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Heterogeneous Nanostructure Design Based on the Epitaxial Growth of Spongy MoSx on 2D Co(OH)2 Nanoflakes for Triple-enzyme Mimetic Activity: Experimental and DFT Studies on the Dramatic Activation Mechanism Yongqi Ding, Guo Wang, Fengzhan Sun, and Yuqing Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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ACS Applied Materials & Interfaces

Heterogeneous Nanostructure Design Based on the Epitaxial Growth of Spongy MoSx on 2D Co(OH)2 Nanoflakes for Triple-enzyme Mimetic Activity: Experimental and DFT Studies on the Dramatic Activation Mechanism

Yongqi Ding1, Guo Wang1, Fengzhan Sun1, Yuqing Lin* Department of Chemistry, Capital Normal University, Beijing 100048, China

*Corresponding author 1

The three authors contributed equally to this paper.

Tel.: +86 1068903047; Fax: +86 1068903047 E-mail address: [email protected] (Y. Lin).

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Abstract In this study, we present a three-in-one catalytic platform for intrinsic oxidase-, peroxidase-, and catalase-like activity, which is enabled by epitaxial growth of the MoSx nanosponge on 2D Co(OH)2 nanoflakes (2D Co(OH)2 NFs) (CoMo hybrids). First, the 2D Co(OH)2 NFs are stripped from hierarchical three-dimensional Co(OH)2 nanoflowers which are synthesized in an eco-friendly way via one-step surfactant-free chemical route. Next, the porous MoSx nanosponge is decorated on the 2D Co(OH)2 NFs’s surface using a solvothermal process forming heterogeneous nanostructured CoMo hybrids. Finally, due to the host-guest interaction, i.e. after the epitaxial growth of spongy MoSx on 2D Co(OH)2 NFs, the heterogeneous nanostructure of CoMo hybrids exhibits unpredictable triple-enzyme mimetic activity simultaneously. The mechanisms of the oxidase-like properties is investigated by density functional theory (DFT) calculations, and it is discovered that a simple reaction/dissociation of O2 absorbed on Co-Mo thin films can explain the enhanced oxidase-like activity of the CoMo hybrids. In addition, the CoMo hybrids is also reproducible, stable, and reusable, i.e. after 10-cycle uses, >90% mimic enzyme activity of the CoMo hybrids is still maintained. The oxidase-like activity of the CoMo hybrids enable it to oxidize 3, 3′, 5, 5′-tetramethylbenzidine (TMB) producing the blue oxTMB, which can selectively oxidize ascorbic acid (AA) and pave a new avenue for colorimetric sensing of AA. The proposed colorimetric strategy has been successfully utilized to measure AA in rat brain during the cerebral calm/ischemia process. Our findings provide in-depth insight into the future research of enzyme-like activities and might help to elucidate the mechanism and understand the role of epitaxial growth on the properties and application of hybrid nanostructures. Keywords: CoMo hybrids, Epitaxial growth, Triple-enzyme mimetic activity, DFT, Colorimetry, Ascorbic acid detection


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1. Introduction Natural enzymes as biological catalysts are highly efficient and specific for catalyzing variety of important reactions. However, natural enzymes have inherent disadvantages including low stability due to easy denaturation in extreme environment, difficulty in preparation, and digestion by proteases.1 Therefore, nanomaterials with artificial enzymes properties (nanozymes) have attracted significant research interest because of their advantages over natural enzymes, including the following: simple preparation, storage, and low cost.2-3 In particular, many nanomaterials can catalyze some redox-like type reactions and exhibit great activity for oxidase-like,4-5 peroxidase-like,6-8 catalase-like,9-10 or superoxide dismutase-like (SOD).11-12 Generally, these materials can be divided into two types based on their chemical composition: carbon-, and metal-based nanomaterials.13 Compared to the previous types, considerable effort has been devoted to metal nanomaterials, with unique electronic and a larger variety of enzyme-like characteristics.4, 6, 11 Recently, the enzyme-mimetic properties of metal nanomaterials have become an area of increasing interest and is widely used in many fields such as biosensing, cancer diagnosis and treatment, neuroprotection, and contaminant removal.5,

7, 14

Several metal-based nanomaterials have been found possessing enzyme-like activity. For instance, Perrett et al. first reported that Fe3O4 nanoparticles exhibit intrinsic peroxidase-like activity similar to horseradish peroxidase (HRP).15 Mugesh et al. showed that V2O5 nanowires possess antioxidant enzyme glutathione peroxidase (GPx-like) mimic activity and can tune the oxidative stress in side the cell by catalytically reducing H2O2 in the presence of glutathione (GSH).16 Cheng et al. developed a method based on the oxidase-like activity of nanoceria (CeO2) for fast and sensitive detection of acetylcholine (AChE) and urease.17 Although the nanomaterials mentioned above possess intrinsic enzyme-like properties and are useful for the detection of small biological molecules, however, these nanomaterials have a relatively fewer enzyme-like activity that limits their further applications in physiological and pathological processes. Consequently, it is fundamentally important



