Co4N Nanowires: Noble-Metal-Free Peroxidase Mimetic with

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CoN Nanowires: Noble-Metal-Free Peroxidase Mimetic with Excellent Salt- and Temperature-Resistant Abilities Yu-Zhen Li, Tong-Tong Li, Wei Chen, and Yan-Yan Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09861 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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ACS Applied Materials & Interfaces

Co4N Nanowires: Noble-Metal-Free Peroxidase Mimetic with Excellent Salt- and Temperature-Resistant Abilities Yu-Zhen Li, a Tong-Tong Li, a Wei Chen,b* Yan-Yan Songa*

a

b

Department of Chemistry, Northeastern University, Shenyang 110004, China

Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian

Province, Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China

KEYWORDS: Cobalt nitride; Peroxidase-like activity; Salt resistance; Temperature resistance; Glucose; Colorimetric sensing

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ABSTRACT: Compared to natural enzymes, nanomaterial-based artificial enzymes have attracted immense attention because of their high stability and cost-effectiveness. In this study, cobalt nitride (Co4N) as a noble-metal-free artificial enzyme exhibiting highly intrinsic peroxidase-like activity and good stability was reported. Kinetic studies revealed that the resultant Co4N nanowires (NWs) exhibited a stronger affinity for 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 than HRP. Compared to Co3O4 NWs, Co4N NWs exhibited highly improved catalytic activities, with H2O2 exhibiting an apparent Km approximately 2 orders of magnitude less than that of Co3O4. In particular, the peroxidase-like activity of Co4N was maintained well over a wide range of temperatures and ionic strength. A Co4N-based method was further developed for the detection of glucose with good sensitivity and reliability. Because of advantages such as easy storage, cost-effectiveness, high sensitivity, and outstanding stability, Co4N NWs demonstrate the potential for replacing noble-metal-based peroxidase mimetics in a wide range of promising applications.

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1 INTRODUCTION Enzymes can catalyze biochemical reactions in cellular metabolic processes and enhance the reaction rates.1,2 Natural enzymes, particularly proteins, demonstrate significant practical applications in medicine, chemical industry, food processing, and agriculture, related to their high substrate specificities and high catalytic efficiencies under mild reaction conditions. Horseradish peroxidase (HRP) as a natural peroxidase enzyme, which is extracted from plants, has been widely used to catalyze the decomposition of peroxides and oxidation of various substrates.3 However, the natural enzyme structure is easily disrupted by heating or using chemical denaturants,4-9 leading to the loss of catalytic activity. Moreover, the intrinsic drawbacks of natural enzymes, including high cost, sensitivity of catalytic activity to the environmental temperature, and low operational stability, further limit their practical applications. Since the first report of the intrinsic peroxidase-like activity of magnetite (Fe3O4) nanoparticles,10 several studies have focused on the design and construction of nanomaterial-based artificial enzymes (nanozymes).11-18 Thus far, several nanoscale noble metals, such as Pt nanoparticles,19 gold nanoparticles,20, 21 Au@Pt hybrid,22 AgM bimetallic nanostructures,23 and nanoclusters,24 have been reported to exhibit peroxidase-like activity. In addition, some metal oxides such as V2O5 NWs,25 CuO NPs,26 Co3O4 NPs,27 Fe3O4 NPs,28 MnO2 nanosheets,29 CoFe2O4 magnetic NPs,30 and CeO2.31, 32 have been reported to exhibit enzyme activity similar to that observed for natural peroxidase. Recently, some metal sulfide nanomaterials, i.e., CuS,33, 34 MoS2,35, 36

