. Introduction The selective oxidation of alcohols to corresponding aldehyde compounds is of great importance in synthetic chemistry [1–3]. One of the biggest challenges is to prevent alcohols from over-oxidation and obtain the important intermediates for fine chemical synthesis. Although some conventional methods are available in aldehydes synthesis, the associated processes usually suffer from intrinsic drawbacks, such as poor atom efficiency and adverse environmental issues [4]. An ideal strategy would be directly oxidize alcohols with molecular oxygen as a benign reagent promoted by reusable catalysts. Therefore, moving away from stoichiometric inorganic or organic oxidants to environmental friendly and reusable catalysts for aerobic oxidation of alcohols becomes the new trend in the practice of green chemistry. Nanoscale enzyme mimics have emerged as a promising candidate to replace natural enzymes in various applications such as biomedical [5], catalysis [6] and molecular sensing [7]. These artificial nanozymes show many advantages over their natural peers, including higher

⁎

stability and super catalytic performance [8]. In this aspect, noble metal nanocatalysts have attracted extensive research attention due to their advantages of convenient preparation, size/surface tunable catalytic activity and excellent stability [9,10]. Particularly, a series of noble metal nanoparticles have been developed as effective catalysts for the selective oxidation of alcohols [1,2,11,12]. Among them, the selective oxidation of benzyl alcohol with molecular oxygen was achieved based on the photothermic effect of electro-spun Au/CeO2 hybrid nanofibers [13]. Jiang et al. proposed a photocatalytic oxidation reaction with molecular oxygen activated by Pt-based nanocatalyst [14]. More recently, Wang et al. presented a nanocatalyst with Pd single atoms anchored on CeO2 supports (Pd1/CeO2), which showed high catalytic activity and chemoselectivity in the oxidation of substituted benzyl alcohols [15]. However, extra energy input either in form of light or heating is required in the above transformations to overcome the oxidation barriers. Methods that do not require additional energy have not been reported yet. In this respect, these enzyme mimics still cannot compete with their nature counterparts, as natural enzyme can often

Corresponding author. E-mail address: [email protected] (Q. Song).

https://doi.org/10.1016/j.apcatb.2020.118725 Received 6 November 2019; Received in revised form 31 January 2020; Accepted 2 February 2020 Available online 03 February 2020 0926-3373/ © 2020 Elsevier B.V. All rights reserved.

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2.3. Synthesis of the Cit-IrNPs

catalyze specific reactions at body temperatures. In last few years, we have focused on the study of iridium nanoparticles (IrNPs) for their enzyme like catalytic properties. It was found that the tannic acid stabilized IrNPs (TA-IrNPs) have peroxidase-like activity, which could be used as peroxidase mimics for hydrogen peroxide detection [16]. The IrNPs stabilized by citrates can catalyze the activation of dissolved oxygen (DO) for the TMB oxidation [17]. On these researches, we focused on developing nanozymes for molecular sensing. In present work, we continued to investigate the mechanistic aspects and more valued applications of IrNPs. Therefore, citrate capped iridium nanoparticles (Cit-IrNPs) with 1.5 nm in diameter are prepared, and their intrinsic catalase, peroxidase and oxidase-like activities were studied, and the kinetics assay and catalytic mechanism were explored in more details. Particularly, we have rationalized the Cit-IrNPs catalyzed production of ·O2¯ from molecular oxygen by a series of experiments. The density functional theory (DFT) calculations also confirmed that the unusual catalytic properties should derive from electron transfer effect from the high-index facet of Cit-IrNPs to the appended O2 molecules. Consequently, Cit-IrNPs were endorsed with a truly enzyme like activities, which allows the highly selective and efficient transformation of aromatic alcohols to corresponding aldehydes in aqueous solution at ambient conditions without need of extra energy input.

Synthesis of the Cit-IrNPs was according to our previous work with some modifications. IrCl3 solution (20 mL, 2 mM) and Na3Cit solution (6 mL, 0.034 M) were mixed together, adjusting the pH to 7–9 with NaOH solution. The mixture was then refluxed under vigorous stirring for 20 min. After that, freshly prepared NaBH4 solution (2 mL, 0.1 M) was added, keeping the mixture stirred for 30 min and cooling to the room temperature. It should be noted the solution color changed gradually from clear yellow to dark blue. The product was collected by drying in vacuum at 45 °C after centrifugation and washing with ethanol for three times. Finally, the obtained black powder was kept in pure acetone further use. 2.4. Peroxidase-like activity assay The steady-state kinetics were studied by recording the absorption spectra at 652 nm in time scan mode at room temperature. The absorption-time profiles were recorded in 2.4 mL of HAc-NaAc buffer (0.01 M, pH 3.86) with 2 ng/mL Cit-IrNPs in the presence of TMB and H2O2. The peroxidase-like activity test was performed by varying the concentrations of TMB from 0.1 to 0.5 mM at a fixed H2O2 concentration of 0.5 mM and vice versa. The Michaelis-Menten constant parameters were calculated by fitting the data to the Lineweaver-Burk plots:

2. Experimental section

1 K 1 1 = m × + v vmax c vmax

2.1. Materials

ν refers to the initial reaction velocity, νmax refers to the maximal conversion rate, c refers to the concentration of substrate and Km refers to the Michaelis − Menten constant.