to investigate catalyst with intrinsically triple/multi-enzyme mimic properties. Previous studies have revealed that a few nanostructured materials have been shown to mimic the activity of peroxidases, oxidase and catalase simultaneously. For example, Dong et al. demonstrated that nickel-palladium hollow nanoparticles possess triple-enzyme mimetic activity and applied it for colorimetric biosensing of glucose.18 Wang et al. developed Co3O4 nanotubes with triple-enzyme mimetic activity, in which the oxidase-like activity was used for the detection of GSH.19 Li also et al. found the V6O13 nanotextiles with interlaced network structure exhibiting triple-enzyme mimetic activity.20 However, the activity of these catalysts needs to be enhanced and the further mechanism needs to be unrevealed. Therefore, there is an urgent requirement to develop more novel nanomaterials with high catalytic properties and explore the mechanism at the same time. Hybrid nanostructures are composed of two or more distinctive components, in which at least one dimension is in the nanometer scale.21-22 The rational design and controlled synthesis of hybrid nanostructures is very important for the tuning of their performance and function. Epitaxial growth is a highly-anticipated method for the synthesis of hybrid with the desired structure, crystalline phase, exposed face and/or interface. Two-dimensional lateral heterostructures can be designed by epitaxial growth of nanoplatelets from the edge sites of seed nanoflakes.23 It is also worth noticing that cobalt hydroxide nanoflakes (Co(OH)2 NFs) have promising mimic enzyme catalytic activity when the surface has a stoichiometric layered configuration.24 The high surface-to-volume ratio of Co(OH)2 NFs is beneficial for hybridization with other substances based on the conventional host-guest interactions and can maximize carrier surface accessibility and metallic catalytic activity. However, the catalytic kinetics and the mass-transfer properties of Co(OH)2 NFs are still poor. To solve the problem, the useful method of mixing transition metal hybrids can significantly affect its catalytic activity, but more and detailed studies involved the mechanism are urgently needed. Amorphous nanomaterials are disordered and have wide prospects in catalysis and energy storage.25 Amorphous MoSx allows its defect-rich sites to absorb reactants and reduce energy barriers during structural


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rearrangements and transitions processes and thus enhance many catalytic properties.26-27 Consequently, the amorphous MoSx may positively affect the mimic enzyme activity of the Co(OH)2 NFs when introducing the MoSx on the surface of the Co(OH)2 NFs. In this work, we decorated Co(OH)2 NFs with amorphous MoSx by solvothermal method to prepare a high-efficiency triple-enzyme mimetic catalysts with layered structure, i.e. CoMo hybrids. The lamellar structure has a relatively uniform thickness, which ensures fast electron transfer and diffusion pathways of reactants and/or products. This may result in excellent enzyme-mimicking catalytic properties.28 The triple enzyme-like activities of the generic CoMo hybrids were studied. The CoMo hybrids had peroxidase-, oxidase- and catalase-like activities (Scheme 1). The triple enzyme-like catalytic activities of CoMo hybrids have not yet been reported. In detail, CoMo hybrids catalyze the rapid oxidation of TMB both in the presence and absence of H2O2 exhibiting the peroxidase- and oxidase-like activity, respectively. Moreover, we also used density functional theory (DFT) calculations to demonstrate that the dissociative adsorption of O2 on the Co-Mo thin film to form single-atomic O adatoms is the necessary endowing the films oxidase-like activity. As a catalase mimic, CoMo hybrids catalyze the decomposition reaction of H2O2 into oxygen and water. The steady-state kinetic assay of CoMo hybrids with peroxidase- and oxidase-like activities was also discussed. In addition, based on the oxidase-like activity of CoMo hybrids, a facile and sensitive colorimetric method for detecting cerebral AA in the brain was established for the first time.



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Scheme 1. Schematic presentation of the fabrication procedure and triple-enzyme mimetic activity CoMo hybrids. 2. Experimental Section 2.1 Chemicals and Materials. Ascorbic acid (AA), glutamic (Glu), dopamine (DA), 5-hydroxytryptamine (5-HT), glutathione (GSH), glycine (Gly), Alanine (Ala), uric acid (UA), histidine (His), 3,4-dihydroxyphenylacetic acid (DOPAC), cysteine (Cys), ethanolamine (C2H7NO), ammonium tetrathiomolybdate (NH4)2MoS4, N, N-dimethylformamide (DMF),

citric

acid

(CA),

3,

3ˊ,

5,

2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic

5ˊ-Tetramethylbenzidine acid

(ABTS),

(TMB), and

2, 1,

2-Diaminobenzene (OPD) were purchased from Sigma (Shanghai, China). Hydrogen peroxide (H2O2), hydrazine hydrate (N2H4·H2O), NaCl, NiCl2·6H2O, KCl, CuCl2·2H2O,

CaCl2·2H2O,

MgCl2·2H2O,

Co(CH3COO)2·4H2O,

FeCl2·4H2O,

FeCl3·6H2O, MnSO4·H2O, Na2S·9H2O, were purchased from Beijing Chemical Reagent Company. 2.2 Apparatus and Characterizations. Absorption spectra were recorded using UV-2550 UV-vis spectrophotometer (Shimadzu, Japan). Scanning electron microscopy (SEM), Mapping images, and


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energy-dispersive spectroscopy (EDX) analyses were recorded on a Hitch SU8010 SEM. Transmission electron micrographs (TEM, JEM-2100F), high-resolution transmission electron microscopy (HRTEM, 200 kV) and selected-area electron diffraction (SAED) was applied for the morphological investigation of samples. X-ray powder diffraction (XRD) patterns were recorded from a Bruker D8 ADVANCE (Germany) X-ray diffractometer with Cu Kα radiation. The X-ray photoelectron spectroscopy (XPS) measurement was performed on an ESCALAB 250 X-ray photoelectron spectrometer. Raman spectroscopy was performed by a RENISHAW instrument. Fourier transform infrared spectroscopy (FT-IR) spectra were measured using an Equinox 55 spectrophotometer (BRUKER, Germany). The O2 generation from H2O2 decomposition was recorded by a VZ8403BZ portable dissolved oxygen meter (Shanghai Hengxin Technology Co., Ltd. China). 2.3 Synthesis of Co(OH)2 NFs. First, cobalt(II) acetate tetrahydrate (Co(CH3COO)2·4H2O, 1.2455 g), and ethanolamine (C2H7NO, 1 mL) was added to H2O (50 mL); then, the mixed solution was continuously stirred for 5 min to form a homogeneous solution. Finally, the mixed solution was sealed and allowed to stand for 12 h at room temperature.33 Subsequently, the pink Co(OH)2 NFs powder was separated from the resulting mixture solution and wasted three times with ethanol, and dried in an oven (60 °C). 2.4 Preparation of the CoMo Hybrids. Co(OH)2 NFs (15 mg) and ammonium tetrathiomolybdate ((NH4)2MoS4, 5.2 mg) were added into DMF (10 mL) and sonicated for 90 min.26 Subsequently, 50 µL of hydrazine hydrate (N2H4·H2O, 80 wt%) was dispersed into the brownish black mixture solution and then stirred vigorously for 30 min. The resulting solution was transferred into a 25 mL Teflon autoclave, heated for 15 h at 200 °C in an oven, and cooled down naturally. The brownish black CoMo hybrids powder was obtained by centrifugation at 7000 rpm, wasted five times with deionized water and ethanol alternately and dried in an oven (60 °C). 2.5 Electrochemistry Measurements. Electrochemical data were recorded by using the CHI 760E electrochemical



