Tandem Synthesis of High Yield MoS 2 Nanosheets and Enzyme Peroxidase Mimicking Properties

: Molybdenum Sulﬁde nanosheets (MoS 2 NSs) have unique properties that allow its use in a wide range of applications. Unfortunately, a lack of green synthesis methods to achieve a high yield remains a challenge after decades. Herein we report a simple, ecofriendly, green and cost-e ﬀ ective approach to synthesize water soluble MoS 2 NSs via probe / Tip sonication method. The sequential batch manner pathway allows us to attain a high yield of MoS 2 NSs (~100%). The prepared MoS 2 NSs were characterized using up-to-date surface science techniques. UV-visible-NIR spectroscopy allowed us to visualize the doublet peaks of pristine MoS 2 at 610 and 680 nm concomitant with the inter-band transitions at 394 nm and 460 nm. Using Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS), the crystallites’ sizes were estimated. X-ray di ﬀ raction (XRD) and Raman Spectroscopy were performed with respect to the bulk MoS 2 . The energy di ﬀ erence between the Raman peaks revealed that our NSs are formed of 5–6 layers. Further, we explored enzyme peroxidase mimetic properties of the synthesized MoS 2 NSs. Results showed that the present MoS 2 NSs o ﬀ er excellent peroxidase mimicking properties. Most importantly, we observed that the optical properties and characteristics of MoS 2 NSs synthesized by the current green method are similar to those of MoS 2 NSs synthesized using conventional harsh methods reported in the literature. So that we strongly assume that the present method is a green alternative for the existing low yield and harsh experimental procedures to achieve water soluble MoS 2 NSs in high yield. The synthesized soluble NSs are promising catalysts for the detection of toxic chemicals in the environment and / or for following enzymatic chromogenic reactions.


Introduction
After the graphene era, there has been significant attention on two dimensional transition metal chalcogenide nanomaterials (TMCs) [1], in particular for MoS 2 , because it has a structural analogue to graphene where two sulfur atoms are sandwiched with one molybdenum atom and these layers are held together by Van der Walls forces [2]. As well, its unique physio-chemical properties make it a promising candidate for various applications, such as catalysis [3], electronics [4], nanomedicine [5], energy and environmental applications [6], beyond graphene. As an advancement of nanotechnology, nano-sized MoS 2 has many more advantages than the bulk MoS 2 because of its enhanced surface area, good optical absorption and extreme flexibility [7,8]. To this end, the vast utilization of MoS 2 NSs in several emerging fields has drawn scientific attention towards synthesis of MoS 2 NSs with high yields using different approaches. Therefore, several methods have been reported in the literature  [23] Liquid exfoliation MoS 2 powders, N-vinyl-2-pyrrolidone Toxic solvents Micro sheets [24,25] Microwave synthesis (NH 4 ) 2 MoS 4 , DMF Toxic solvents [26] Mechanical exfoliation High temperature, transfer process High temperature, transfer process [9] Chemical Vapor Deposition (CVD) MoO 2 , SiO 2 , Sulfur [10] Ionic liquid assisted grinding exfoliation MoS 2 powder, BMIMPF 6 , DMF Toxic solvents [27] Ball milling and chemical intercalation MoS 2 powders, DMF, NMP Toxic solvents, Disordered NSs, lower yields [16] Morrison method MoS 2 Bulk, Hexane, n-buthyllithium, N2 atmosphere Toxic solvents, inert condition [28] Ultra-sonication followed by hydrothermal ((NH 4 ) 2 MoS 4 , DMF and water Toxic solvents [29] Probe sonication (tandem process) MoS 2 powder, PAA and water High yield, water soluble NSs, batch manner Present work In this work, we report a simple and green tip/probe sonication approach to synthesize water soluble MoS 2 NSs. The sequential batch manner/tandem synthesis allowed us to achieve MoS 2 NSs in high yield (estimated yield is~100%) than the conventional methods reported in literature. Further, we assessed the optical and enzymatic peroxidase mimicking properties of the synthesized MoS 2 NSs. To the best of our knowledge, this is the first report on the synthesis of MoS 2 NSs via the batch manner pathway to accelerate the yields.  Figure 1 illustrates the batch synthesis method of MoS 2 NSs via probe sonication method. Further, the morphology of the as-synthesized B1/B2/B3-MoS 2 NSs was examined by TEM analysis. As shown in Figure 2, low and high resolution TEM images revealed that B1-MoS 2 NSs were uniformly distributed with the clear morphology of nanosheets. After probe sonication, all the MoS 2 NSs synthesized in different batches (B1/B2/B3-MoS 2 NSs) exhibited less size as compared to the bulk MoS 2 , showing 2-3 µm size (as provided by the manufacturer). These sizes are further discussed from the DLS results in Section 2.2. HR-TEM images of MoS 2 NSs show the clear lattice fringes resulting from a good crystalline nature. The same features, such as clear morphology and crystalline nature, were also observed for B2-MoS 2 NSs and B3-MoS 2 NSs (Figure 2). EDS analysis revealed the presence of both elements (Mo and S) in the synthesized MoS 2 NSs as seen in Figure S1.