Iridium trichloride (IrCl3), trisodium citrate dehydrate (Na3Cit), sodium hydroxide (NaOH), sodium borohydride (NaBH4), 3,3,5,5-tetramethyl benzidine (TMB), ascorbic acid (AA), sodium acetate anhydrous (NaAc), acetic acid (HAc) and luminol were purchased from Aladdin Chemicals Co. Ltd (Shanghai, China). Benzoquinone, 1,3-diphenylisobenzofuran (DPBF), tryptophane, tannic acid (TA), benzyl alcohol, terephthalic acid (TPA), isopropyl alcohol, dimethylformamide (DMF), polyvinylpyrrolidone (PVP, average MW58000, K29–32) and hydrogen peroxide (H2O2, 30 %) were obtained from Sinopharm Chemical Reagent Co. Ltd. (China). DMPO (5,5-dimethyl-1-pyrroline Noxide, 97 %) was bought from Energy Chemical Co. Ltd. (Shanghai, China). Milli-Q ultra-pure water (18.2 MΩ cm) was used throughout the experiments.

2.5. Catalase-like activity assay In the catalase-like activity assay, the variations of DO concentration were monitored with a multi-parameter analyzer at room temperature. Cit-IrNPs solution (20 μL, 3 mg/mL) were added to 3 mL water in the presence of varied concentrations of H2O2. The effects of pH on the activity of Cit-IrNPs was studied by varying pH from 1 to 12 with a fixed H2O2 concentration at 0.1 M. The same reaction system in the absence of Cit-IrNPs was performed as the control.

2.2. Characterizations 2.6. Oxidase-like activity assay

The fluorescence and the absorption spectra were recorded with an Edinburgh Instruments FS5 fluorescence spectrophotometer and a Shimadzu UV-2700 spectrophotometer, respectively. Fourier infrared spectrometer (Nicolet iS50 FT-IR, Thermo Fisher Scientific, USA) was used to record the transmission fourier transform infrared spectra (FTIR) and total reflectance infrared spectra (ATR-IR). The surface morphology was inspected by transmission electron microscope (TEM, JEOL, JEM-2100plus). X-Ray photoelectron spectroscopy (XPS) measurement was performed by a PHI Quantum 2000 Scanning ESCA Microprobe. X-ray diffraction (XRD) pattern was measured on XRD spectrometer (D8, Bruker AXS, Germany) with Cu Kα radiation source over the 2θ range of 5–90°. Zeta potential analyzer (Brookhaven instruments Corporation) was employed to determine the Zeta potential and hydrodynamic size of NPs. Electron spin resonance (ESR) experiments were carried out on a Bruker EMXplus-10/12 spectrometer. All products in the catalytic oxidation reaction were identified and quantified by gas chromatography (GC9790II with a 0.25 mm × 60 m SE-54 capillary column). The pH and dissolved oxygen were recorded by DZS708 L Multiparameter analyzer (Shanghai INESA Scientific Instrument Co. Itd). A laboratory-built flow system was used for CL detection with the Remex MPI-E ECL analyzer (Xi’an Remax Electro-Science and Technology Co. Ltd., China).

To study the steady-state mechanism of the reaction, experiments were carried out in reaction buffer (2.4 mL, 0.01 M HAc-NaAc, pH 3.86) with various concentrations of TMB while Cit-IrNPs concentration was kept constant (2 ng/mL). To study the effects of pH, the reaction was carried out pH 2–11, in the presence of 0.2 mM TMB or AA with 2 ng/ mL Cit-IrNPs. The reaction kinetics for the catalytic oxidation of TMB or AA were monitored at 652 or 265 nm in time scan mode at room temperature, respectively. The kinetic parameters, νmax and Km could be calculated by fitting the data to the Lineweaver-Burk plots. 2.7. TMB oxidation An amount of TMB (100 μL, 10 mM) was mixed with 3 mL of HAcNaAc buffer solution (0.01 M, pH 3.68). Then an aqueous suspension of 10 μL of Cit-IrNPs (3 mg/mL) was added into the reaction mixture after bubbling different gas (O2, N2 or air) for 10 min. The samples were detected at 652 nm in time scan mode for UV–vis measurements. To verify the type of reactive oxygen species (ROS), different ROS scavengers (1 mM) such as tryptophane, benzoquinone and isopropyl alcohol were added into the solution prior to the UV–vis measurements. 2

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Fig. 1. Structural characterizations for Cit-IrNPs. (A) TEM image (insert particle size distributions), (B) HRTEM image and (C) XRD pattern of Cit-IrNPs. (D) Comparison of ATR-IR spectra of Cit-IrNPs and Na3Cit. (E) Ir 4f XPS spectra of Cit-IrNPs. (F) The schematic diagram for the surface chemistry.

nanoparticles, which would be reused in the subsequent runs. The obtained solution was analyzed by GC with n-dodecane as the internal standard.