FeS, and FeSe,37 also have been reported to exhibit peroxidase-like activity. 3



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However, during the storage of nanozymes under ambient conditions, their surface properties can be altered by oxygen, humidity, and the environmental temperature. Moreover, most of the reported nanomaterial-based peroxidase mimetics exhibit a poor affinity for H2O2 compared to natural peroxidase. Thus, to ensure the accuracy and reliability of peroxidase nanomimetics for future practical applications, it is challenging to develop peroxidase mimetics with good stability and high affinity for H2O2. Cobalt nitride, as one of the binary nitride systems for 3d metals, has been regarded as a metallic interstitial compound.38 The nitrogen atoms incorporated into the interstices of the cobalt-based framework are covalently bonded to the cobalt atoms, furnishing metal-like properties.39 Currently, metallic Co4N has been employed as highly efficient electrochemical catalysts for the oxygen evolution reaction in alkaline solutions.40 To the best of our knowledge, thus far, no study has reported the potential of Co4N as nanozymes. In this study, highly crystalline metallic Co4N NWs were synthesized by simple nitridation without the use of any scaffold or template. The Co4N NWs exhibited good peroxidase-like activity, outstanding stability, and higher affinity for TMB and H2O2 compared to HRP and other reported non-metal peroxidase nanomimetics. In addition, Co4N was further successfully used as an artificial peroxidase to establish a simple and sensitive colorimetric assay for the detection of blood glucose.

2 EXPERIMENTAL SECTION 4


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2.1 Materials and Reagents Na2HPO4, NaH2PO4·2H2O, H2O2 (30%), ethanol, dimethyl sulfoxide (DMSO), Co(NO3)2·6H2O, NH4F, and urea were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Terephthalic acid (TA) was purchased from Aladdin Reagent Company (Shanghai, China). 3,3′,5,5′-Teramethylbenzidine (TMB), glucose, and glucose oxidase (GOx) were purchased from Sigma-Aldrich (St. Louis, USA). Other reagents and chemicals were of analytical reagent grade and used without further purification. All solutions were prepared using deionized (DI) water (>18 MΩ). 2.2 Apparatus The morphologies of Co4N and the precursor were observed by field-emission scanning electron microscopy (FE-SEM; Hitachi S-4800, Tokyo, Japan) and transmission electron microscopy (TEM; JEOL 2000). X-ray diffraction (XRD) patterns were recorded on an X’pert XRD spectrometer (Philips, Guildford, Surrey, UK) with a Cu Kα X-ray source. UV–Vis absorption spectra were recorded on a Lambda Bio/XLS+ UV–vis spectrophotometer (Perkin Elmer, USA). X-ray photoelectron spectroscopy (XPS) profiles were recorded on a Perkin-Elmer Physical Electronics 5600 spectrometer using Al Ka radiation at 13 kV as the excitation source. All XPS peaks were calibrated by using C 1s (284.6 eV) as the reference. 2.3 Preparation of Co4N NWs Co4N NWs were prepared by nitridation using Co(OH)(CO3)0.5·xH2O as the precursor.41-45 Briefly, Co(NO3)2·6H2O (0.291 g), NH4F (0.093 g), and urea (0.30 g) 5



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were dissolved in 35 mL of deionized water under vigorous stirring for 30 min. Second, the solution was transferred to a Teflon-lined stainless-steel autoclave, and the temperature was maintained at 120°C for 6 h. The obtained precursor Co(OH)(CO3)0.5·xH2O was washed three times with DI water and dried in vacuum at 60°C. Nitridation was carried out by treating Co(OH)(CO3)0.5·xH2O at 500 °C for 2 h in a tube furnace under flowing NH3, affording 50 mg Co4N powder in the crucible. 2.4 Peroxidase-like activity of Co4N NWs The peroxidase-like activity of Co4N NWs was investigated by the catalytic oxidation of the peroxidase substrate TMB in the presence of H2O2. In a typical experiment, catalytic oxidation was carried out at 25 °C by mixing 15 µL of Co4N (2.0 mg mL−1) with 2.0 mL of phosphate buffer solution (PBS, 20 mM, pH 4.0) in a quartz cuvette, followed by the addition of 80 µL of TMB (20 mM) and 30 µL of H2O2 (2.0 M). The mixture was stirred with an extremely small magnetic bar for 10 min, affording a blue color solution. After stirring was completed, Co4N NWs settled to the bottom of the cuvette, and the upper clear blue solution was used for absorption measurement. The blue color was spectrophotometrically monitored by measuring the absorbance change at 652 nm. All catalytic experiments were carried out under daylight, unless otherwise stated. 2.5 Kinetic analysis Kinetic analysis was carried out in the time-course mode by monitoring the absorbance at 652 nm. Measurements were carried out by varying the TMB concentrations at a constant H2O2 concentration (30 mM) or varying the H2O2 6