workstation of a three-electrode configuration (Shanghai Chenhua Instrument Co. Ltd.). The modified glassy carbon electrodes (GCE, 3.0 mm in diameter), platinum wire, and Ag/AgCl (KCl-saturated) were used as the working electrode, auxiliary electrode, and reference electrode, respectively. First, the GCE were polished with alumina slurry, rinsed and sonicated in deionized water. Then, the GCE was dried by blowing air. Subsequently, CoMo hybrids slurry was synthesized by mixing 2.5 mg of CoMo hybrids powder, 730 µL of deionized water, 250 µL of ethanol, and 20 µL of Nafion solution and then ultrasonication for 30 min at room temperature. 10 µL of the resulting suspension was dropped onto a polished GCE and dried under a halogen lamp. 2.6 Steady-State Kinetic Analysis of CoMo Hybrids as Peroxidase/Oxidase Mimetics. Kinetic experiments were implemented in a reaction with volume of 3.0 mL mixture solution (pH 4.0) containing 1 mL 100 µg mL-1 CoMo hybrids, 1 mL 10 mM H2O2, and 1 mL 2.5 mM TMB. The mixture solutions were incubated for 1 min and the absorbance change at 652 nm were monitored using a UV-2550 UV-vis spectrophotometer. The Michaelis-Menten constant was calculated by using a Lineweaver-Burk plot: 1/V = Km/Vmax (1/[S] + 1/Km), where V is the initial velocity, Vmax is the maximal reaction velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant. 2.7 Colorimetric detection of Cerebral AA based on CoMo Hybrids Oxidase Mimic. Surgery for in vivo microdialysis was performed as reported previously.29 Briefly, the male Sprague-Dawley rats (350-400 g) were purchased from the Peking University School of Medicine. Rats with guide cannula were anesthetized with intraperitoneal injection of 10% chloral hydrate (345 mg/kg, i.p.). The probe was inserted into the left striatum and perfused with a microdialysis probe (BAS; 4 mm in dialysis length, 0.24 mm in diameter) at 3 µL min-1 for 90 min and then the microdialysate for AA sensing was collected. To evaluate the sensitivity toward AA based on CoMo hybrids oxidase mimic, a typical colorimetric analysis was performed


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as follows: (1) 1 mL aliquot of CoMo hybrids (100 µg mL-1) and 1 mL TMB (2.5 mM) were incubated (pH 4.0) at room temperature for 5 min. (2) 0.5 mL of different concentrations of AA solution were added into the above mixture solution, and then, the supernatant was subjected to absorbance spectroscopy measurement. The sensing of cerebral AA of rats were performed in a manner similar to that described above.

3. Results and Discussion 3.1. Morphology and Structural Characterization of CoMo Hybrids. The surface morphology and size characteristics of the CoMo hybrids were characterized by SEM, TEM, HRTEM, and SAED (Figure 1). Figure 1A and B show the SEM images of CoMo hybrids at different magnifications. The CoMo hybrids were dispersed nicely into lamellar structures with smaller size nanoparticles. With intense ultrasonic assistance, the large stacked flakes were broken down into relatively dispersed smaller flakes with a greater exposed surface area. This step greatly increases the number of catalytic active sites and promotes electron conduction for TMB. The thickness of the Co(OH)2 NFs is 60 nm from SEM (Inset of Figure S1A in the Supporting Information), while the thickness of the CoMo hybrids is approximately 92.5 nm in Figure 1B. Compared to the thickness of Co(OH)2 NFs and CoMo hybrids, we propose that the nanosponge-like MoSx (Figure S1D-F) is successfully encapsulated on Co(OH)2 NFs (Figure S1A-C) through a three-step process: 1) The Co(OH)2 nanoflakes were synthesized which possess catalytically active octahedral MO6 structures; 2) Individual nanoflakes were stripped from the stacked Co(OH)2 nanoflakes by using intense ultrasonic technology and broken them down into smaller nanoflakes as well as uniformly attach MoS42- ions on the nanoflakes; 3) MoS42- ions were decomposed into amorphous MoSx on the Co(OH)2 nanoflakes by hydrothermal process.26 Figure 1C clearly reveals that the only lattice spacing of the CoMo hybrids is 0.27 nm and could be indexed to the (100) plane of Co(OH)2 NFs. The only (100) plane of the Co(OH)2 NFs is also shown in the SAED image (Figure 1D). These HRTEM and SAED data show that Co(OH)2 NFs maintains their crystal structure in the hybrid, whereas the MoSx has an amorphous structure.



Figure 1. SEM images of CoMo hybrids with low (A) and high (B) magnifications; (C) TEM image of CoMo hybrids (Inset: HRTEM image of CoMo hybrids); (D) SAED pattern of CoMo hybrids.