2.8. Fluorescence measurement for superoxide anion The classical probe molecules DPBF been used to detect superoxide anion. Stock solution of DPBF (2 mg/mL) was made in DMF, and stored at 4 °C in the dark before use. Then 100 μL of the probe solution was mixed with 3 mL of H2O with or without addition of Cit-IrNPs suspension (3 mg/mL). The fluorescence of DPBF was monitored with 450 nm excitation light.

2.11. DFT calculations The density functional theory (DFT) calculations were performed to study the interaction between IrNPs surface and O2 molecule. The VASP package (PAW) was employed in all calculations. The plane wave cutoff energy was set at 400 eV. The electronic exchange and correlation were simulated with the revised Perdew-Burke-Ernzerhof (RPBE) functional. The Methfessel–Paxton technique was used for electron smearing with a smearing width consistent to 0.2 eV. The force convergence criteria less than 0.03 eV/Å was used to realize the structure optimization. The adsorption energy was defined as

2.9. ESR trapping measurements ESR measurements were carried out for the detection of spin adducts at room temperature. Sample solution was sealed in the glass capillary tube, which was inserted into the ESR cavity and measured at selected time. The ESR instrument parameters were set as follows: microwave power 0.1 mW, scan range 120 G, modulation amplitude 2 G and time constant 0.3 s. As for the ·O2¯ trapping tests, an amount of 100 μL Cit-IrNPs aqueous suspension (3 mg/mL) and 20 μL of DMPO was mixed with 0.5 mL of H2O. The mixed solution was monitored by ESR spectrometer immediately. ESR was also used to investigate the catalase activity of CitIrNPs. Tubes containing 50 mM DMPO and 5 mM H2O2 in the present and absent Cit-IrNPs were exposed to UV light (355 nm) for 10 min and characterized immediately.

Eads = E(adsorbate

+ surface)

- E(adsorbate) - E(surface)

E(adsorbate + surface) refers to the total energy of the adsorbate interacting with the surface; E(adsorbate) is the energies of the free adsorbate in gas phase and E(surface) represents the energies in the bare surface. The exothermic adsorption results in a negative value and more negative means stronger binding. 3. Results and discussion

2.10. Alcohol oxidation reactions

3.1. Characterization of Cit-IrNPs

The catalyst solution was prepared by adding 9 mg Cit-IrNPs powder to 3 mL water. To ensure no aggregation of Cit-IrNPs, the solution was sonicated for 5 min. A typical concentration of benzyl alcohol used in the experiments was 33 mM, which is below the saturation concentration of benzyl alcohol (4.29 g/100 mL in 20 °C) [18]. In the presence of Cit-IrNPs and the help of vortex effect, a uniform solution can be prepared at room temperature. All the reactions were carried out in a 20 mL bottle as the reactor stirred by a magnetic bar. The reactor was sealed with a balloon filled with O2 gas at ambient temperature. Then the mixture was diluted by ethanol and then centrifuged to separate the

The Cit-IrNPs were prepared by the reduction of IrCl3 precursor with NaBH4 in the presence of Na3Cit as the stabilizers. The as-synthesized IrNPs were obtained in form of dark blue solution. After centrifugation, washing and drying, the IrNPs powders were obtained. As presented in Fig. 1A, the IrNPs exhibited quasi-spherical morphology with 1.5 nm diameter based on a statistical analysis of 100 nanoparticles. The obtained nanoparticles were uniform and stable during preparation and storage. When re-dispersed in water, the suspension shows an absorption hump peak at 565 nm in UV–vis spectrum (Fig. S1), which could be assigned to the intense surface plasmon resonance 3

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G. Jin, et al.

the variation of pH, demonstrating a high stability of Cit-IrNPs. This anti-reunion property to condition changes is important for the practical applications of nanoparticles.

