Few-layered MoSe₂ nanosheets as an efficient peroxidase nanozyme for highly sensitive colorimetric detection of H₂O₂ and xanthine

Abstract

Molybdenum-containing and selenium-containing enzymes are generally prevalent in the biosphere, and the active sites of these involve molybdenum and selenium respectively. Herein, we for the first time demonstrated that few-layered molybdenum selenide (MoSe₂) nanosheets possess intrinsic peroxidase-like activity. The few-layered MoSe₂ nanosheets were prepared by a simple liquid exfoliation method. The catalytic process of the MoSe₂ nanosheets accords with the Michaelis–Menten behavior and they have a higher affinity to both TMB and H₂O₂ compared to HRP. In addition, we established that the MoSe₂ nanosheets catalyze the oxidation of TMB by promoting the electron transfer process from TMB to H₂O₂. More importantly, we applied the MoSe₂ nanosheets to the field of biological detection by developing a highly sensitive and selective colorimetric detection of H₂O₂ and xanthine concentration. With these advantages, the few-layered MoSe₂ nanosheets have critical potential in the diagnostics and biomedical fields.

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Introduction

Nanozymes are artificial enzymes with the characteristics of inorganic nanomaterials.¹ Since the first report that Fe₃O₄ nanoparticles possess an intrinsic peroxidase activity,² an emerging class of nanomaterials such as metal oxides,^2,3 metal-based nanomaterials,⁴ carbon-based nanomaterials,^5,6 transition metal chalcogenides⁷ and others⁸ has been exploited as nanozymes. Nanozymes are equipped with numerous advantages such as high stability, low cost, high versatility and durability to harsh environments,⁹ making them potential alternatives for natural enzymes in various fields including the food industry, agriculture, biosensors and environmental treatment.^10,11

Since the discovery of graphene was reported in 2004,¹² increasing attention has been paid to 2D materials and their various potential applications.^13–17 Transition metal dichalcogenides (TMDs),¹⁸ as a newly emerging class of 2D materials, possess abundant active edges and large surface areas, as well as unique electrical, chemical, mechanical and thermal properties.¹⁹ On the basis of these characteristics, TMDs harbor the potential to serve as nanozymes and have been applied to biological detection such as glucose detection.^20–22 Moreover, xanthine is a kind of metabolite intermediate of purine nucleotides and deoxynucleotides in animals.²³ An extremely abnormal level of xanthine will give rise to gout and hyperuricaemia.²⁴ Hence, the use of TMDs as a nanozyme in xanthine detection is quite significant and indispensable.

As we know, molybdenum-containing enzymes and selenoenzymes have considerable universality around the biosphere. For almost all molybdenum-containing enzymes including dimethylsulfoxide (DMSO) reductase, xanthine oxidase, sulfite oxidase and aldehyde ferredoxin oxidoreductase (AOR),²⁵ the catalytic site is the molybdenum cofactor (Moco) in different variants.²⁶ Moco plays an important role in the transfer of an oxygen atom which ultimately is derived from or incorporated into water to or from a substrate.²⁵ In addition, there are more than 50 selenoprotein families that consist of selenoenzymes including glutathione peroxidases, thioredoxin reductases and iodothyronine deiodinases.^27,28 Their structure apparently ensures that the functions of most selenoproteins should be involved in redox-related reactions and selenocysteine (Sec) is an active-site residue essential for catalytic activity.²⁸ Motivated by the critical importance of molybdenum and selenium, we propose that molybdenum selenide (MoSe₂) nanosheets are likely to possess significant potential to be enzyme mimics.

In this contribution, we report few-layered MoSe₂ nanosheets that were obtained via a liquid exfoliation method for their ability to catalyze peroxidative reactions in aqueous media.²⁹ In the presence of H₂O₂, MoSe₂ nanosheets can rapidly catalyze the oxidation of peroxidase substrate TMB to form blue oxTMB, and show peroxidase mimetic activity. The kinetics investigations show that the catalytic process follows Michaelis–Menten behavior, and their kinetic parameters are comparable to HRP. Furthermore, the catalytic pathways of peroxidase-like activity were also studied to clarify the catalytic mechanisms. Based on such characteristic activity of MoSe₂ nanosheets, and given the selective catalytic oxidation of xanthine to produce H₂O₂ with xanthine oxidase, a colorimetric method to detect H₂O₂ and subsequently xanthine was developed. Therefore, these results demonstrated that this assay is sensitive and accurate.