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concentration at a fixed TMB concentration (0.8 mM). The apparent kinetic parameters were calculated by the Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation,10 1/V = Km/Vm(1/[S] + 1/Km), where V and Vm represent the initial and maximal reaction velocities, respectively, [S] represents the substrate concentration, and Km represents the Michaelis constant. 2.6 Glucose sensing Glucose (200 µL) of different concentrations was mixed with GOx (40 µL, 1 mg mL−1). After the mixture was incubated at 37 °C for 30 min, Co4N (15 µL, 2 mg mL−1), TMB (80 µL, 20 mM), and phosphate buffer (1665 µL, 20 mM, pH 4.0) were added. The mixtures were incubated for another 10 min at 25 °C. The color of the product was measured on a Lambda Bio/XLS+ UV–visspectrophotometer (Perkin Elmer, USA) at 652 nm. 2.7 Detection of ·OH The ·OH was detected by the addition of TA into the H2O2–Co4N system. Briefly, 2.0 mL of the PBS solution (pH 4.0) containing 50 mM H2O2, and 3 mM of TA was incubated with 30 µg of Co4N NWs at different times. The FL spectra of the solution were measured at an excitation wavelength of 315 nm after the reaction period.

3 RESULTS AND DISCUSSION 3.1 Structural characterization

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Figure 1. (A) SEM, (B) TEM, and (C) HRTEM images of Co(OH)(CO3)0.5·xH2O; (D) SEM, (E) TEM, and (F) HRTEM images of Co4N.

Figure 1A shows the scanning electron microscopy (SEM) image of the precursors synthesized by the hydrothermal reaction. Wire-like nanostructures with the length of approximately 6 µm are obtained. The TEM image (Figure 1B) revealed smooth surfaces for these NWs. The XRD pattern (Figure 2) indicated the presence of orthorhombic Co(OH)(CO3)0.5·xH2O (JCPDS#00-048-0083) precursors. Well-defined lattice fringes with a spacing of 5.06 Å, corresponding to the (020) planes of Co(OH)(CO3)0.5·xH2O,

are

observed

(Figure

1C).

The

transfer

of

the

Co(OH)(CO3)0.5·xH2O precursor to Co4N was based on equation (1):

24Co(OH)(CO3)0.5·xH2O + 16NH3=6Co4N + (36+x)H2O + 12CO2+5N2

(1) 8


(220)

(200)

(111)

Co4N (412) (450)

(340)

(220) (121) (300) (221) (040) (301) (231)

(020)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intensity (a.u.)

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Co(OH)(CO3)0.5•xH2O 20

30

40

50

60

70

80

2θ (degree) Figure 2. XRD patterns of Co(OH)(CO3)0.5·xH2O and Co4N.

Figure 1D and Figure 1E show the NWs after treatment under flowing NH3 at 500°C. Nitridation has been demonstrated to be effective for preparing specific cobalt nitrides.46-49 Notably, slightly curled NW structures are observed after nitridation at high temperature. In the XRD pattern of the product, new peaks are observed at 2θ of 43.84, 51.04, and 74.90°, corresponding to the (111), (200), and (220) planes of cubic Co4N, respectively (Figure 2).50 The HRTEM image in Figure 1F further shows a distinct lattice fringe of 0.207 nm, corresponding to the (111) lattice plane of Co4N. These results clearly demonstrate that Co(OH)(CO3)0.5·xH2O precursors are successfully converted to Co4N by nitridation.

9


Co2p Co2p

Intensity (a.u.)

A

O1s C1s

N1s

Co4N

B

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Co 2p3/2

Co 2p1/2

Co-N

Co 2p3/2 Co 2p1/2

Co4N

Co(OH)(CO3)0.5•xH2O 1000

900

800

700

600

500

400

300

Co(OH)(CO3)0.5•xH2O

200

810

805

Binding Energy (eV)

C

N1s

800

795

405

400

395

Binding Energy (eV)

785

780

775

D

770

O1s

Co4N

Co(OH)(CO3)0.5•xH2O

Co4N 410

790

Binding Energy (eV)

Intensity (a.u.)

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Intensity (a.u.)


540

535

530

525

Binding Energy (eV)

Figure 3. XPS spectra of Co(OH)(CO3)0.5·xH2O and Co4N: (A) full spectra. (B–D) high resolution of (B) Co 2p peak, (C) N 1s peak, and (D) O 1s peak.