(SPR) absorption of IrNPs [19]. To gain a better understanding of the surface chemistry, the assynthesized Cit-IrNPs were characterized with various techniques. HRTEM image in Fig. 1B shows the lattice spacing on the surface of CitIrNPs, the well-defined lattices can be attributed to the crystal faces {111}, {200}, {220} and {311} of the metallic Ir. The corresponding electron diffraction (Fig. S2) and XRD patterns (Fig. 1C) could also be indexed to the theoretical values of typical lattice planes, which are in line with the results from the HRTEM. These high-index facets are critical to ensure the high catalytic activity of Cit-IrNPs. In addition, only broad peaks are observed in the XRD patterns, indicating the small sizes of Cit-IrNPs. Fig. 1D exhibits the ATR-IR spectra, which reveals the structural details of citrate anions adsorbed on the Cit-IrNPs. The two curves with similar profiles expressively indicate that the major structures of citrate molecule are retained after the formation of nanoparticles. More useful information could be obtained from the spectra. For instance, the ν(CeO)alch of the oxhydryl (alch) group [20] were observed at 1193, 1156, 1077 and 1064 cm−1 for Na3Cit, while only the peak at 1065 cm−1 was observed for the Cit-IrNPs (Fig. S3A). This change could be used as the evidence for the alcohol coordination to metal nanoparticles. Compared with the peak at 3256 cm−1 assigned to ν(OeH)alch [21] for Na3Cit, the disappearance of this peak in Cit-IrNPs spectra could be attributed to the breakage of the alcohol band (OeH) and the formation of a new band of O]Ir&+ in Cit-IrNPs. FT-IR spectroscopy was carried out to study the interaction between the carboxylate and metal ions (Fig. S3B). The two bands appeared at 1636 and 1381 cm−1 could be attributed to νasy(COO) and νsym(COO) stretching vibrations [22] that coordinating on Ir surface. The separation (Δν), νasy (COO)−νsym(COO) is generally representative of the coordination type of the carboxylate with the metal ions [23]. The Δν value here is 155 cm−1, which confirms that the Ir ions take on the bidentate structure coordination with carboxylate groups (Fig. S3C). XPS data can provide detailed information about coordination groups on the nanoparticles surface. In Fig. 1E, the 4f7/2 (41.67 %) and 4f5/2 (31.22 %) components of Ir (0) appear at 60.57 and 63.6 eV respectively [24]. And the peaks at 62.21 and 65.4 eV correspond to the 4f7/2 (15.5 %) and 4f5/2 (11.61 %) of Ir&+-O respectively [25]. These results suggest the zero valent Ir is the dominant species in Cit-IrNPs. As described in Fig. S4B, the C1s spectrum of Cit-IrNPs exhibits four peaks, which can be ascribed respectively to COOH or COO− (288.58 eV) [26], CeOHalch (287.86, 285.9 eV) [27] and CeC or CeH (284.74 eV). In Table 1, the binding energy at 287.86 eV could be ascribed to the coordinated carboxylates (COOeIr), which agrees well with the IR analysis given above. As for O1s spectrum of Cit-IrNPs (Fig. S4C), the two peaks at 533.6 and 531.45 eV correspond to Ir&+-O and C]O, whereas three peaks are observed in the O1s spectrum of Na3Cit (Fig. S4E). On the basis of above characterizations, the formation of the CitIrNPs can be simply understood as the carboxyl groups of citrate anions are adsorbed on the IrNPs surface by O-Ir conjunction (Fig. S5), preventing unlimited growth and agglomeration of Cit-IrNPs during the reduction of Ir3+ into Ir(0) by NaBH4. The surface structure of Cit-IrNPs is schematically summarized in Fig. 1F. Additionally, the Zeta potential of Cit-IrNPs in water was determined to be −35.8 mV, suggesting the presence of free carboxyl (COO−) on the surface of Cit-IrNPs. The effect of pH on effective diameter and Zeta potential was further investigated. As shown in Fig. S6, no obvious changes in diameter or Zeta potential were observed with