Figure 2A illustrates the typical X-ray diffraction (XRD) spectra of the Co(OH)2 NFs and CoMo hybrids powder samples. A series of typical peaks at 18.8º, 32.3º, 37.8º, and 51.05º are corresponded to the (001), (100), (011), and (102) planes of Co(OH)2 (PDF#74-1057).30 The Co(OH)2 NFs display no obvious diffraction peaks after being hybridized with poorly crystalline MoSx. Figure 2B shows the EDX spectrum and elemental mappings (Figure S3) of the CoMo hybrids. These results show the presence of O, S, Co and Mo elements.. Two elements (Mo and S) coexist on the nanoflakes again suggesting that MoSx is successfully developed on the surface of Co(OH)2 NFs. The disordered structure of the MoSx nanosponge makes it possible to reduce the energy barrier during possible structural rearrangements and electron transformations.25 Therefore, the growth of the MoSx nanosponge onto Co(OH)2 NFs


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to form heterogeneous nanostructure may accelerate the electron transport capability for the mimic enzyme catalyzed kinetic activities.

Figure 2. (A) XRD patterns, (B) EDX spectra of CoMo hybrids, (C) FT-IR spectra, and (D) Raman spectra.

The surface structures and composition of Co(OH)2 NFs and CoMo hybrids were further investigated by the Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy. The FT-IR spectrum of the Co(OH)2 NFs is shown in Figure 2Ca; the peaks at ~ 3627 and 1619 cm-1 are attributed to the H-O-H bending and O-H stretching vibrations, respectively, indicating the presence of surface-absorbed water and hydrogen-bound hydroxyl groups (-OH).31-32 The band at ~ 488 cm-1 is assigned to the Co-O stretching and Co-O-H bending vibrations.33 The broad band of CoMo hybrids at ~ 3345 cm-1 corresponds to H-bound hydroxyl groups (Figure 2Cb). The peak at ~ 1640 cm-1 is assigned to O-H stretching vibrations due to surface-absorbed water in the CoMo hybrids. The peak at ~ 1132 cm-1 is derived from the asymmetric



stretching vibrations of the surface tetrahedral SO4.34 The peak at ~ 855 cm-1 is attributed to the Mo-O-Mo stretching vibrations of bridging oxygen,35 which indicates that MoS2 produced is oxidized during the hydrothermal process, which agree well with the results obtained from the FT-IR spectrum of pure MoSx (Figure S2B). The new peaks at ~ 614 cm-1 corresponds to the Co-O2- complex in the oxide.36 Figure 2D shows the Raman spectrum of the Co(OH)2 (black curve) and CoMo (red curve) hybrid powder sample. The peaks at ~ 259, 432, 503, and 566 cm-1 correspond to Eg(T), A1g(T), A2u(R), and Eg(R) modes, respectively.37-38 The band at ~ 335 cm-1 is attributed to the Mo-S vibrations and this result also demonstrates the existence of bridging S22- ligands. The bands at ~ 468 cm-1, ~ 672 cm-1 and ~ 933 cm-1 correspond to the Mo-S vibrations in a lower symmetry environment, the mode of the Mo-O-Mo and the O=Mo=O, respectively.39 The partial oxidation of the resulting Mo(IV) is verified through the Raman measurement results of CoMo hybrids. All of the above results confirm the development of MoSx on the Co(OH)2 nanoflakes, i.e., the successful preparation of CoMo hybrids. The composition and elemental chemical states of CoMo hybrids were studied using the X-ray photoelectron spectroscopy (XPS) measurements in Figure 3. The survey spectra (Figure 3A) indicates that the as-prepared materials mainly contain O, S, Mo and Co elements and confirmed the development of MoSx on Co(OH)2 NFs. High resolution XPS spectra of CoMo hybrids were collected to further confirm the Co 2p, Mo 3d, and S 2p (Figure 3B, C and D, respectively). The binding energy peaks located at ~ 797.41 and 781.50 eV correspond to Co 2p1/2 and 2p3/2 spin-orbit splitting (Figure 2B). This result mainly indicates the existence of Co2+ oxidation state.40 Compared with pure Co(OH)2 NFs in Figure S4, the electronic binding energy of the Co 2p spectrum of the CoMo hybrids clearly shows a downward shift of ~0.75 eV. In addition, the relatively lower oxidation state of cobalt atoms is mainly due to the electron transfer behavior from MoSx to Co(OH)2 nanoflakes, thus this difference in the electron binding energy is proposed.38


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Figure 3. Survey scan of the CoMo hybrids (A); High-resolution Co 2p (B), Mo 3d (C), and S 2p (D) XPS spectra of CoMo hybrids.

The high resolution spectrum of Mo 3d shows a doublet whose the peaks are at ~ 232.59 and 235.72 eV, which corresponds to Mo 3d5/2 and 3d3/2, respectively (Figure 3C). The binding energy and the splitting width (∆Mo 3d = 3.13 eV) agree well with Mo6+.41 The peaks at ~ 228.4 and 231.2 eV correspond to the Mo(IV) 3d5/2 and 3d3/2.26 Furthermore, the existence of Mo(IV) also indicates that some MoS2 may be present in MoSx. Figure 3D shows that the lower binding energy peaks at ~ 162.14 and 163.36 eV are due to S 2p3/2 and 2p1/2.42 The MoSx nanostructures contain the enriched unsaturated sites and have been confirmed by the fitting area of the S atom. they are consistent with the amorphous properties, and these unsaturated sites can be used as an active region for the absorption of the reactants. These results confirm that nanoparticles grown on the surface of Co(OH)2 NFs were amorphous MoSx, i.e., the CoMo hybrids were successfully fabricated.