Characterization of MoS 2 NSs
To further identify the -OH formation on MoS2 NSs, TA assay was performed. Briefly, to the buffer solution (pH = 5), 50 mM H2O2, 1 mM TA and 100 μL of MoS2 NSs (500 μg mL −1 ) added. Further, the reaction mixture was incubated for 1 h at room temperature and then after the catalyst was centrifuged. The final solutions were subjected to the fluorescence measurements at excitation 315 nm. Figure 1 illustrates the batch synthesis method of MoS2 NSs via probe sonication method. Further, the morphology of the as-synthesized B1/B2/B3-MoS2 NSs was examined by TEM analysis. As shown in Figure 2, low and high resolution TEM images revealed that B1-MoS2 NSs were uniformly distributed with the clear morphology of nanosheets. After probe sonication, all the MoS2 NSs synthesized in different batches (B1/B2/B3-MoS2 NSs) exhibited less size as compared to the bulk MoS2, showing 2-3 μm size (as provided by the manufacturer). These sizes are further discussed from the DLS results in Section 3.2. HR-TEM images of MoS2 NSs show the clear lattice fringes resulting from a good crystalline nature. The same features, such as clear morphology and crystalline nature, were also observed for B2-MoS2 NSs and B3-MoS2 NSs ( Figure 2). EDS analysis revealed the presence of both elements (Mo and S) in the synthesized MoS2 NSs as seen in Figure S1.  The changes in size of MoS2 during the synthesis were monitored using Dynamic Light Scattering (DLS) analysis. Bulk MoS2 is approximately ~2-3 μm, which is in agreement with the size provided by Sigma-Aldrich. After the probe sonication for 3 h, the size of B1-MoS2 NSs was dramatically reduced to 235 ± 5 nm. Similarly, the size of B2-MoS2 NSs and B3-MoS2 NSs were  The changes in size of MoS 2 during the synthesis were monitored using Dynamic Light Scattering (DLS) analysis. Bulk MoS 2 is approximately~2-3 µm, which is in agreement with the size provided by Sigma-Aldrich. After the probe sonication for 3 h, the size of B1-MoS 2 NSs was dramatically reduced to 235 ± 5 nm. Similarly, the size of B2-MoS 2 NSs and B3-MoS 2 NSs were reduced to 189 ± 6 nm and 185 ± 5 nm, respectively, as shown in Figure 3A. However, the size of the residual content R1-MoS 2 and R2-MoS 2 were 850 ± 70 nm and 535 ± 10 nm, respectively ( Figure S2). Further, we postulated the yields of B1/B2/B3-MoS 2 NSs by vacuum drying the resulting NSs. Weighing the dry powder allowed for the estimation of the yields by using amount/weight of starting precursor at each batch. The approximate yields are as follows:~50%,~30% and~20% of MoS 2 NSs were achieved for B1-MoS 2 NSs, B2-MoS 2 NSs and B3-MoS 2 NSs, respectively. Overall, the present batch manner pathway offers a high yield (100%) water soluble MoS 2 NSs in a green manner within three tandem cycles. The changes in size of MoS2 during the synthesis were monitored using Dynamic Light Scattering (DLS) analysis. Bulk MoS2 is approximately ~2-3 μm, which is in agreement with the size provided by Sigma-Aldrich. After the probe sonication for 3 h, the size of B1-MoS2 NSs was dramatically reduced to 235 ± 5 nm. Similarly, the size of B2-MoS2 NSs and B3-MoS2 NSs were reduced to 189 ± 6 nm and 185 ± 5 nm, respectively, as shown in Figure 3A. However, the size of the residual content R1-MoS2 and R2-MoS2 were 850 ± 70 nm and 535 ± 10 nm, respectively ( Figure S2). Further, we postulated the yields of B1/B2/B3-MoS2 NSs by vacuum drying the resulting NSs. Weighing the dry powder allowed for the estimation of the yields by using amount/weight of starting precursor at each batch. The approximate yields are as follows: ~50%, ~30% and ~20% of MoS2 NSs were achieved for B1-MoS2 NSs, B2-MoS2 NSs and B3-MoS2 NSs, respectively. Overall, the present batch manner pathway offers a high yield (100%) water soluble MoS2 NSs in a green manner within three tandem cycles.

