2D CTAB-Mose2 Nanosheets and 0D Mose2 Quantum Dots

nanomaterials

Article 2D CTAB-MoSe2 Nanosheets and 0D MoSe2 Quantum Dots: Facile Top-Down Preparations and Their Peroxidase-Like Catalytic Activity for Colorimetric Detection of Hydrogen Peroxide

1,2, , 1, 1 1,2 Da-Ren Hang * † , Ya-Qi Pan †, Krishna Hari Sharma , Mitch M. C. Chou , Sk Emdadul Islam 3 , Hui-Fen Wu 4 and Chi-Te Liang 3,* 1 Department of Materials and Optoelectronic Science, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; [email protected] (Y.-Q.P.); [email protected] (K.H.S.); [email protected] (M.M.C.C.) 2 Center of Crystal Research, National Sun Yat-sen University, Kaohsiung 80424, Taiwan 3 Department of Physics, National Taiwan University, Taipei 10617, Taiwan; [email protected] 4 Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; [email protected] * Correspondence: [email protected] (D.-R.H.); [email protected] (C.-T.L.); Tel.: +886-7-5252000 (ext. #4066) (D.-R.H.); +886-2-33665129 (C.-T.L.) These authors contributed equally to this work. † Received: 29 September 2020; Accepted: 13 October 2020; Published: 16 October 2020

Abstract: We report the facile and economic preparation of two-dimensional (2D) and 0D MoSe2 nanostructures based on systematic and non-toxic top-down strategies. We demonstrate the intrinsic peroxidase-like activity of these MoSe2 nanostructures. The catalytic processes begin with facilitated decomposition of H2O2 by using MoSe2 nanostructures as peroxidase mimetics. In turn, a large amount of generated radicals oxidizes 3,3,5,5-tetramethylbenzidine (TMB) to produce a visible color reaction. The enzymatic kinetics of our MoSe2 nanostructures complies with typical Michaelis–Menten theory. Catalytic kinetics study reveals a ping–pong mechanism. Moreover, the primary radical responsible for the oxidation of TMB was identiﬁed to be O˙ 2− by active species-trapping experiments. Based on the peroxidase mimicking property, we developed a new colorimetric method for H2O2 detection by using 2D and 0D MoSe2 nanostructures. It is shown that the colorimetric sensing capability of our MoSe2 catalysts is comparable to other 2D materials-based colorimetric platforms. For instance, the linear range of H2O2 detection is between 10 and 250 µM by using 2D functionalized MoSe2 nanosheets as an artiﬁcial enzyme. Our work develops a systematic approach to use 2D materials to construct novel enzyme-free mimetic for a visual assay of H2O2, which has promising prospects in medical diagnosis and food security monitoring.

Keywords: MoSe2 quantum dots; peroxidase-like activity; hydrogen peroxide; few-layer MoSe2 nanosheets; colorimetric detection

1. Introduction

The development of convenient and sensitive detection of hydrogen peroxide (H2O2) is in high demand in the ﬁelds of food security, environmental monitoring and biochemical analysis. H2O2, produced from the incomplete reduction of O2, can be found as a byproduct in diverse biological processes. Higher amounts than normal of cellular H2O2 have been linked to the risk of a few diseases including Parkinson’s disease and cancer development [1,2]. Thus, it is of practical importance to analyze and detect H2O2 by a simple, sensitive and economic method. So far, various