To further confirm the formation of Co4N, XPS was carried out to characterize the precursor before and after nitridation. Exclusive signals corresponding to Co, O, and C are observed for the precursor (Figure 3A, Figure S2). The signal corresponding to N 1s is detected for the product. A new Co 2p3/2 peak located at 779.0 eV, corresponding to the binding energy of the Co–N bond, is observed after nitridation. Correspondingly, a peak at 397.2 eV is observed after nitridation in the N 1s spectrum (Figure 3C), suggesting the successful conversion of the precursor to Co4N. Co4N has been reported to exhibit a low alloying property with 12 Co coordinators, and the Co–Co distance is slightly longer than that of Co0, but shorter 10


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than that of Co3O4. Thus, the Co atoms on the Co4N NW surface have been partially oxidized to Co3O4.47 Therefore, the O 1s signals with two small peaks are detected at 529.7 eV and 531.7 eV after nitridation (Figure 3D). In addition, the corresponding HRTEM images in Figure S1 indicated that the NW comprises a crystalline core and shell (~7–10 nm). Notably, the Co4N core is surrounded by a thin Co3O4 shell, as evidenced by the presence of the (222) and (311) facets of Co3O4. However, because of an extremely thin cobalt oxide shell, cobalt oxides are not detected in the XRD pattern (Figure 2).

3.2 Peroxidase-like activity of Co4N 3.0 2.5

Absorbance

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b

2.0 1.5

c

1.0 0.5

a

0.0 300

350

400

450

500

550

600

650

700

750

800

Wavelength (nm) Figure 4. UV–vis spectra of the reaction solution containing 0.8 mM TMB and 30 mM H2O2 in the absence of Co4N (curve a), in the presence of 15 µg mL−1 Co4N (curve b), and after quenching with the addition of 500 µM of H2SO4 (curve c). Inset image: the corresponding photographs of the solutions corresponding to curves a−c.

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To investigate the peroxidase-like activity of Co4N, the catalysis of the peroxidase substrate TMB was tested. TMB is oxidized to TMB(ox) by H2O2 from colorless (curve a) to bright blue (curve b) with the maximum absorbance at 652 nm in the presence of Co4N (Figure 4). This color change is negligible in the absence of Co4N. These results suggested that Co4N NWs function as natural peroxidase enzymes toward the typical substrate TMB, affording a blue charge-transfer complex (chromogen), such as that observed for the natural enzyme HRP. This color reaction can be quenched by the addition of H2SO4 to form a yellow product, i.e., oxidized diimine, with a new absorption peak at 450 nm (curve c). It is crucial to rule out the possibility that Co4N, rather than cobalt ions leached from Co4N in the acidic solution, is responsible for the observed peroxidase-like activity. To investigate this point, Co4N NWs were incubated in a standard reaction buffer (PBS at pH 4.0) for 6 h, and the Co4N NWs were removed from solution by centrifugation. Next, the activity of the leaching solution was compared with that of Co4N NWs under the same conditions. The leaching solution exhibits no activity, indicating that the observed peroxidase-like activity originates from Co4N NWs (Figure S3). To demonstrate that the high peroxidase-like activity originates from Co4N and not Co3O4, the precursor was also annealed in air. The XRD pattern of this sample in Figure S4A demonstrates the formation of Co3O4 by annealing the precursor in NH3 at 500 °C for 2 h. By comparing the peroxidase-like activity of Co(OH)(CO3)0.5·xH2O, Co3O4 with Co4N under the same experimental conditions (Figure S4B), a higher peroxidase-like activity is observed for the Co4N sample. This 12


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result indicated that the observed peroxidase-like activity mainly originates from the strong intrinsic peroxidase-like activity of Co4N. 3.3 Dependence of pH, temperature, and H2O2 0.40 0.36

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.32 0.28 0.24 0.20 0.16 0

5

10

15

20

25

30

-6

H2O2 Concentration (×10 M) Figure 5. Calibration plot showing the absorbance at 652 nm as a function of the H2O2 concentration.