3.2. The Triple-enzyme Mimetic Activity of CoMo Hybrids. To demonstrate the enzyme mimetic catalytic activity of CoMo hybrids, a series of experiments were carried out. In the presence of H2O2, the CoMo hybrids could catalyze the oxidation of the typical peroxidase substrates TMB, ABTS, and OPD which were blue, green, and yellow, respectively (Figure 4A). The results suggest that CoMo hybrids possessed peroxidase-like activity. Meanwhile, in the absence of H2O2, the CoMo hybrids could also catalyze the direct oxidation of TMB to produce oxTMB, indicating intrinsic oxidase-like activity of CoMo hybrids (Figure 4B).

Figure 4. (A) CoMo hybrids catalyzing the oxidation of various substrates to produce different colors in the presence of H2O2: ABTS, OPD, and TMB respectively. Inset: the corresponding photograph of different substances. (B) Effect of O2 concentration on the direct oxidation of TMB by CoMo hybrids without H2O2. Experimental conditions: 1 mL 2.5 mM TMB and 1 mL 100 µg mL-1 CoMo hybrids solutions were incubated (pH 4.0) for 5 min. The TMB and CoMo hybrids solution were bubbled with N2 or O2 for 30 min, respectively. Inset: the corresponding photograph of different reaction conditions. (C) Effect of the CoMo hybrids concentration on the O2


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generation from H2O2 decomposition. Experimental conditions: 50 mM H2O2 and different concentrations (0, 10, 25, 50, 70, and 100 µg mL-1) of CoMo hybrids were incubated (pH 4.0). (D) The effect of CoMo hybrids concentration on the formation of the hydroxyl radical with terephthalic acid as a fluorescent probe (concentration of CoMo hybrids: 0, 10, 25, 50, 100, and 200 µg mL-1).

To investigate the oxidation of TMB catalyzed by CoMo hybrids with oxidase-like activity, different concentrations of oxidizing agent (i.e. dissolved oxygen) in the TMB-O2-CoMo hybrids system were explored. Compared to the bubbling of N2 into the system, the absorbance value of the oxTMB was significantly increased after saturation with O2. Increasing the concentration of oxygen can enhance the oxidase-like activity of CoMo hybrids. The electrochemical polarization method also demonstrates the oxidase-like activity of CoMo hybrids.43 Figure S5 shows that the modified GCE exhibited a relatively more negative balance potential and higher current density than the bare GCE. The modified GC is more likely to react with TMB than the bare electrode further indicating that TMB was oxidized by the CoMo hybrids even in the absence of H2O2.44 Meanwhile, the CoMo hybrids also exhibit catalase-like activity that catalyzes the decomposition of H2O2 to generate oxygen. To verify the catalase-like activity of the CoMo hybrids, the oxygen generated from the decomposition of H2O2 via the CoMo hybrids is measured by a dissolved oxygen meter in concentration-dependent manner. Figure 4C shows that the oxygen concentration increased with increasing CoMo hybrids concentrations. Furthermore, the electrocatalytic behaviors of the bare and CoMo hybrids-modified GC towards the electrochemical reduction of H2O2 were also investigated. As shown in Figure S6A, versus the bare GC, the CoMo hybrid-modified GC has an obvious reduction of current in the presence of H2O2. Figure S6B shows that the reduction current response increased gradually and reached the steady-state by adding an aliquot of H2O2 at -0.6 V. These results indicated that CoMo hybrids have a great electrocatalytic activity of reducing H2O2. In previous literature, the terephthalic acid (TA) could easily react with ·OH from the



decomposition of H2O2 to form a highly fluorescent 2-hydroxyterephthalic acid (Figure S7).45 Figure 4D shows that the fluorescence intensity gradually increases with increasing CoMo hybrids concentrations (from bottom to top: 0, 10, 25, 50, 100, and 200 µg mL-1). This indicates the increasing amount of ·OH generated.46 The results further suggest that the CoMo hybrids have the catalytic ability to decompose H2O2. This verified the catalytic-like activity of the nanohybrids. To evaluate the role of each constituent material in the triple-enzyme mimetic activity, a series of control experiments were conducted as shown in Figure 5. The MoSx could not catalyze the oxidation of TMB in the presence (Figure 5A) or absence (Figure 5B) of H2O2, and the MoSx could hardly catalyze the decomposition of H2O2 (Figure 5C). However, though the Co(OH)2 NFs had a triple-enzyme mimetic activity, each type of enzyme mimetic activity is lower versus the CoMo hybrids, even under the same reaction conditions. These results indicate that the triple-enzyme mimetic activity of CoMo hybrids is mainly from the Co(OH)2 NFs. Additionally, the MoSx also had an indispensable effect on improving the enzyme-like behaviors, even if there was no visible triple-enzyme mimetic catalytic activity for MoSx itself.47-48 The CoMo hybrids had a secondary lamellar nanostructure after nanosponge MoSx growth, which offer a large surface area with active sites for the catalytic reaction and could improve probability of the collisions of the active molecules when TMB and H2O2 were trapped in an uneven lamellar structure, resulting in a high enzyme-like catalytic activity of the CoMo hybrids. As shown in Figure S8, the Co/Mo molar ratio was screened, and the results indicate that the CoMo hybrids with a Co/Mo molar ratio of 8 exhibit the best catalytic activity. In Figure 5D, to investigate whether the catalytic activity of CoMo hybrids itself or the supernatant coexisting with the CoMo hybrids, CoMo hybrids were incubated (pH 4.0) for 10 min, and then removed by centrifugation to collect the leaching solution. The activity of the leaching solution in Figure 5D (a) was then compared to that of the intact CoMo hybrids in Figure 5D (b), showing the supernatant has no significant enzyme-like catalytic activity even under the same conditions. These results demonstrated that the high triple-enzyme mimetic catalytic activity was evolved from the CoMo hybrids themselves rather than the


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possible leaching of Co or Mo ions from CoMo hybrids.