Experimental section

Materials preparation

MoSe₂ powders were purchased from Alfa Aesar. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD) were bought from Sigma-Aldrich. 3,3′,5,5′-Tetramethylbenzidine (TMB), NaOH, xanthine and xanthine oxidase were purchased from Aladdin Industrial Corporation. Anhydrous ethanol, acetic acid, acetic acid sodium salt, and hydrogen peroxide (30%) were bought from Guangzhou Chemical Reagent Factory. Terephthalic acid (TA) was purchased from Tokyo Chemical Industry Co. Ltd. Milli-Q water (18.2 MΩ cm) was used for all solution preparations.

The few-layered MoSe₂ nanosheets were prepared from commercial MoSe₂ flakes via a simple liquid exfoliation method. Briefly, commercial MoSe₂ powders (2 g) were dissolved in a 45 vol% ethanol/water mixture (200 mL) and ultrasonicated for 8 h at 80% amplitude at 10 °C. Then the solution was centrifuged two times at 5000 rpm for 5 min and the supernatant was obtained by decantation. Suction filtration was carried out to remove ethanol and the filtered residue was redispersed into DI water by bath sonication for 10 min. This was followed by centrifugation at 3000 rpm for 3 min, and the supernatant was collected as the few-layered MoSe₂ nanosheet solution for further characterization.

Material characterization

X-ray powder diffraction (XRD) was performed using a Rigaku D/Max-IIIA X-ray diffractometer with Cu Kα radiation (λ = 1.54056 Å, 40 kV, 20 mA) at a scanning rate of 2° s⁻¹. The ultraviolet-visible (UV-vis) absorption spectra of the samples were obtained using a UV 3150 spectrophotometer (Shimadzu, Japan). Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were acquired by using an FEI Tecnai G2 F30 transition electron microscope equipped with a field-emission gun. The Raman spectra were collected by a Renishaw inVia spectrometer using a 514 nm laser for excitation. The thickness profile was acquired on a Bruker Multimode 8 atomic force microscope (AFM). Fluorescence spectroscopy was performed on a Hitachi F-4500 fluorescence spectrophotometer.

Peroxidase-like activity assay

Experiments were carried out at room temperature in a 1 mL tube. 50 μL of 0.15 mg mL⁻¹ MoSe₂ nanosheets and 100 μL of 1 M H₂O₂ were added into 750 μL of 200 mM acetate buffer (pH 2.5–7). Immediately after this 100 μL of 10 mM TMB was added, and the color of the reactions was observed.

The steady-state kinetic analysis of MoSe₂ nanosheets with H₂O₂ and TMB as the substrates was performed by varying the concentrations of TMB at a fixed H₂O₂ initial concentration of 1 M and vice versa at a fixed TMB initial concentration of 10 mM in acetate buffer (200 mM, pH 3.5). The apparent kinetic parameters were calculated based on the Michaelis–Menten equation: v₀ = V_max × [S]/(K_m + [S]), where v₀ is the initial velocity, V_max is the maximal reaction velocity, [S] is the concentration of the substrate and K_m is the Michaelis constant.

Detection of hydroxyl radicals

Terephthalic acid (TA) was used as a fluorescent probe for the tracking of ˙OH and the generation of 2-hydroxy terephthalic acid (TAOH) emitted a unique fluorescence at around 430 nm. 0.2 mL of 0.15 mg mL⁻¹ MoSe₂ nanosheets, 0.4 mL of 1 M H₂O₂ and 0.4 mL of 25 mM TA were added into 3.0 mL of acetate buffer (pH 5.5). After 12 h of incubation in the dark at room temperature, fluorescence measurement was observed.

Cytochrome C electron transfer experiment

Cytochrome C (Cyt C) was used as an active reactant in the electron transfer process to understand the electron transfer mediator role of the MoSe₂ nanosheets. 40 μL of 0.15 mg mL⁻¹ MoSe₂ nanosheets and 25 μL of 0.4 mM Cyt C were added into 120 mL acetate buffer (pH 3.5). Following 18 h of incubation in the dark at room temperature, the reaction solution was centrifuged at 8000 rpm for 5 min and then the supernatant was measured by using a UV-vis absorbance spectrometer.

H₂O₂ and xanthine detection

H₂O₂ detection was carried out at room temperature in a 1 mL tube. 50 μL of 0.15 mg mL⁻¹ MoSe₂ nanosheets and 100 μL of H₂O₂ of different concentrations were added into 750 μL of 200 mM acetate buffer (pH 3.5). Immediately after this 100 μL of 10 mM TMB was added, and the color of the reactions was observed.