Optical Properties of MoS2 NSs
Light absorption properties of as-synthesized MoS2 NSs were studied by Ultraviolet-visible-near infrared (UV-vis-NIR) spectrophotometry. As shown in Figure 3B, B1-, B2-and B3-MoS2 NSs exhibited good light absorption in the spectral region 200-1000 nm. Commonly, the spectral absorption of MoS2 NSs exhibits four kinds of excitonic/electronic transitions, which are assigned as A, B, C, and D excitons. Briefly, the peaks are positioned at 680 nm and 610 nm and correspond to the doublet peaks of pristine MoS2, and are assigned as A and B excitonic inter-bands transitions. Spin-orbit splitting transition is responsible for the arising doublet peaks at the K peak. The C and D

Optical Properties of MoS 2 NSs
Light absorption properties of as-synthesized MoS 2 NSs were studied by Ultraviolet-visible-near infrared (UV-vis-NIR) spectrophotometry. As shown in Figure 3B, B1-, B2-and B3-MoS 2 NSs exhibited good light absorption in the spectral region 200-1000 nm. Commonly, the spectral absorption of MoS 2 NSs exhibits four kinds of excitonic/electronic transitions, which are assigned as A, B, C, and D excitons. Briefly, the peaks are positioned at 680 nm and 610 nm and correspond to the doublet peaks of pristine MoS 2 , and are assigned as A and B excitonic inter-bands transitions. Spin-orbit splitting transition is responsible for the arising doublet peaks at the K peak. The C and D transitions at 460 nm and 394 nm originated from the inter-band transition between occupied dz2 orbital and the unoccupied dxy, dx2−y2, and dxz,yz orbitals [30]. In addition, two other peaks appeared at 394 nm and 460 nm, which can be attributed to the optical absorption of smaller sized nanosheets due to the quantum effects in MoS 2 NSs [31]. Moreover, the absorption spectra of the synthesized MoS 2 NSs are in agreement with spectral properties reported in the literature [20,27].
Additionally, we used PAA as a biocompatible/water soluble ligand, which is believed to be surface coordinated with MoS 2 NSs. To confirm the presence of PAA on MoS 2 NSs, we carried out Zeta potential measurements. As shown in Figure 4A, the synthesized B1-, B2-and B3-MoS 2 NSs displayed negative surface charge corresponding to the carboxylic group (COO − ) in PAA, which was surface bonded on MoS 2 NSs. Besides zeta potential measurements, we also performed the FTIR analysis to confirm the presence of PAA on MoS 2 NSs. As shown in Figure S3, absorption bands of PAA on B1/B2/B3-MoS 2 NSs were identified at 2951, 1714, and 1453 cm −1 , which correspond to the vibrational modes of CH 2 , C=O, and C-O, respectively. Whereas for the FT-IR spectra of bulk MoS 2 , we did not observe these characteristic peaks corresponding to the PAA. The successful surface attachment of PAA greatly offered a water soluble nature to MoS 2 NSs. The optical images of water soluble B1-, B2-and B3-MoS 2 NSs are shown in Figure 4B. It is readily seen from Figure 4B that the three prepared NSs exhibit high dispersibility in water.
PAA on B1/B2/B3-MoS2 NSs were identified at 2951, 1714, and 1453 cm , which correspond to the vibrational modes of CH2, C=O, and C-O, respectively. Whereas for the FT-IR spectra of bulk MoS2, we did not observe these characteristic peaks corresponding to the PAA. The successful surface attachment of PAA greatly offered a water soluble nature to MoS2 NSs. The optical images of water soluble B1-, B2-and B3-MoS2 NSs are shown in Figure 4B. It is readily seen from Figure 4B that the three prepared NSs exhibit high dispersibility in water.  