Figure 5. (A) The absorbance intensity of TMB and 50 mM H2O2 upon the addition of different materials. Experimental conditions: 1 mL 100 µg mL-1 CoMo hybrids, MoSx, or Co(OH)2 NFs, 1 mL 2.5 mM TMB and 50 mM H2O2 were incubated (pH 4.0) for 5 min (Inset: digital photos of different colorimetric reaction systems). (B) Time-dependent absorbance of TMB at 652 nm varied with different catalysts used. Experimental conditions: 1 mL 2.5 mM TMB; 1 mL 100 µg mL-1 CoMo hybrids, MoSx, Co(OH)2 NFs; measured at room temperature. (C) O2 generation from H2O2 decomposition with different catalysts. Experimental conditions: 50 mM H2O2 and 100 µg mL-1 each catalyst (MoSx, Co(OH)2 NFs, or CoMo hybrids) were incubated (pH 4.0). (D) The catalytic activity comparision of CoMo hybrids and the supernatant coexisting with the CoMo hybrids. Experimental conditions: 1 mL 2.5 mM TMB, 1 mL 100 µg mL-1 CoMo hybrids.

3.3. DFT Study of the Oxidase-Like Activity of CoMo Hybrids.



First principle calculations were performed in order to elucidate the mimic enzyme catalytic activity of the CoMo hybrids. The oxidase-like activity was taken as an example. The calculations were carried out with the projector-augmented wave basis in the Viena ab initio simulation package.49 The energy cutoff is 400 eV. The revised density functional RPBE50 that fulfils the Lieb-Oxford criterion locally was used to obtain an improved description of small molecules on metal surfaces. Since there are 3d Co metal atoms in the CoMo hybrids, the GGA+U method with U-J=3.3 eV51 for Co atoms was used to describe the strongly correlated localized 3d electrons. Spin polarization was considered throughout the calculations because there are unpaired electrons in Co atoms. Since the catalytic reaction occurs at the surface of the catalyst, two-dimensional models are needed. For the bare Co(OH)2 surface, a 4×4 of rectangular supercells along the surface direction was constructed. The lattice parameters (12.77 and 18.63 Å) are larger than 10 Å. Therefore, the influence from the adsorbed small molecules in the neighbor cells can be omitted. The vacuum layer perpendicular to the surface direction is also approximately 10 Å. The 3×2 Monkhorst-Pack k-point sampling was used along the two directions. As indicated in Figure 6(A), each layer contains sixteen Co atoms in a unit cell. Four layers were used to represent the thin film. The top two layers were allowed to be relaxed during geometric optimization in order to reflect the interaction between the surface and the adsorbed small molecules, while the bottom two layers were kept fixed to represent the influence from the inner structure. Bulk β-Co(OH)2 has an antiferromagnetic electronic state.52-53 After the calculation, the two-dimensional Co(OH)2 model also has antiferromagnetic electronic state that it inherits from the parent structure. The only difference is that the OH- groups at the surface become vertical. For the oxidase-like activity, we follow the mechanism in which an oxygen molecule was firstly adsorbed on the surface of the catalyst, and then it dissociates to yield oxygen atoms, and finally it abstracts hydrogen atoms from other species. The adsorption energy of an oxygen molecule on the surface is a convenient descriptor to investigate the catalytic activity.54 For this reason, an oxygen molecule adsorbed on the surface of the Co(OH)2 was calculated, as shown in Figure


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5(A). Although it is a stationary point, the structure is energetically unfavorable. The adsorption energy is a quite large positive value (2.27 eV). After adsorption, an O atom forms a bond with a Co atom with bond length of 2.34 Å. The bond length in the oxygen molecule increases slightly from 1.24 Å in its free state to 1.26 Å. Another oxygen atom forms a hydrogen bond with an adjacent OH- group. In its free state, an oxygen molecule has triplet ground state. The reaction of a close shell TMB molecule with an oxygen molecule involves a conversion of oxygen molecule from triplet to singlet state. Thus the magnetic moment on each O atom is another important parameter54 that affects the oxidase-like activity. After adsorption, the magnetic moment decreases slightly from 1 to 0.7 µB. The convention is not accomplished. Therefore, the oxidase-like activity of the bare Co(OH)2 surface is not high.

(A)

(B)

(C)

Figure 6. Unit cells of an oxygen molecule adsorbed on the surfaces of (A) the bare Co(OH)2, (B) Mo-doped Co(OH)2 and (C) Co-Mo thin film models.

For the CoMo hybrids, two types of models were considered. One is doping model. As shown in Figure 6(B), a Co atom was replaced by a Mo atom and four adjacent OH- groups were replaced by four S atoms, so that the penta-coordinated nature does not change for both Mo and Co atoms at the surface and the oxidation number of the Co atoms remains unchanged. For the Mo-doped Co(OH)2, three adsorption sites were considered. In Figure 6(B), the oxygen molecule is adsorbed on the Mo atom with bond length of 1.85 Å. The bond length in the oxygen molecule increases from 1.24 to 1.30 Å, which is longer than that adsorbed on the bare Co(OH)2. The oxygen molecule with longer bond length should be more active. Another oxygen atom also makes a hydrogen bond with an adjacent OH- group. After