3.2. Peroxidase-like activity and catalase-like activity of Cit-IrNPs The peroxidase-like behavior of Cit-IrNPs was evaluated by using TMB as a chromogenic substrate in acidic buffer (pH 3.86). The steadystate kinetic parameters can be calculated by fitting the data to Lineweaver-Burk equation [16] (Fig. 2A and B). It can be seen in Table 2, the Km value of Cit-IrNPs is 0.27 mM by using H2O2 as the substrate, which demonstrates that Cit-IrNPs have a significantly higher affinity for H2O2 than HRP and other nanomimics [16,28,29]. When using TMB as the substrate, the Km value of Cit-IrNPs is 0.0906 mM, which is slightly greater than that of other Ir nanoparticles (Table 2). This might due to the small size of Cit-IrNPs and the hydrophilic groups on the NPs surface that could have repulsion to TMB. The obtained νmax and kcat are also listed in Table 2. Evidently, the kcat value of Cit-IrNPs for H2O2/TMB is much higher than that of other mimic enzymes or HRP, which could be ascribed to the higher surface-to-volume ratio of Cit-IrNPs, thus providing more binding sites. In addition, the nanoparticles exhibit intrinsic catalase-like activity, Fig. 2C shows that Cit-IrNPs could accelerate H2O2 decomposition under various pH environments. The DO content as a function of H2O2 concentrations was monitored to evaluate the enzymatic parameters (Fig. 2D). The Km value of Cit-IrNPs with H2O2 was calculated to be 21.09 mM, much smaller than that of the reported mimic enzymes [28,30] (Table 2). Therefore, Cit-IrNPs can be considered as a promising candidate of catalase mimic particularly in high pH conditions. Furthermore, ESR was used to evaluate the catalase-like activity of CitIrNPs. As expected, addition of Cit-IrNPs can effectively reduce the ·OH radical production through catalyzing the decomposition of H2O2 into H2O and O2, hence the generation of ·OH in the H2O2/UV system was substantially reduced (Fig. S7). The catalytic efficiency k of Cit-IrNPs as a catalase mimic and a peroxidase mimic were calculated. As demonstrated in Fig. S8, we could find the k value on the peroxidase-like pathway was higher than that for the catalase-like one, demonstrating the rate of TMB oxidation faster than the decomposition of H2O2 catalyzed by Cit-IrNPs. The Fenton-like reactions are widely accepted to explain the catalytic mechanism of peroxidase mimics, considering that H2O2 molecules and ·OH radicals have a close connection [31]. The mechanism of peroxidase-like activity of Cit-IrNPs was further investigated. However, the results showed that Cit-IrNPs did not enhance ·OH generation as evidenced by using TPA as the probe [32] (Fig. S9). Three radical scavengers (benzoquinone [33] for O2¯, tryptophane [34] for 1O2 and isopropyl alcohol [35] for OH) were employed to investigate the specific ROS generated in the presence of H2O2 and Cit-IrNPs. In contrary to the results obtained with tryptophane and isopropyl alcohol probes, the peroxidase-like activity of Cit-IrNPs was substantially suppressed in the addition of benzoquinone in the Cit-IrNPs–H2O2–TMB system, indicating the involvement of O2¯ in the oxidation reaction (Fig. 3A). Electron spin resonance (ESR) is considered as a reliable assay for ROS detection [36]. In this practice, DMPO/OH spin adduct was not observed in the presence of Cit-IrNPs and H2O2. Nevertheless, the DMPO/ ·OOH spin adduct and a three-line ESR spectrum (1:1:1 triplet one) were obtained in Fig. 3B, which is resemble to the ESR spin characterization in the oxidase-like activity of Cit-IrNPs in the section 3.3. In addition, gas bubbles were observed in the Cit-IrNPs–TMB–H2O2 system. Based on the above observations, we suggested this peroxidaselike reaction was the cascade one, as H2O2 was first catalytically decomposed and forms O2 and H2O. Then the O2 is enzymatically transformed to O2¯, which further oxide the TMB to oxTMB.

Table 1 C1s Binding Energy of Cit-IrNPs and Trisodium Citrate.

Cit-IrNPs Na3Cit

COH/CH2

COO−Ir3+(maybe)

285.9 285

287.86

COO−Na+

free COO(H)

286.31

288.58 288.08

4

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Fig. 2. Double-reciprocal plots of peroxidase-like activity Cit-IrNPs in HAc-NaAc buffer (0.1 M, pH 3.86) at room temperature. (A) The H2O2 concentration was fixed at 0.5 mM and TMB was varied. (B) TMB concentration was fixed at 0.5 mM and H2O2 was varied. (C) Effect of pH on the catalase-like activity Cit-IrNPs. (D) Doublereciprocal plots of catalase-like activity of Cit-IrNPs versus varying concentration of H2O2. Table 2 Kinetic Parameters of Catalase- and Peroxidase-like Activities of Cit-IrNPs and Other Reported Enzyme Mimic. enzyme

[E] (M)

substrate

Km (mM)

νmax (10−3 mM/s)

kcat (103 s−1)

ref

peroxidase activity Cit-IrNPs

∼3.4 × 10−7 ∼1.97 × 10−9

0.0906 0.27 0.02 266 0.03 18.02 0.103 174 0.434 3.7

1.7 1.5 0.108 0.385 0.17 0.81 0.256 0.189 0.1 0.871

0.5 0.44 0.055 0.196

this work

PVP-IrNPs

TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 TMB H2O2 H2O2 H2O2 H2O2

21.09 297 126

TA-IrNPs −9

Co3O4 NPs

2.53 × 10

HRP

2.5 × 10−11

catalase activity Cit-IrNPs PVP-IrNPs Ac-G9/PtNPs

[28] [16]

0.101 0.0747 0.4 0.348

[29] [29]

this work [28] [30]

[E] is the particle concentration, Km is the Michaelis constant, νmax is the maximal reaction velocity, and kcat is the catalytic constant, where kcat = νmax / [E].

calculated to be 0.287 mM and 0.587 μM/s, respectively. In addition, the experiments of TMB oxidation were performed in various atmosphere conditions (N2, O2, and air). The results proved that DO should be the only oxidant for the catalytic oxidation reactions (Fig. 4C). To optimize the pH for O2 activation, the absorbance changes of TMB were monitored at different pH. As can be seen from Fig. 4D, TMB can only be oxidized in the acidic environment and the optimal pH is about 3–4. When ascorbic acid (AA) was employed, however, a different phenomenon was observed. The catalytic activity of Cit-IrNPs increased gradually with the increase of pH up to 5. It should be noted