Figure 5A shows the crystallinity and phase purity of the synthesized MoS2 NSs by x-ray diffraction (XRD). Further, the synthesized MoS2 NSs XRD patterns were compared with the bulk MoS2. The XRD results clearly revealed that the formation of nanosheets results from reducing the (002) diffraction plane in all synthesized MoS2 NSs as compared to the bulk MoS2. All the synthesized B1/B2/B3-MoS2 NSs presented diffraction peaks of (002), (100), (103), (104), (105) and (008), which reflected the pure hexagonal phase of MoS2 NSs (magnified XRD spectra shown in Figure S4). These results are in alignment with the reported literature [20,27]. It is worth mentioning that we did not observe any peaks corresponding to impurities, indicating that as synthesized MoS2 NSs possessed good crystallinity with high purity. Likewise, Raman spectra of the as-synthesized B1-MoS2 NSs were recorded. Two peaks corresponding to the E2g (the in-plane vibration mode) and the A1g (out-ofplane vibration mode) were observed for the MoS2 Nanosheets. These peaks were observed for the as-prepared B1/B2/B3-MoS2 NSs at 381 cm −1 , 384 cm −1 , 382 cm −1 (for E2g) and 406.8 cm −1 , 408.7 cm −1 , 406 cm −1 (A1g), respectively, as shown in Figure 5B. These values correspond to the characteristic peaks of MoS2. Moreover, based on the Raman spectra, the reduction in bulk MoS2 to NSs clearly shows that the distance between two peaks E2g and A1g is shorter than bulk MoS2 [32]. The energy difference between the two peaks is in the range ~24.2-25.8 cm −1 which corresponds to the presence of 5-6 layers in the synthesized MoS2 NSs [20,33]. Lee et al. worked on the weakness of the van der Waals interlayers interaction and how these forces can affect the bonding and the lattice vibrations of few layered MoS2 [33]. Compared to graphene, MoS2 presents highly stable layers that do not present complexities associated to the non-adiabatic electron-phonon coupling as it is in the case of graphene. Further, we compared our results with the existing ultra-sonication methods. As shown in  Figure 5A shows the crystallinity and phase purity of the synthesized MoS 2 NSs by X-ray diffraction (XRD). Further, the synthesized MoS 2 NSs XRD patterns were compared with the bulk MoS 2 . The XRD results clearly revealed that the formation of nanosheets results from reducing the (002) diffraction plane in all synthesized MoS 2 NSs as compared to the bulk MoS 2 . All the synthesized B1/B2/B3-MoS 2 NSs presented diffraction peaks of (002), (100), (103), (104), (105) and (008), which reflected the pure hexagonal phase of MoS 2 NSs (magnified XRD spectra shown in Figure S4). These results are in alignment with the reported literature [20,27]. It is worth mentioning that we did not observe any peaks corresponding to impurities, indicating that as synthesized MoS 2 NSs possessed good crystallinity with high purity. Likewise, Raman spectra of the as-synthesized B1-MoS 2 NSs were recorded. Two peaks corresponding to the E2g (the in-plane vibration mode) and the A1g (out-of-plane vibration mode) were observed for the MoS 2 Nanosheets. These peaks were observed for the as-prepared B1/B2/B3-MoS 2 NSs at 381 cm −1 , 384 cm −1 , 382 cm −1 (for E2g) and 406.8 cm −1 , 408.7 cm −1 , 406 cm −1 (A1g), respectively, as shown in Figure 5B. These values correspond to the characteristic peaks of MoS 2 . Moreover, based on the Raman spectra, the reduction in bulk MoS 2 to NSs clearly shows that the distance between two peaks E2g and A1g is shorter than bulk MoS 2 [32]. The energy difference between the two peaks is in the range~24.2-25.8 cm −1 which corresponds to the presence of 5-6 layers in the synthesized MoS 2 NSs [20,33]. Lee et al. worked on the weakness of the van der Waals interlayers interaction and how these forces can affect the bonding and the lattice vibrations of few layered MoS 2 [33]. Compared to graphene, MoS 2 presents highly stable layers that do not present complexities associated to the non-adiabatic electron-phonon coupling as it is in the case of graphene. Further, we compared our results with the existing ultra-sonication methods. As shown in Table S1, most of the ultra-sonication assisted methods extensively utilize toxic organic solvents, such as NMP/Isopropanol (IPA)/ethanol, for the exfoliation of bulk MoS 2 to MoS 2 NSs. In some other reports, even though offering a greener pathway, longer exfoliation times and lower yields are still considered as limiting factors. Consequently, the present Tandem process method can significantly overcome the mentioned limitations and produce high yield water soluble MoS 2 NSs.