adsorption, the magnetic moments of the O atoms in the oxygen molecule are reduced to zero. The conversion from triplet to singlet state is achieved, which is favorable to the catalytic activity. The adsorption energy is -0.14 eV. When the oxygen molecule is placed on the Co atom adjacent to or far away from the Mo atoms (not shown in Figure 6), the distance between the O and Co atom is as long as 3.44 or 2.98 Å. The adsorption and the spin state conversion are not achieved. Therefore, the oxygen molecule should be adsorbed on the Mo atom in the doping model. In another model, the MoSx layer is adsorbed on the Co(OH)2 surface to form a Co-Mo thin film. MoS2 has good crystalline form,55 while MoS3 has amorphous structure. For the amorphous MoSx, no crystal structure has been reported as far as we know. For the structural information, we referred to the theoretically predicted crystal structure of MoS3.56 In the optimized two-dimensional structure of MoS3, the Mo atoms are sandwiched between two S layers. There is no place for adsorption of oxygen molecule, which is similar to the situation for MoS2. For the case of MoSx layer adsorbed on the surface of Co(OH)2, a MoS3 nanoribbon adsorbed on the two-dimensional Co(OH)2 was constructed as shown in Figure 6(C). In this model, two OH- groups were deleted and another two were replaced by two O atoms, so that the oxidation number of the Co atoms does not change. After adsorption, the Mo atoms are more exposed for the adsorption of oxygen molecules. The two bond lengths between the O and Mo atoms are 1.95 and 2.13 Å. The bond length in oxygen molecule increases to 1.41 Å. The adsorption energy is -0.55 eV. The magnetic moments on the two O atoms are reduced to zero, which is favorable to the catalytic activity. When the oxygen molecule is placed on the Co atom adjacent to the MoS3 nanoribbon (not shown in Figure 6), the triplet state of the oxygen molecule does not change and the adsorption energy is near zero. The reason should be similar to the situation for the bare Co(OH)2 surface. Therefore, the oxygen molecule should be also adsorbed on the Mo atom in the thin film model. The adsorption energy, the bond length, and the magnetic moment on the oxygen molecule are factors that affect the oxidase-like activity. For the Co-Mo hybrids, the magnetic moment reduces to zero both in the doping and thin film model, when the


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oxygen molecule is adsorbed on the Mo atom. The adsorption energy is more negative and the bond length is longer in the thin film model. These imply that the model in Figure 6(C) is more favorable to the oxidase-like activity. The adsorbed MoSx thin film provides a platform for the adsorption of oxygen molecules. Thus, the Co-Mo thin film has great oxidase-like activity.

3.4. Steady-State Kinetic Assay of CoMo Hybrids. To further investigate the mechanism of the peroxidase-like activity of CoMo hybrids, the apparent steady-state kinetic parameters were determined by changing the concentrations of TMB and H2O2 in the system, respectively (Figure S9). The Michaelis-Menten constant (Km) and maximum initial velocity (Vmax) were calculated by using Lineweaver-Burk plots (Figure S9B, D). Versus with other Co or Mo-based similar catalysts and HRP (Table S1), the CoMo hybrids showed excellent catalytic affinity for TMB and H2O2, i.e., smaller and larger values of Km and Vmax, respectively. This result indicates that amorphous MoSx could enhance the mimic enzyme activity of the Co(OH)2 NFs.25-26 Since CoMo hybrids exhibited great oxidase-like activity, the kinetic parameters were recorded by using different concentrations of TMB. The results reveal that the catalytic oxidation of the CoMo hybrids follows a typical Michaelis-Menten behavior toward TMB (Figure 7A). Figure 7B shows the Km and Vmax obtained from a Lineweaver-Burk plot. The values of Km and Vmax for the CoMo hybrids with TMB were 0.1773 mM and 19.5 × 10-8 M s-1, respectively. Compared with the pure Co(OH)2 NFs, the Km and Vmax values of CoMo hybrids with TMB as the substrate were lower and higher than that of its (0.5557 mM, 9.68 × 10-8 M s-1), respectively (Figure 7C, D). A low Km implies the strong affinity of the enzyme to the substrates and vice versa. Therefore, these results revealed that the CoMo hybrids had higher affinity for TMB than that of pure Co(OH)2 NFs and explained an explanation for the enhanced oxidase-like activity of CoMo hybrids, which was consistent with the above theoretical calculation.



Figure 7. Steady-state kinetic assay of oxidase-like activity of CoMo hybrids and pure Co(OH)2 NFs. Michaelis-Menten curve of CoMo hybrids and pure Co(OH)2 NFs with TMB (A) and (C); (B) and (D) double reciprocal plots of activity of CoMo hybrids and pure Co(OH)2 NFs, respectively. Error bars represent the standard deviation values of three parallel measurements.

3.5. AA Detection Using the TMB-O2-CoMo Hybrids System. The optical properties of the TMB-O2-CoMo hybrids system were illustrated to investigate the interaction between ascorbate acid (AA) and the TMB-O2-CoMo hybrids system. Figure 8A shows that the CoMo hybrids could catalyze the oxidation of TMB to form a typical blue color product (oxTMB) (curve a); in the absence of CoMo hybrids, the control experiment (curve c) showed no blue product indicating the oxidase-like activity of the CoMo hybrids. Furthermore, the introduction of AA into the TMB-O2-CoMo hybrids system resulted in a rapid (less than 5 s) absorbance decrease in the oxTMB production (Figure 8B). The strong reduction of AA is derived from the enediol group that reduces oxTMB to colorless TMB (curve b) and such


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redox reaction can be explored to detect AA. Based on of the intrinsic oxidase-like activity of CoMo hybrids, a simple colorimetric method was developed to detect AA by using the TMB-O2-CoMo hybrids system. Under the optimal conditions discussed above, different concentrations of AA were added to the TMB-O2-CoMo hybrids system for UV-vis spectroscopy

measurements.

From

Figure

8C

and

D,

the

typical

AA

concentration-response curve shows that the system reveals a good response to AA with the AA concentration increases. The absorbance is linearly correlated (R2 = 0.9914) with AA concentrations from 0.5 to 50 µM (A = -0.012 [AA]/µM + 0.7026) and limit of detection (LOD) of 0.25 µM at a signal-to-noise of 3. Compared to those previously reported nanomaterials for the detection of AA (Table S2), the CoMo hybrids exhibited higher sensitivity and lower detection limits. As we know, sensors with high sensitivity permit sufficient sample dilution during the assay period, which can benefit the reduce of interference in complex substrates (physiological species),57-58 which provides the basic conditions for sensing the extracellular AA in the brain.