3.3. Oxidase-like activity of Cit-IrNPs Apart from the peroxidase-like activity, Cit-IrNPs also exhibit oxidase-like activity, which was manifested by the catalytic oxidation of TMB only in the presence of DO. The oxidase-like activity was investigated by performing steady-state kinetic analysis. After addition of Cit-IrNPs, the process of TMB oxidation was visually observable due to the formation of the blue colored products. As shown in Fig. 4A, a typical Michaelis-Menten behavior was followed in the TMB oxidation. Based on the data in Fig. 4B, the Km and νmax of Cit-IrNPs were 5

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Fig. 3. (A) The UV–vis absorbance spectra for the TMB oxidation with H2O2 over Cit-IrNPs in the presence of different scavengers. (B) ESR spectra of the samples after mixing DMPO with and without Cit-IrNPs in the present of H2O2.

To investigate the mechanism of the oxidase-like activity, various scavengers including benzoquinone, tryptophane and isopropyl alcohol were employed to evaluate what specific ROS are generated in the presence of DO and Cit-IrNPs. As shown in Fig. 5A, the oxidation of TMB was only suppressed when benzoquinone was added to the system, suggesting that O2¯ oxidation might be the pathway for the oxidase-like activity of Cit-IrNPs. To further confirm this mechanism, the DPBF

that the catalytic activity was still maintained for AA even in the basic condition. These results tend to confirm that the pH-dependent catalytic activity is mainly related to the substrate rather than the nanoparticles. Interestingly, we found TMB was mainly oxidized to the intermediate by DO (Fig. S10), suggesting the formation of a ROS with moderate oxidation ability, which is essential for selective oxidation of primary alcohols to aldehydes rather than carboxylic acids.

Fig. 4. (A) Steady state kinetic assay of oxidase-like activity of Cit-IrNPs in HAc-NaAc buffer (20 mM, pH 3.86) versus varying concentration of TMB at room temperature. (B) Double-reciprocal plots of oxidase-like activity of Cit-IrNPs. (C) Time-dependent absorbance changes at 652 nm of TMB over Cit-IrNPs in various gas environments at room temperature and pH 3.65. The reaction rate after bubbling with N2 for 20 min is greatly reduced. (D) Effects of pH on the catalytic activities of Cit-IrNPs for TMB and AA oxidation. 6

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Fig. 5. (A) Time-dependent absorbance changes at 652 nm for the TMB oxidation product over Cit-IrNPs in the presence of different scavengers. (B) Fluorescence spectrogram of DPBF solution with various concentrations of Cit-IrNPs. (C) Kinetic curves and CL intensity (inset) of the CL system under different conditions: (a) luminol + Cit-IrNPs; (b) luminol + Cit-IrNPs after bubbled N2 for 10 min; (c) luminol + Cit-IrNPs + benzoquinone. Experimental conditions: luminol, 0.01 M in 0.1 M NaOH; Cit-IrNPs, 0.3 mg/mL; benzoquinone, 0.1 mM. (D) ESR spectra of the samples after mixing DMPO with and without Cit-IrNPs (◼corresponded to oxDMPO and ●corresponded to DMPO/OOH), and simulated (sim) total oxDMPO spectrum and simulated DMPO/·OOH spectrum.

fluorescence quenching method was conducted for the detection of O2¯ [37]. As expected, the fluorescence intensity decreased dramatically upon the addition of Cit-IrNPs into the DPBF solution (Fig. 5B). These results suggest that O2¯ plays the key role during the Cit-IrNPs catalyzed oxidation reaction. Chemiluminescence (CL) is a very useful method for the ROS detection [38]. As a widely used CL reagent, luminol can be oxidized by ROS to produce a CL reaction. Fig. 5C displays the CL kinetic curves obtained from different conditions. As can be seen, a strong CL was only observed in the presence of Cit-IrNPs and DO. After bubbling the solution by N2 for 10 min, the CL intensity decreased accordingly, suggesting the CL is due to the catalytic ROS generation by Cit-IrNPs rather than direct reaction between luminol and DO or CitIrNPs. Furthermore, the addition of benzoquinone substantially quenched the CL signal. To verify the possible involvement of other ROS species (1O2 or OH), the experiments were conducted with 1O2 or ·OH indicators. TPA, for example is a good indicator of OH. Fig. S11 shows ·OH was only detected in the contrast experiment (i.e., in the photolytic decomposition of H2O2), thus we rule out the production of OH in our catalytic system. Similarly the production of 1O2 can be sensitively detected by monitoring its intrinsic light emission [39]. In present work, no CL signal was detected when Cit-IrNPs was continuously merged with the oxygen saturated solution in a two-line flow system, hence we can also exclude the generation of 1O2 (Data was not shown). The mechanism of Cit-IrNPs as oxidase mimetic was verified using the ESR method. Herein, DMPO was employed as the spin trap for the analysis of O2¯. As illustrated in Fig. 5D, no spins were captured for pure