XRD and Raman Spectra Analysis of MoS 2 NSs
Catalysts 2020, 10, x FOR PEER REVIEW 7 of 11 Table S1, most of the ultra-sonication assisted methods extensively utilize toxic organic solvents, such as NMP/Isopropanol (IPA)/ethanol, for the exfoliation of bulk MoS2 to MoS2 NSs. In some other reports, even though offering a greener pathway, longer exfoliation times and lower yields are still considered as limiting factors. Consequently, the present Tandem process method can significantly overcome the mentioned limitations and produce high yield water soluble MoS2 NSs.

Peroxidase Mimetic Properties of MoS2 NSs.
Various nanomaterials were successfully explored in environmental and biomedical applications [34][35][36]. Recently, more research was focused on nano-enzymes-particularly enzyme peroxidase mimicking properties-due to its great promise in biomedical applications, such as

Peroxidase Mimetic Properties of MoS 2 NSs
Various nanomaterials were successfully explored in environmental and biomedical applications [34][35][36]. Recently, more research was focused on nano-enzymes-particularly enzyme peroxidase mimicking properties-due to its great promise in biomedical applications, such as excellent substrate specificity and superior catalytic power. However, unfortunately, low sensitivity, low operational stability, and high costs of catalyst preparation and purifications limit their practical usage [37,38]. Therefore, herein we examined the enzyme peroxidase-like activities of present green and water soluble B1-MoS 2 NSs by catalytic oxidation of TMB to oxidized TMB (ox-TMB) in the presence and absence of H 2 O 2 at room temperature. TMB is a chromogenic peroxidase probe which can react with ·OH radicals to form its oxidation product during the enzymatic degradation of H 2 O 2 [39]. Further, ox-TMB is blue, and was monitored by measuring the absorbance at 652 nm. The detailed schematic illustration of peroxidase activity on MoS 2 NSs is shown in Figure 6A. As shown in Figure 6B, the buffer + TMB + H 2 O 2 + MoS 2 NSs group exhibited excellent peroxidase properties and resulted in the radical enhancement detected by monitoring the absorption peak of ox-TMB at 652 nm. In contrast, other groups, such as buffer + TMB, buffer + TMB+ MoS 2 NSs, buffer + H 2 O 2 + MoS 2 NSs, did not show significant absorption enhancement. Notably, in the buffer + TMB + H 2 O 2 group, a small absorption enhancement was observed that might be due to the slight decomposition of H 2 O 2 at room temperature. Subsequently, we also measured the time dependent peroxidase activity of MoS 2 NSs at standard condition (buffer + TMB + H 2 O 2 + MoS 2 NSs). Figure 6C shows that the absorption of oxidized TMB (ox-TMB) at 652 nm gradually increased with prolonged reaction time. The excellent peroxidase activity can be visualized by the blue color. As shown in Figure 6D, the buffer + TMB + H 2 O 2 + MoS 2 NSs group displayed bright blue color compared to other control groups, confirming the formation of ox-TMB product. Besides B1-MoS 2 NSs, we also evaluated the peroxidase activity of B2-and B3-MoS 2 NSs at identical conditions (i.e., same concentration of NSs, 100 µL of MoS 2 NSs (~500 µg mL −1 )). Our results show that B2-and B3-MoS 2 NSs also exhibit an excellent peroxidase activity, similar to B1-MoS 2 NSs ( Figure S5).