Figure 8. (A) Typical absorption spectra for TMB in different reaction systems: (a) TMB-O2-CoMo hybrids, (b) TMB-O2-CoMo hybrids + AA, (c) TMB solution (Inset: the color change with the different solutions). (B) Time-dependent absorbance of TMB-O2-CoMo hybrids + AA. Experimental conditions: 1 mL 2.5 mM TMB; 1 mL 100 µg mL-1 CoMo hybrids; 0.5 mL 40 µM AA; measured at room temperature. (C) Absorption spectra of TMB with different AA concentrations (from top to bottom: 0, 0.5, 1, 5, 10, 20, 25, 30, 35, 40, and 50 µM). (D) The good linear calibration plots for AA detection. Error bars represent the standard deviation values of three parallel measurements.

Furthermore, the specificity of the TMB-O2-CoMo hybrids system was investigated using some coexisting metal ions, anions, and biological molecules (K+, Na+, Ca2+, Mg2+, Cu2+, Mn2+, S2-, Ni2+, Fe3+, Fe2+, UA, DOPAC, Ala, His, 5-HT, GSH, Cys, Gly, Glu (Glutamic), and DA). As shown in Figure 9A, a significant increase in absorbance change at 652 nm was observed with AA relative to any of the interfering ions. These results indicate the relatively good specificity of the colorimetric method for detecting AA. Previous work has shown that the approximate oxidation potential of the common reducing substances, i.e., AA, DA and DOPAC were 0.2 V and that of UA was 0.3 V vs Ag/AgCl,59 indicating that DA and DOPAC may have a similar reduction activity with AA and oxTMB redox reaction. However, in this study, the TMB-O2-CoMo hybrids system exhibits a high selectivity toward AA against others substances, which could be attributed to a relatively fast dynamic reaction process between oxTMB and AA compared to those of oxTMB and DA, DOPAC, and UA. The proposed solvothermal method for preparing CoMo hybrids is simple, robust, and reproducible. In addition, the stabilities and reusabilities of the CoMo hybrids were also examined. Table S3 shows that the reproducibility among seven batches of the prepared CoMo hybrids and the relative standard deviation (RSD) is 90% (Figure S9B), indicating their


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high stability during long-term storage. These results described above reveal that the CoMo hybrids possess good stability and high catalytic activity.

Figure 9. (A) Determination of the selectivity of AA detection. Different final concentration of species: 1 mM K+, Na+, Ca2+, and Mg2+ and 10 µM for Cu2+, Mn2+, S2-, Ni2+, Fe3+, Fe2+, UA, DOPAC, Ala, His, 5-HT, GSH, and AA and 1 µM for Cys, Gly, Glu (Glutamic), and DA; (B) Detection of AA in the microdialysate of rat brains under different physiological conditions: normal, cerebral ischemia. Error bars represent the standard deviation values of three parallel measurements.

3.6. Detection of AA in Rat Brain Microdialysates During the Cerebral Calm/Ischemia Process. To explore the practical application of the system for the detection of AA in complex brain environment, a rat brain calm/ischemia process model was designed to verify its feasibility. The cerebral dialysate containing AA was obtained by microdialysis during the normal/ischemia process. Figure 9B shows that the basal level of AA was 2.26 ± 0.39 µM under the calm period (60 min); during the preischemia surgery at ~ 20 min, the level of AA in the microdialysates was 3.62 ± 0.35 µM, while at 60 min after cerebral ischemia, the cortex AA level kept fast increased rapidly to 17.47 ± 0.95 µM, which was almost identical to the previously reported literature,

60

Such information may benefit to explore the neurochemical

processes, and again validate the TMB-O2-CoMo hybrids system can be a promising sensing platform for detecting AA in the rat brain.



4. Conclusions An eco-friendly synthetic approach for CoMo hybrids through a solvothermal strategy is provided, and these hybrids possess good stability and high catalytic activity. During the synthetic process, the sponge MoSx is epitaxially grown on the two-dimensional cobalt hydroxide nanoflakes to form bumpy hierarchical nanostructures CoMo hybrids. For the first time, the CoMo hybrids is found have outstanding intrinsic triple-enzyme activity including peroxidase-like, oxidase-like, and catalase-like activity. Furthermore, we investigated the mechanisms of oxidase-like activity for the CoMo hybrids via density functional theory (DFT) calculations. A simple reaction-dissociation of O2-supported on Co-Mo thin film can deeply account for the oxidase-like activity of the CoMo hybrids. Compared with different catalysts (Co(OH)2 NFs, MoSx, and CoMo hybrids), the classless enzyme activity of the MoSx nanosponge can enhance the nanozyme activity of Co(OH)2 NFs due to exposing the plenty of active sites and the large accessible surface area formed in the heterogeneous nanostructure. A colorimetric method for the detection of cerebral AA in the brain following the calm/ischemia was developed based on the oxidase-like activity of the CoMo hybrids. In addition, this work develops a new strategy to synthesis functionalized bimetallic composites based on the 2D Co(OH)2 NFs and will promote the study of the nanomaterials as triple-enzyme mimics.

Acknowledgments This work was financially supported by National Natural Science Foundation (21575090), Beijing Municipal Natural Science Foundation (2162009), Scientific Research Project of Beijing Educational Committee (KM201810028008), Youth Innovative Research Team of Capital Normal University and Capacity Building for Sci-Tech Innovation-Fundamental Scientific Research Funds (025185305000/195).

Compliance with ethical standards.


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Coupled with On-line Electrochemical Detection. Brain Res. 2009, 1253, 161-168.

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