Xanthine detection was carried out at room temperature. First, 10 μL of 0.5 U mL⁻¹ xanthine oxidase aqueous solution and 40 μL xanthine with different concentrations were added into 50 μL of 200 mM acetate buffer (pH 7.0). Following 20 min of incubation, 750 μL of 200 mM acetate buffer (pH 3.5), 50 μL of 0.15 mg mL⁻¹ MoSe₂ nanosheets and 100 μL of 10 mM TMB were successively added to the xanthine reaction solution and the color of the reactions was observed.

Results and discussion

The crystal structure of the as-exfoliated MoSe₂ was studied by XRD, as shown in Fig. 1A. The XRD patterns of the bulk powders and the as-exfoliated MoSe₂ exhibit a major peak at a 2θ value of 13.6 corresponding to the (002) plane of crystalline 2H-MoSe₂. After exfoliation, the (002), (004), (006) and (008) peaks were still present while the others disappeared, indicating the potent exfoliation of MoSe₂.

Fig. 1 (A) XRD spectra of bulk MoSe₂ and the corresponding exfoliated MoSe₂ nanosheets prepared by liquid exfoliation. (B) UV-vis spectra and (inset) a photograph of MoSe₂ nanosheets dispersed in water. (C) TEM image, (D) high resolution TEM image and the (inset) corresponding SAED pattern, (E) AFM topographic image and (F) Raman spectra of MoSe₂ nanosheets.

As illustrated in Fig. 1B, the UV-vis spectra of the as-exfoliated MoSe₂ exhibit typical characteristic peaks at 808 and 693 nm which originate from the energy split of the valence-band and spin-orbital coupling, indicating the generation of 2H-phase few-layered MoSe₂ nanosheets which correspond to the XRD patterns.³⁰ The inset of Fig. 1B shows the photograph of the dispersion of MoSe₂ nanosheets stabilized in water.

The microscopic features of the as-exfoliated MoSe₂ were first studied by TEM. As exhibited in Fig. 1C and Fig. S1 (ESI†), the morphology of the nanomaterials presents as two-dimensional thin sheets. Extremely thick nanosheets were rarely observed, indicating that the liquid-exfoliation process was highly efficient for obtaining few-layered sheets. In the HRTEM image (Fig. 1D), individual layer edges can be observed near the periphery of these nanosheets. The inset of Fig. 1D shows the corresponding SAED pattern which revealed the high crystallinity of the MoSe₂ nanosheets and the crystal planes of (100) and (110).

The Raman spectrum is illustrated in Fig. 1F. Typical peaks of MoSe₂ nanosheets at 240.2 cm⁻¹, 285.2 cm⁻¹ and 350.4 cm⁻¹ were observed, which corresponded to the out-of-plane A_1g mode, in-plane E¹_2g mode and B¹_2g mode.³¹ The red shift of the A_1g mode by approximately 2 cm⁻¹ compared to the bulk MoSe₂, the blue shift of the E¹_2g mode, and the coexistence of all three modes indicate the formation of few-layered MoSe₂ nanosheets.^31,32

The thicknesses of the as-exfoliated MoSe₂ were monitored through AFM examination. The AFM image of MoSe₂ (Fig. 1E and Fig. S1, ESI†) exhibits thin polydispersed wrinkled nanosheets corresponding to few-layered nanosheets given that the thickness of a MoSe₂ monolayer is about 0.65 nm. All the aforementioned results confirmed that the few-layered MoSe₂ nanosheets had been produced.

Kinetics investigations of peroxidase-like catalysis activities of the few-layered MoSe₂ nanosheets

In the first set of experiments, the intrinsic peroxidase catalytic activity of the MoSe₂ nanosheets was first investigated. As the result shows in Fig. 2A, when the MoSe₂ nanosheets, H₂O₂ and TMB coexisted in the reaction solution, the absorbance at 652 nm increased rapidly with time. In the absence of H₂O₂ or MoSe₂ nanosheets, the increase in absorbance was negligible, revealing that the MoSe₂ nanosheets actually possess intrinsic peroxidase catalytic activity, facilitating the oxidation of TMB in the presence of H₂O₂. Under the same conditions, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD) were adopted as the substrates for the catalytic experiment. However, only the former shows an obvious color change, indicating that the MoSe₂ nanosheets possess substrate specificity³³ (Fig. S2, ESI†). Given the redox-related selenoproteins, a hypothesis was proposed that to some degree selenium contributes to the peroxidase-like activity. The inset of Fig. 2A exhibits the color reaction aforementioned, showing that TMB was oxidized to blue oxTMB. Similar to HRP, the catalytic activities of the MoSe₂ nanosheets show pH and H₂O₂ concentration dependence.³⁴ Different buffer types and pH values as well as sizes of the MoSe₂ nanosheets lead to different catalytic velocities. As can be seen in Fig. 2B and Fig. S3 (ESI†), the MoSe₂ nanosheets show optimal activity at around pH 3–3.5, which is very close to the value for HRP.² The acetate buffer and MES buffer can maintain the maximum catalytic ability of the MoSe₂ nanosheets. Thus, acetate buffer at pH 3.5 was adopted as the standard condition for subsequent analysis of MoSe₂ nanosheet activity.