Peroxidase Activity Mechanism
To further understand the peroxidase activity mechanism of the present MoS2 NSs in the oxidation of TMB to ox-TMB in the presence of H2O2, we carried out some mechanistic controls. One possible mechanism for the peroxidase-like property of the MoS2 NSs might be its ability to

Peroxidase Activity Mechanism
To further understand the peroxidase activity mechanism of the present MoS 2 NSs in the oxidation of TMB to ox-TMB in the presence of H 2 O 2 , we carried out some mechanistic controls. One possible mechanism for the peroxidase-like property of the MoS 2 NSs might be its ability to decompose H 2 O 2 species to produce hydroxyl radicals (-OH). To further confirm the generated -OH, here we used Terephthalic acid (TA) assay [40]. As shown in Figure 7A, TA can react with -OH and produce highly fluorescent 2-hydroxy terephthalic acid (2-TAOH), which shows an emission intensity around 430 nm after excitation at 315 nm. As shown in Figure 7B, the emission intensity increased in the presence of TA + H 2 O 2 + MoS 2 NSs; whereas other control groups, such as TA alone and TA + MoS 2 NSs, did not exhibit any significant enhancement in emission intensity. These results clearly indicate that the generation of hydroxyl radicals in the presence of MoS 2 NSs are key species to decompose the H 2 O 2 to water and oxygen. Notably, the TA + H 2 O 2 group also shows a small enhancement in the emission intensity. This can be attributed to the H 2 O 2 decomposition at room temperature, which is well aligned with our TMB oxidation assay results. Overall, from TMB oxidation and terephthalic acid assay results, we strongly believe that the present synthesized water soluble MoS 2 NSs exhibits an excellent peroxidase enzyme, mimicking by itself without coupling it with other co-catalyst as is the actual case. Thus, present MoS 2 NSs can be useful to detect environmentally toxic chemicals, such as H 2 O 2 , via naked eye detection, as well as in biomedical cancer diagnostic applications.

Conclusions
In summary, high yield water soluble MoS2 NSs were successfully synthesized from bulk MoS2 material by using a simple probe sonication method. The sequential batch/tandem process could significantly improve the yield of MoS2 NSs and reach up to 100% in a green manner. Subsequently, synthesized B1/B2/B3-MoS2 NSs were well characterized. Moreover, the optical properties of present synthesized MoS2 NSs still remains the same as the MoS2 NSs, which were synthesized by reported harsh condition methods. Thus, it is a clearly evident that the present method is a good and green alternative for the existing low yield and non-green methods. Furthermore, MoS2 NSs exhibited excellent intrinsic peroxidase mimetic enzyme activities at ambient conditions. Mechanistic studies revealed that the MoS2 NSs playing a key role in generation of hydroxyl radicals by decomposing the H2O2 efficiently. Overall, we strongly envision that the present cost effective, green and eco-friendly approach is able to seize significant attention to fabricate a high yield synthesis of metal chalcogenide nanosheets for the future energy, environment and biomedical applications.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Energy dispersive spectroscopy (EDS) elemental analysis of B1-MoS2 NSs, Figure S2: DLS size distribution analysis of bulk, R1 and R2-MoS2, Figure S3: FTIR spectra of bulk MoS2 and as synthesized B1, B2 and B3-MoS2 NSs, Figure  S4: XRD spectra of as synthesized B1, B2 and B3-MoS2 NSs, Figure S5: TMB oxidation assay for evaluating the peroxidase activity of B2 and B3-MoS2 NSs at identical conditions and Table S1: Literature summary of methods for synthesizing the water soluble MoS2 nanosheets via sonication assisted methods by using bulk MoS2.