water in the presence of DMPO. However, the radical signals with an intensity ratio of 1:1:1:1 were substantially intensified in the present of Cit-IrNPs, confirming the generation of O2¯. In between the characteristic peaks of DMPO/·OOH, several unexpected peaks with an intensity ratio of 1:1:1 are present, which could be attributable to the nitro product through the opening of pyrroline ring by O2/O2¯ absorbed on the Cit-IrNPs surface [40] (Scheme S1). With seven electrons in 5d orbital, metal Ir can adsorb oxygen molecule and catalyze the reactions of oxidation. In present work, the high-index facets on Cit-IrNPs would reinforce such effect. Based on above experiment observations and relevant literature reports, DFT calculations were also conducted trying to have a theoretical insight into the catalytic process. By comparing the d-band center values of the four crystal faces of Cit-IrNPs to Fermi energy level in Fig. S13, it revealed that the oxygen molecule was, mostly, physisorbed on the stoichiometric Ir (220) surface. Moreover, according to the calculation, O2 can be adsorbed on the Ir (220) crystal plane, forming O-Ir bond (Scheme S2), and the Bader charge of O2 species adsorption is −0.79 e. This Bader charge value indicates that the O2 species adsorbing on the Ir (220) crystal plane contribute to O2¯ [41], which proves the formation of superoxide species in theory. Capping agents are usually introduced to improve the stability of nanoparticles, however, different stabilizers may have different influence on the catalytic activity. Herein we adopted small molecule sodium citrate as the stabilizer. The contrast experiments were carried out by using IrNPs prepared with other stabilizers such as tannic acid (TAIrNPs) [16] and PVP (PVP-IrNPs) [28]. As shown in Fig. S12, when TMB 7

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was chosen as the substance, only the small molecule capped nanoparticles Cit-IrNPs exhibit super oxidase-like activity compared with those of large molecule stabilized nanoparticles. According to the DFT calculation, the Ir (220) crystal plane on Cit-IrNPs surface should play the key role, as TA-IrNPs and PVP-IrNPs have shown no Ir (220) crystal plane on the surface. Furthermore, the superficial Ir atoms of IrNPs should conduce to the catalytic activity as the catalytic center. Thus, the surface modification, especially the thickness of coating, influences the catalytic abilities of nanozymes [42]. With the increase of molecular weight of capping agents, the catalytic centers of nanozymes may be shielded, leading to a decrease of collision probability between the substance molecules and catalytic centers. In contrast, capping with small molecules (e.g., citrate) is more permeable, which would promote the transfer of substrate molecules to the catalytic centers when compared with that capped with large molecules (e.g., TA, PVP).

Table 3 Oxidation of various aromatic alcohols to corresponding aldehydes over CitIrNPs.a.

Time (h)

Conversionb (%)

Selectivelyc (%)

1

10

94

98

2

12

95

98

3

5

92

97

4

6

94

98

5

6

93

95

6

10

95

96

7

14

92

97

8

12

94

96

Enty

Substrate

3.4. Aerobic alcohol oxidation with enzyme mimic Cit-IrNPs Based on their effective production of O2¯ from DO, Cit-IrNPs are exploited for selective alcohol oxidation reactions. As one of the most widely studied substrates, benzyl alcohol was selected here as the model compounds [43]. In the presence of Cit-IrNPs, benzyl alcohol was completely converted to benzaldehyde at room temperature in water. The other byproducts were not detected by GC, indicating a nearly 100 % selectivity. To gain a deeper insight into the catalysis, time-activity profile for the oxidation was performed with benzyl alcohol (Fig. S14). The conversion rate of benzyl alcohol increased with the increase of reaction time, and a complete conversion can be reached at about 10 h. Moreover, with the increase of reaction temperature from 25 to 45 °C, the reaction time for the complete conversion of benzyl alcohol to benzaldehyde shorten obviously. However, further increase in temperature cannot shorten the time for complete conversion. A likely reason is that with the increase of temperature, the DO content would decrease accordingly. Fig. 6A shows that the variation of pH has little influence on the catalytic activity of Cit-IrNPs in alcohol oxidation, suggesting a good stability of the Cit-IrNPs catalyst. The recycling stability of Cit-IrNPs was also tested here. As shown in Fig. 6B, the catalytic activity maintained pretty well even after 6 cycles. The sample of recovered catalyst Cit-IrNPs was studied using TEM and XPS. No obvious change of CitIrNPs was observed after the reaction (Fig. S15), suggesting the excellent recyclability and stability of Cit-IrNPs. Fig. S16 shows the XPS spectra of Cit-IrNPs remain unchanged after the reaction, indicating the Cit-IrNPs still kept high catalytic activity and can be used as catalyst with good conversion rate. Satisfied with the excellent catalytic

a Reaction conditions: catalyst (9 mg Cit-IrNPs), alcohol (0.1 mmol), 3 mL of H2O, O2 (1 atm), 25 °C. b Conversion (%) of aldehyde was analyzed by GC, and n-dodecane was used as the internal standard. c Selectivity = yield/conversion, mol %.