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techniques for H2O2 determination have been explored, such as fluorometry [3,4], cellular imaging [5], electrochemistry [6,7], and the colorimetric method [8,9]. Among these approaches, the colorimetric method has drawn a lot of attention due to its convenient operation, visibility, facile miniaturization, and low cost [10,11]. In this respect, natural enzymes were extensively used for the detection of H2O2 due to its catalysis capability under mild conditions. Nevertheless, these conventional enzymes usually suffer from the disadvantages of low stability against harsh conditions and high expenditures for preparation and purification. Consequently, researchers actively sought artificial enzyme-mimic materials without these shortcomings. Nanomaterials are currently regarded as a rich source to synthesize desired alternative mimic enzymes with the benefits of low cost, plentiful raw materials, and ease in purification and storage. Many nanomaterials with intrinsic enzyme-mimetic activity analogous to that of natural enzymes were fabricated, such as metal organic frameworks [12], Pt nanoclusters [13], silver nanoparticles [14], and gold nanoparticles [15]. Although enormous progress has been made, the discovery and development of novel promising artificial peroxidase mimics is still in urgent need. With the persistent advancement of nanotechnology and materials science, two-dimensional (2D) nanomaterials beyond graphene have received much attention because of many fascinating chemical and physical properties. The transition metal dichalcogenides (TMDs), a family of layered compound materials consisting of 2D sheets weakly bound by van der Waals interactions, is the most renowned group of emerging 2D materials. They have shown huge promise in a wide range of applications. In particular, TMD nanostructures have shown good potential in biomedical applications due to their large surface area, low cytotoxicity, and higher structural rigidity than other 2D nanomaterials. For instance, it was found that TMDs exhibited lower cytotoxicity than typical graphene and its analogues [16]. As for structural rigidity, commonly used graphene and hexagonal boron-nitride have 2 relatively low flexural rigidity around 3.5 eV Å /atom. On the other hand, these values for MoS2 and 2 2 WS2 are 27 eV Å /atom and 30 eV Å /atom, respectively [17]. These properties should make 2D TMDs appropriate for biomedical applications. Finally, TMDs can remain stable in liquid due to the lack of dangling bonds on the surface, which supports their use in biomedical applications. Molybdenum disulfide (MoS2), the most prominent member of the TMD family, possesses distinctive properties and has found diverse successful applications in electronics [18], energy devices [19], photocatalysis [20], and sensors [21,22]. In particular, the peroxidase-like catalytic ability of a few MoS2 nanostructures has been shown by researchers [23,24]. While a large amount of investigations is devoted to MoS2, considerably less attention has been devoted to molybdenum diselenide (MoSe2)[25]. 2H-MoSe2, also with a graphene-like lamellar structure, is a semiconductor whose bandgap energy increases from ~1.1 eV in bulk to ~1.55 eV in ultrathin form with atomic thickness. In a previous comparative study, Gholamvand and coworkers concluded that MoSe2 is the most effective electrocatalyst among TMDs [26]. Recent studies also showed that few-layered MoSe2 nanosheets (NSs) could be a promising candidate with peroxidase-like activity and good biocompatibility [27,28]. Moreover, inspired by the fact that selenium-containing enzymes are generally prevalent in the biosphere and their active sites usually involve selenium, we thus turned our attention to the investigation of nanoscale MoSe2 in this respect. Even though MoSe2 is expected to function as efficient peroxidase mimetics for colorimetric detection, so far little progress has been made in this respect. Its extended topics, such as surface modification and variation in dimensionality, were rarely studied for use in colorimetric detection. Here, we intend to fill the gap along this exploration. For instance, TMD in quantum dot (QD) form deserves more investigation because their pronounced quantum confinement effects (QCE) and edge effects further aid applications in catalysts and sensing [29,30]. Significant enhancement in photoluminescence (PL) quantum efficiency by QCE is favorable to develop a sensor by optical means [31,32]. Moreover, a few optical properties in the strong coupling regime for semiconductor QDs and nanostructures could be implemented to strengthen the functionality of biosensors [33–35]. Nanomaterials 2020, 10, 2045 3 of 19