Fig. 2 (A) Time-dependent absorbance of TMB at 652 nm in different reaction systems and (inset) photographs of oxidation of TMB by MoSe₂ nanosheets in the presence of H₂O₂. (B) pH dependent peroxidase-like activity of MoSe₂ nanosheets in acetate buffer and the maximum point was set as 100%. (C–F) Steady-state kinetic assay of MoSe₂ nanosheets. Michaelis–Menten curve fit for (C) variation of TMB concentration and keeping H₂O₂ concentration constant and (D) variation of H₂O₂ concentration and maintaining the TMB concentration constant and the corresponding Lineweaver–Burk plots of MoSe₂ nanosheets with (E) TMB as a substrate and (F) H₂O₂ as a substrate.

For a better understanding of the MoSe₂ nanosheet catalytic activities, a steady-state kinetics analysis was performed to acquire the kinetic parameters. Within the suitable range of TMB or H₂O₂ and at the same time fixing the other concentration, the Michaelis–Menten curves were obtained as shown in Fig. 2C and D. And the kinetic parameters including the Michaelis–Menten constant (K_m) and the maximal velocity (V_max) were obtained according to the Lineweaver–Burk plot (Fig. 2E and F). The apparent K_m value is an index that measures the affinity of the substrates to enzymes and the lower the K_m value is, the higher the affinity the substrate bears. Table 1 lists the K_m and V_max values for the MoSe₂ nanosheets and HRP. For both TMB and H₂O₂ as the substrates, the apparent K_m values of HRP are 30 times and 22.8 times higher than those of the MoSe₂ nanosheets respectively, suggesting that the MoSe₂ nanosheets bear a higher affinity for both TMB and H₂O₂ than HRP. The reasons will be summarized in the following two contributions. Firstly, the plentiful active edges and defects on the surface as well as large the surface areas of the 2D materials provide the MoSe₂ nanosheets with numerous bonding points to TMB and H₂O₂.³⁵ Secondly, taking into consideration the functions of molybdenum in Moco and selenium in selenocysteine, an assumption was proposed that molybdenum and selenium are apt to absorb the substrate which needs further research in the future.

Table 1 Comparison of the kinetic parameters of MoSe₂ nanosheets and HRP. K_m is the Michaelis constant, V_max is the maximal velocity

Catalyst	Substrate	K m (mM)	V max (10^−8 M s^−1)	Ref.
HPR	TMB	0.434	10.0	2
	H2O2	3.70	8.71
MoSe2 nanosheets	TMB	0.014	0.56	This work
	H2O2	0.155	0.99

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Reaction mechanism

In order to figure out the mechanism with which MoSe₂ nanosheets catalyze the oxidation of TMB in the presence of H₂O₂, two experiments were designed.³⁶ On the one hand, the hydroxyl radicals (˙OH) generated by H₂O₂ were detected to find out whether the ˙OH radicals oxidise TMB. Terephthalic acid (TA) was used as a fluorescence probe for the tracking of ˙OH and the generation of 2-hydroxy terephthalic acid (TAOH) emitted a unique fluorescence at around 430 nm. As illustrated in Fig. 3A, after 12 h of incubation in the dark, the fluorescence at 430 nm decreased with the increased concentration of MoSe₂ nanosheets, indicating the ˙OH consumption due to the presence of MoSe₂ nanosheets, excluding that the catalytic action was caused by ˙OH. On the other hand, reduced cytochrome C (Cyt C) was used as an active reactant in the electron transfer process to understand the electron transfer mediator role of the MoSe₂ nanosheets. After 18 h of incubation in dark in the presence of MoSe₂ nanosheets, the absorbance of Cyt C at typically 520 and 550 nm disappeared while the evolution of a new peak at 530 nm appeared (Fig. 3B). The change from the reduced state to the oxidized state of Cyt C, reveals the electron transfer from Cyt C to the MoSe₂ nanosheets.