performance of Cit-IrNPs in the oxidation of benzyl alcohol, we proceeded to investigate the broader utility of the catalyst for the oxidation of primary alcohols with a wide range of substrates under the same reaction condition. It can be seen in Table 3, efficient and selective oxidation was achieved with a high yield of the aldehydes for all the tested benzyl alcohol derivatives. In particular, the conversion rate for benzyl alcohol oxidation with electron-withdrawing groups is substantially faster than that with electron donating substituents, which agrees well with previous report [44]. To prove the multi-enzyme activities of Cit-IrNPs, the experiments with H2O2 as sole oxidizing agent

Fig. 6. Oxidations of benzyl alcohol (A) at different pH. (B) with 6 cycles. Reaction conditions: alcohol substrate (0.1 mmol), 3 mL H2O and 9 mg Cit-IrNPs, reaction time 12 h, reaction temperature 30 °C. 8

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Scheme 1. Proposed mechanism of the oxidation of benzyl alcohol to benzaldehyde.

intriguing multifunctional nanozyme properties of Cit-IrNPs, which we believe could inspire further studies in vitro/vivo monitoring and catalytic therapy in the future.

were performed. Evidently, Cit-IrNPs can catalyze the oxidation of these substrates in the presence of H2O2 (Fig. S17). In the proposed mechanism, O2 was first activated on Cit-IrNPs to form O2¯ absorbed on the surface of Cit-IrNPs while the electron was transferred to the oxygen molecule from the Ir (0). A Irδ+-alkoxide species was afforded through the oxidative addition of an OeH bond from alcohol to the Irδ+. Then, a Irδ+-hydride species and the corresponding aldehyde would be produced undergoing a β-hydride elimination. Finally, the Irδ+-hydride species could reductively eliminate O2¯ to regenerate the Ir (0) species, along with the formation of H2O2. As H2O2 could be decomposed to O2 and H2O by Cit-IrNPs, the nanocatalysts are thereby able to activate O2 molecule from H2O2 for O2¯ generation in the next cycle (Scheme 1). Thus, in theory the oxidation reaction would naturally proceed as long as O2 is available. In this process, O2¯ plays an important role to remove adsorbed H atoms on Ir surface, which will allow the smooth proceeding of the aerobic oxidation and obtaining a high product yield. The mild oxidation ability of O2¯ is critical for the selective conversion of alcohol [1]. The presence of aromatic alcohols may also contribute to the high selectivity, as it was reported that benzyl alcohol can effectively intercept the generation of acylperoxy radical, which plays the key role in the oxidation of benzaldehyde to benzoic acid [45]. Thus a highly active and reusable catalyst was obtained for the aerobic oxidation of a wide variety of benzylic alcohols in aqueous media without need of extra energy input.

Author contributions statement The first author, Mr. Guangxia Jin carried out most of the experiments and characterizations. He also contributed to write the first draft of the manuscript. Mrs. Guangxia Ran contributed to the ESR test and the result discussion. Mr. Jie Liu and Dr. Bing Liu are mainly involved in the DFT calculations. Dr Chan Wang and Dr. Wenxiu Gu contributed to the experiment design and result discussions. Prof. Qijun Song supervised the whole project and contributed to the final revision of the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work is supported by the Chinese National first-class discipline program of Food Science and Technology (JUFSTR20180301), the Fundamental Research Funds for the Central Universities, China (NO. JUSRP11708) and the 111 Project, China (B13025).

4. Conclusions This work investigated the multi-enzyme activities of Cit-IrNPs with high-index facets. The characterization results obtained from TEM, ATR-IR, XRD and XPS provide a blueprint of the conformation of citrate layers on Cit-IrNPs surfaces. The Cit-IrNPs can be facilely prepared in mild condition and short time, and their catalytic activities were substantially superior to those reported nanoenzymes as the catalytic reactions can proceed in ambient condition. The Cit-IrNPs could not only catalyze oxygen and hydrogen peroxide reduction but also produce oxygen through the dismutation decomposition of hydrogen peroxide. The TMB oxidation by DO in room temperature suggests that the CitIrNPs can also be considered as a new kind of oxidase-mimics. Furthermore, a nearly 100 % selectivity was achieved in the oxidation of aromatic alcohols to aldehyde products by using Cit-IrNPs as the aerobic catalyst. The results presented in this work demonstrated the

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