Liquid-phase synthesis routes are suitable to produce TMD nanostructures in large quantity at low cost. In general, solution-based synthesis approaches can be divided into “top-down” methods and “bottom-up” methods. For bottom-up wet-chemical synthesis methods, specific precursors are needed and a high-temperature and high-pressure environment is required. Among top-down approaches, liquid phase exfoliation (LPE) is a powerful technique to efficiently exfoliate various types of layered crystals into few-layer nanosheets or even QDs [36,37]. The basic protocol of LPE technique is very general and only parent crystal is needed instead of the need for specific precursors in bottom-up chemical methods. In this paper, we prepared two types of low-dimensional MoSe2 nanostructures based on top-down techniques. In the first case, LPE-derived 2D MoSe2 nanosheets were functionalized with cetyltrimethyl ammonium bromide (CTAB). It is expected that the CTAB surfactant could aid exfoliation efficiency and prevent 2D nanosheets from restacking or agglomeration [38]. Secondly, 0D MoSe2 QDs were obtained based on top-down exfoliation approaches. For usual 0D TMD QDs derived from LPE, longer ultrasonication time and higher power were typically adopted, which could easily deform the microstructure and result in higher density of surface traps states. As surface-to-volume ratio is rather large for QDs, these deep trap states pose negative impact to many applications. In our work, a novel and efficient ultrasonication-assisted solvothermal exfoliation technique is firstly introduced for preparing small size and high-quality MoSe2 QDs. In the initial probe-assistant ultrasonication exfoliation phase, MoSe2 bulk is broken into nanosheets or nanoparticles by the acoustic cavitation effect. The sonication time is kept short in this stage. Next, the solvothermal treatment with a polar solvent continuously weakens the van der Waals forces of thinned MoSe2 and break it up into small 0D QDs. We show that our CTAB-modified MoSe2 NSs and 0D MoSe2 QDs are able to efficiently catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a colored product. On this basis, we have successfully demonstrated novel platforms for colorimetric detection of H2O2. It is found that the sensing capability of our MoSe2 systems is comparable to those of published 2D materials-based platforms. As far as we know, it is the first time the potential of CTAB-functionalized MoSe2 nanosheets and 0D MoSe2 QDs have been explored for colorimetric detection of H2O2. Toxic and high boiling point solvents were not used in our synthesis methods thus our protocols also provide a non-toxic and systematic way to fabricate new 2D nanomaterials for construction of novel colorimetric sensors and for use in extended applications.

2. Materials and Methods

2.1. Materials and Reagents

MoSe2 powder (99.9%), 3,3,5,5-tetramethylbenzidine (TMB), and isopropanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cetyltrimethyl ammonium bromide (CTAB) was purchased from Millipore (Burlington, MA, USA). Acetic acid, sodium acetate anhydrous, hydrochloric acid, and hydrogen peroxide (35%) were obtained from Alfa Aesar (Tewksbury, MA, USA). These chemicals were of analytical purity and were used as received. Deionized water (DI water) was used as a solvent throughout.

2.2. Methods

2.2.1. Preparation of Surfactant Modiﬁed Two-Dimensional (2D) MoSe2 Nanosheets (NSs)

The few-layer CTAB-MoSe2 NSs synthesis protocol is based on the grinding-assisted liquid phase exfoliation approach [37]. The synthesis protocol of 2D CTAB-MoSe2 NSs is illustrated in Figure1a. First, 100 mg of MoSe2 powder and 50 mg of CTAB were ground for 30 min. The mixture was subsequently dispersed in 20 mL of DI water and stirred for 1 h at 90 ◦C in a beaker. The solution was probe sonicated for 3 h with a horn sonic tip (Qsonica CL-334) at a power output of 125 W in a water-cooled bath at 20 ◦C. Residual sediment and thick ﬂakes were further removed by centrifugation Nanomaterials 2020, 10, 2045 4 of 19 at 4000 rpm for 20 min. The upper portion of the supernatant was taken for the next centrifugation for 20 min at the speed of 9000 rpm. The upper portion of the resultant supernatant was transferred to a refrigerator at 4 ◦C for storage. Finally, the 2D CTAB-MoSe2 NS product was collected by another centrifugationNanomaterials 2020 at, 10 the, x FOR speed PEER of REVIEW 9000 rpm. 5 of 19

FigureFigure 1.1. TheThe schematicschematic illustrationsillustrations forfor thethe preparationspreparations ofof ((aa)) two-dimensionaltwo-dimensional (2D)(2D) cetyltrimethylcetyltrimethyl ammoniumammonium bromidebromide (CTAB)-MoSe(CTAB)-MoSe22 nanosheetsnanosheets andand ((bb)) 0D0D MoSeMoSe22 quantumquantum dotsdots (QDs).(QDs).