Besides the intrinsic peroxidase-like ability, the catalytic ability of Co4N, similar to other nanomaterial-based peroxidase mimetics and HRP, is also dependent on pH, temperature, and the concentration of H2O2 in solution. Figure S5A clearly shows the relationship between the absorbance of the product TMB(ox) at 652 nm and the Co4N concentration. Clearly, the speed of the colorimetric reaction linearly increases with the catalyst concentration at a relatively low Co4N concentration range (2–20 µg mL−1). If the Co4N concentration exceeds 20 µg mL−1, the reactants could be depleted in a short time. The reaction speed is not only affected by catalyst concentration but also by substrate concentration;4,51 hence, the relationship becomes sublinear. In 13



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Figures S5B–S5D, the peroxidase-like activity of Co4N was further examined by the variation in the pH from 2.0 to 10.0, the reaction temperature from 15 °C to 60 °C, and the H2O2 concentration from 0.01 to 2.0 M. The optimal pH and temperature was pH 4.0 and 25 °C. Thus, for the subsequent analysis of the Co4N NW activity, a Co4N NW concentration of 15 µg mL−1, a pH of 4.0, and a temperature of 25 °C are utilized. In contrast to that observed for HRP,10 no inhibition effect was observed for Co4N-NW-catalyzed reaction at high H2O2 concentration, suggesting that compared to the natural peroxidase activity, the peroxidase-like activity of Co4N NWs is more stable at high H2O2 concentration. Remarkably, the absorbance of TMB(ox) gradually increases with increasing H2O2 concentration at low H2O2 concentration. A linear relationship is obtained between the absorbance and H2O2 concentration in the range of 5.0 × 10−7 M to 3.0 × 10−5 M (Figure 5). The limit of detection is 2.4 × 10−8 M at a signal-to-noise ratio of 3σ (where σ is the standard deviation of the blank solutions). Specially, the colorimetric method involving Co4N, which mimics the peroxidase activity, exhibits a relatively wider linear range, and a lower limit of detection (LOD) compared to the other reported artificial peroxidase mimetics (Table S1) at a convenient working temperature (25 °C). This low LOD suggested that Co4N is sensitive for the detection of trace amounts of H2O2.

3.4 Steady-State Kinetics and Reactive Mechanism

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A

-8

8.0x10

7

3.5x10

7

3.0x10

7

2.5x10

7

2.0x10

7

1.5x10

7

1.0x10

7

-7

8.0x10

-8

B 7

2.4x10

7

2.2x10

-8

7

)

2.0x10

-1

-1

-8

4.0x10

6.0x10

4.0x10

1/v (sM

-8

6.0x10

1/v (sM )

-1

4.0x10

1.0x10

v (Ms-1)

-7

1.0x10

v (Ms )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-8

7

1.8x10

7

1.6x10

7

1.4x10

7

1.2x10

-8

2.0x10

0

200

400

600

800

1000

2.0x10

-8

7

1.0x10 3 1x10

1/CH2O2 (M-1)

3

2x10

3

3x10

3

4x10

3

3

5x10

6x10

3

7x10

1/CTMB (M-1)

0.0

0.0

0.00

0.02

0.04

CH2O2 (M)

0.06

0.08

0.0

4.0x10

-4

8.0x10

-4

1.2x10

-3

1.6x10

-3

CTMB (M)

Figure 6. Steady-state kinetic assay of Co4N nanowires. The reaction velocity was measured using 15 µg mL–1 Co4N in a 20 mM phosphate buffer (pH 4.0) at 25 °C. (A) The TMB concentration was 0.8 mM, and the H2O2 concentration was varied. Inset: double-reciprocal plots of the activity of Co4N at a constant TMB concentration as a function of the varying concentration of the second substrate for H2O2. (B) The H2O2 concentration was 30 mM, and the TMB concentration was varied. Inset: double-reciprocal plots of the activity of Co4N at a constant H2O2 concentration versus varying TMB concentrations.

To better understand the peroxidase-like activity of Co4N, the steady-state kinetics for the TMB oxidation was investigated. The oxidized product of TMB was calculated on the basis of absorbance data (ɛ=39000 M−1 cm−1 at 652 nm).52 Typical Michaelis–Menten curves are obtained for Co4N NWs within the suitable range of concentrations for H2O2 and TMB (Figure 6). In Figure 6A, the reaction rate sharply increases at low H2O2 concentration (

Co4N Nanowires: Noble-Metal-Free Peroxidase Mimetic with

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