Synthesis of MoS 2 NSs
Commercially available bulk MoS 2 with an average diameter of 2-3 µm (Sigma-Aldrich, St. Louis, MO, USA) was used as a starting precursor. To attain MoS 2 NSs with more biocompatible/water soluble, Poly (acrylic acid) (PAA) was added prior to the probe sonication. First, aqueous solution of bulk MoS 2 (300 mg) and PAA (100 mg) were subjected to probe sonication for a period of 3 h (QSONICA probe sonicator, 25 W, 31 amplitude). Subsequently, the solution was centrifuged (5000 rpm, 3 min), and the supernatant solution containing MoS 2 NSs and the residue were collected separately and noted as batch-1 MoS 2 NSs (B1-MoS 2 NSs) and residue 1 MoS 2 (R1-MoS 2 ), respectively. Successively, we added the appropriate amount of PAA to the R1-MoS 2 and performed probe sonication for the period of another 3 h to achieve a batch-2 MoS 2 NSs (B2-MoS 2 NSs) in supernatant and the residue 2 MoS 2 (R2-MoS 2 ). Finally, R2-MoS 2 NSs were subjected to sonication for 3 h by adding PAA and Catalysts 2020, 10, 1009 8 of 11 achieved a batch 3 MoS 2 NSs (B3-MoS 2 NSs). Overall, the present successive batch manner process significantly offered a high yield of MoS 2 NSs (100%) within three tandem cycles of probe sonication. Finally, samples were stored in water for further applications.

Characterization
The morphology and elemental analysis of synthesized MoS 2 NSs was examined by using HR-TEM and Elemental Dispersive Spectroscopy (EDS) (JEOL JEM-2100, 200 kV, Tokyo, Japan), respectively. MoS 2 NSs sample droplets were dried on a Lacey carbon grid for TEM analysis. A UV-visible-NIR analysis of MoS 2 NSs aqueous solution was carried out by using a UV-vis-NIR spectrophotometer (JASCO, Tokyo, Japan). DLS Nanotrac Wave (Microtrac Inc., Philadelphia, PA, USA) was used for particle size analysis and zeta potentials of MoS 2 NSs. Raman spectra were measured by using micro-Raman spectrometer (HORIBA, LabRAM, HR800, Kyoto, Japan). FT-IR data were recorded on a Bruker Tensor 27 FT-IR (DTGS detector and KBR beam splitter, Bruker, Vertex 80v and Tensor 27, Billerica, MA, USA).

Enzyme Peroxidase Activity Measurement
Enzyme peroxide mimetic properties of MoS 2 NSs were evaluated by using TMB assay. Briefly, 50 µL of 50 mM H 2 O 2 and 100 µL of 50 mM TMB were mixed in 3.0 mL of sodium acetate buffer (0.2 M, pH 4). To this solution, 100 µL of MoS 2 NSs (500 µg mL −1 ) solution were added and incubated for 30 min at room temperature. Afterwards, samples were centrifuged (10,000 rpm, 5 min) and measured the absorbance at 652 nm by using UV-visible spectrophotometer. In a similar way control experiments were carried out such as buffer + TMB, buffer + TMB+ MoS 2 NSs, buffer + H 2 O 2 + MoS 2 NSs.

Identification of Hydroxyl Radicals (•OH)
To further identify the -OH formation on MoS 2 NSs, TA assay was performed. Briefly, to the buffer solution (pH = 5), 50 mM H 2 O 2 , 1 mM TA and 100 µL of MoS 2 NSs (500 µg mL −1 ) added. Further, the reaction mixture was incubated for 1 h at room temperature and then after the catalyst was centrifuged. The final solutions were subjected to the fluorescence measurements at excitation 315 nm.

Conclusions
In summary, high yield water soluble MoS 2 NSs were successfully synthesized from bulk MoS 2 material by using a simple probe sonication method. The sequential batch/tandem process could significantly improve the yield of MoS 2 NSs and reach up to 100% in a green manner. Subsequently, synthesized B1/B2/B3-MoS 2 NSs were well characterized. Moreover, the optical properties of present synthesized MoS 2 NSs still remains the same as the MoS 2 NSs, which were synthesized by reported harsh condition methods. Thus, it is a clearly evident that the present method is a good and green alternative for the existing low yield and non-green methods. Furthermore, MoS 2 NSs exhibited excellent intrinsic peroxidase mimetic enzyme activities at ambient conditions. Mechanistic studies revealed that the MoS 2 NSs playing a key role in generation of hydroxyl radicals by decomposing the H 2 O 2 efficiently. Overall, we strongly envision that the present cost effective, green and eco-friendly approach is able to seize significant attention to fabricate a high yield synthesis of metal chalcogenide nanosheets for the future energy, environment and biomedical applications.