2.2.2.3. Results Preparation and Discussion of 0D MoSe 2 QDs

The 0D MoSe2 QDs were obtained according to the ultrasonication-assisted solvothermal exfoliation3.1. Structural technique. Studies Figure1b depicts the synthesis procedure of 0D MoSe 2 QDs. Typically, 100 mg of MoSe powder was dispersed in 60 mL of 50 vol% Isopropyl alcohol (IPA) DI water mixture The microstructure2 of the resultant MoSe2 nanomaterials is characterized/ by transmission inelectron a beaker. microscopy Then, the (TEM). solution As was shown probe in sonicated Figure 2a, for the 1 has with-obtained a horn MoSe sonic2 tipQDs (Qsonica reveal a CL-334) spherical at ashape power without output noticeable of 150 W inaggregation, a water-cooled indicating bath atthe 20 successful◦C. Afterward, formation the resultantof highlydispersions dispersive QDs. was furtherThe statistic transferredal analysis to a 60 of mL particle Teflon-lined size distribution autoclave and was reacted conducted at 200 ◦ byC for counting 24 h. After 700 the QD autoclave profiles cooledmeasured naturally, by TEM. the The supernatant outcome is containing displayed MoSe by the2 QDshistogram was centrifuged in Figure 2b for along 30 min with at its the calculated speed of 9000Gaussian rpm. fitting After that,curve. the The upper average portion size of of the the supernatant 0D QDs was was determined collected for to secondbe 4.5 nm centrifugation and up to 80% for 15QDs min have with their the same diameters rotation in speed.the narrow Finally, range the MoSe from2 3QD to product6 nm. A was high collected-resolution and thenTEM stored(HRTEM) in a refrigeratorimage of a single at 4 ◦C MoSe for use.2 QD in the inset of Figure 2a reveals that the lattice spacing of the synthesized QD was 0.23 nm, which coincides with the (103) plane of hexagonal MoSe2 [40]. 2.3. Characterization Transmission electron microscopy (TEM) images and high-resolution TEM (HRTEM) images were taken by using a JEOL-3010 transmission electron microscope at an accelerating voltage of 200 kV (Tokyo, Japan). The elemental composition and bonding configuration analysis were carried out by an ultrahigh vacuum JEOL JPS-9010 X-ray photoelectron spectrometer (XPS) equipped with a multi-channel detector. The detected binding energies were calibrated to the C1 s peak at 284.8 eV of the surface adventitious carbon. The ultraviolet–visible (UV–vis) spectra were recorded with a Jasco V-730 spectrophotometer (USA) with a standard 10-mm path length quartz cuvette (Easton, MD, USA). The photoluminescence spectra were measured using a Hitachi F-4500 florescence spectrophotometer connected to a 150 W Xenon lamp as the excitation source. The Raman spectra were recorded in ambient conditions using a confocal microscope linked to a Horiba iHR320 spectrometer (Piscataway, NJ, USA) [39].

Figure 2. (a) Transmission electron microscope (TEM) image of synthesized MoSe2 QDs. The inset

shows representative high-resolution TEM (HRTEM) image of the MoSe2 QD. (b) Statistical analysis

of the size of MoSe2 QDs measured by TEM and its Gaussian fitting curve.

Next, Figure 3a shows the representative TEM image of the as-prepared 2D CTAB-MoSe2 nanosheet, in which a sheet-like structure can be found. The HRTEM image in the inset of Figure 3a resolves lattice fringes with lattice spacing of 0.28 nm, which is in agreement with the (100) plane of 2H-MoSe2. As shown in Figure 3b, the selected-area electron diffraction (SAED) pattern again verifies

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2.4. Peroxidase-Mimetic Activity of MoSe2 Quantum Dots (QDs) and 2D Cetyltrimethyl Ammonium Bromide (CTAB)-MoSe2 NSs To evaluate the catalytic peroxidase-like properties, a blue product was generated by the peroxidase substrate TMB in the presence of H2O2. In a typical experiment, 600 µL 0.1 mg/mL MoSe2 catalyst was incubated with 500 µL acetate buffer solution (0.1 M, pH 3.6), 200 µLH2O2 (10 mM), 200 µL TMB (5 mM) and 60 µLH2O at room temperature for 15 min. Then, the absorbance of the mixture was measured by a Jasco V-730 UV-visible spectrophotometer (Easton, MD, USA). For H2O2 detection, different contents of H2O2 were incubated with 600 µL 0.1 mg/mL MoSe2 catalyst, 500 µL acetate bufferFigure solution 1. The (0.1 schematic M, pH 3.6) illustrations and 200 forµL the TMB preparations (5 mM) at of room (a) two temperature-dimensiona forl (2D 15) min,cetyltrimethyl and then the absorbanceammonium at 652 bromide nm was (CTAB recorded.)-MoSe2 nanosheets and (b) 0D MoSe2 quantum dots (QDs).