In addition, considering the function of Moco that catalyzes the transfer of an oxygen atom and redox-related selenoproteins with selenocysteine as the active-site residue, molybdenum and selenium are likely to facilitate electron transfer and redox-related reactions. The mechanism of the MoSe₂ nanosheets acting as a peroxidase was proposed as follows. First, TMB is absorbed to the surface of the MoSe₂ nanosheets and transfers lone-pair electrons in the amino groups to the MoSe₂ nanosheets. Because of the large specific surface area of the 2D MoSe₂ nanosheets, plenty of active sites are available for the transfer process, leading to the oxidation of TMB and increase of the MoSe₂ nanosheet electron density. Then, the electron-rich MoSe₂ nanosheets transfer their electrons to nearby H₂O₂ and reduce H₂O₂ to water. The electron concentration gradient accelerates the electron transfer from the MoSe₂ nanosheets to H₂O₂ and consequently the decrease in electron concentration of the MoSe₂ nanosheets promotes the electron transfer from TMB to the MoSe₂ nanosheets. As a result, TMB is oxidized to blue oxTMB and H₂O₂ is reduced to water. In short, the MoSe₂ nanosheets catalyze the process which is similar to the case of graphene oxide,⁵ that is, MoSe₂ nanosheets promote the electron transfer from TMB to H₂O₂, producing blue oxTMB and water.

Colorimetric detection of H₂O₂ and xanthine

On the basis of the peroxidase catalytic activity of the MoSe₂ nanosheets, a colorimetric detection method for H₂O₂ and xanthine concentration was designed and a schematic of the colorimetric methods was proposed as illustrated in Fig. 4A. As mentioned above, the catalytic activities of MoSe₂ nanosheets show H₂O₂ concentration dependence, which shows the potential for MoSe₂ nanosheets to detect H₂O₂ concentration. Fig. 4B shows the increased time-course absorbance at 652 nm with different H₂O₂ concentrations, suggesting that the oxidation rate is in proportion to the H₂O₂ concentration. The corresponding H₂O₂ concentration–response curve is shown in Fig. 4C. A linear relationship was obtained in the inset of Fig. 4C and the H₂O₂ concentration was in the range of 0.01 mM to 0.16 mM with a detection limit of 0.408 μM. Given the H₂O₂ production of the xanthine oxidation, a colorimetric detection method for xanthine was established consequently. The same as the detection of H₂O₂, the increased time-course absorbance at 652 nm is in proportion to the xanthine concentration as shown in Fig. 4D and E. The inset of Fig. 4E illustrates the linear relationship and the xanthine concentration is in the range of 0.01 mM to 0.32 mM with a detection limit of 1.964 μM. This method based on MoSe₂ nanosheets possessed a larger linear range compared to other nanomaterials^37,38 (Table S1, ESI†) and showed a high selectivity for xanthine (Fig. S4, ESI†). In addition, a serum sample was detected using this method with the result 0.215 ± 0.009 mM, indicating the practicability of this method.

Fig. 3 (A) Fluorescence at 430 nm of 2-hydroxy terephthalic acid (TAOH), and the generation of terephthalic acid (TA) tracking ˙OH. (B) Electron transfer experiment from cytochrome C (Cyt C) to MoSe₂ nanosheets.

Fig. 4 (A) Schematic illustration of colorimetric detection of H₂O₂ and xanthine. (B and D) Time-dependent absorbance of TMB at 652 nm in the presence of increased H₂O₂ or xanthine concentration. (C and E) The corresponding H₂O₂ or xanthine concentration–response curve using MoSe₂ nanosheets as a nanozyme and (inset) the linear calibration plot for H₂O₂ or xanthine.

Conclusion

In summary, few-layered MoSe₂ nanosheets were prepared via a liquid exfoliation method and these MoSe₂ nanosheets exhibit a high peroxidase-like activity in that they can oxidize TMB in the presence of H₂O₂, producing a color reaction. The catalytic process complies with the Michaelis–Menten behavior and the steady-state kinetics analysis indicates that MoSe₂ nanosheets have a higher affinity for both TMB and H₂O₂ compared to HRP. The catalytic mechanism assays illustrate that the MoSe₂ nanosheets promote the electron transfer process from TMB to H₂O₂. On the basis of the color reaction, a detection method with high sensitivity and selectivity for H₂O₂ and xanthine concentration was designed. The detection of H₂O₂ and xanthine was in a linear range from 0.01 mM to 0.16 mM and 0.01 mM to 0.32 mM respectively, opening up possibilities for few-layered MoSe₂ nanosheets as peroxidase mimics in the diagnostics and biomedical fields.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The National Basic Research Program of China (2014CB931700) and the State Key Laboratory of Optoelectronic Materials and Technologies supported this work.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7tb02434g

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