3. Results and Discussion

3.1. Structural Studies The microstructure of the resultant MoSe nanomaterials is characterized by transmission electron The microstructure of the resultant MoSe2 2 nanomaterials is characterized by transmission microscopy (TEM). As shown in Figure2a, the as-obtained MoSe QDs reveal a spherical shape without electron microscopy (TEM). As shown in Figure 2a, the as-obtained2 MoSe2 QDs reveal a spherical noticeableshape without aggregation, noticeable indicating aggregation, the successfulindicating formationthe successful of highly formation dispersive of highly QDs. dispersive The statistical QDs. analysisThe statistic of particleal analysis size distribution of particle size was distribution conducted by was counting conducted 700 QD by proﬁles counting measured 700 QD by profiles TEM. Themeasured outcome by TEM. is displayed The outcome by the is histogram displayed in by Figure the histogram2b along in with Figure its calculated2b along with Gaussian its calculated ﬁtting curve.Gaussian The fitting average curve. size The of theaverage 0D QDs size was of the determined 0D QDs was to be determined 4.5 nm and to up be to 4.5 80% nm QDs and haveup to their80% diametersQDs have intheir the diameters narrow range in the from narrow 3 to 6range nm. Afrom high-resolution 3 to 6 nm. A TEM high (HRTEM)-resolution image TEM of(HRTEM) a single MoSe QD in the inset of Figure2a reveals that the lattice spacing of the synthesized QD was 0.23 nm, image2 of a single MoSe2 QD in the inset of Figure 2a reveals that the lattice spacing of the synthesized which coincides with the (103) plane of hexagonal MoSe [40]. QD was 0.23 nm, which coincides with the (103) plane of2 hexagonal MoSe2 [40].

Figure 2.2. ((a)) TransmissionTransmission electronelectron microscopemicroscope (TEM)(TEM) imageimage ofof synthesizedsynthesized MoSeMoSe22 QDs.QDs. The insetinset shows representativerepresentative high-resolution high-resolution TEM TEM (HRTEM) (HRTEM image) image of of the the MoSe MoSe2 QD.2 QD. (b )(b Statistical) Statistical analysis analysis of theof the size size of MoSeof MoSe2 QDs2 QDs measured measured by by TEM TEM and and its its Gaussian Gaussian ﬁtting fitting curve. curve.

Next, Figure3 3aa shows shows the the representative representative TEM TEM image image of of the the as-prepared as-prepared 2D 2D CTAB-MoSe CTAB-MoSe22 nanosheet, in which a sheet-likesheet-like structure can bebe found.found. The HRTEM image in the inset of Figure3 3aa resolves lattice fringes with with lattice lattice spacing spacing of of 0.28 0.28 nm, nm, which which is isin in agreement agreement with with the the (100) (100) plane plane of of2H 2H-MoSe-MoSe2. As2. shown As shown in Figure in Figure 3b, the3b, selected the selected-area-area electron electron diffraction di ffraction (SAED) (SAED) pattern patternagain verifies again verifies the diffraction pattern from the 2H-MoSe2 crystal and demonstrates the good crystallinity of the exfoliated nanosheets. Atomic force microscopy (AFM) measurement was adopted to further confirm the 2D nature of CTAB-MoSe2 nanosheets. A representative AFM image is shown in Figure3c and the height profile along the black line was measured. It was found that the nanosheet thickness ranges from 4.2 to 4.7 nm, which confirms the 2D few-layered structure. Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 19 the diffraction pattern from the 2H-MoSe2 crystal and demonstrates the good crystallinity of the exfoliated nanosheets. Atomic force microscopy (AFM) measurement was adopted to further confirm the 2D nature of CTAB-MoSe2 nanosheets. A representative AFM image is shown in Figure 3c and theNanomaterials height profile2020, 10 along, 2045 the black line was measured. It was found that the nanosheet thickness ranges6 of 19 from 4.2 to 4.7 nm, which confirms the 2D few-layered structure.

Figure 3. ((a)) TEM image of 2D CTAB-MoSeCTAB-MoSe2 prepared by liquid phase exfoliation ( (LPE).LPE). The inset shows itsits HRTEMHRTEM image image ( b()b The) The selected selected area area electron electron diﬀ diffractionraction pattern pattern (SAED). (SAED). (c) The (c) atomicThe atomic force

forcemicroscopy microscopy image image of as-obtained of as-obtained 2D CTAB-MoSe 2D CTAB2 -andMoSe the2 and corresponding the corresponding height proﬁle height along profile the along black theline black in the line image. in the image.

3.2. Surface Surface Elemental and Valence State Analysis To further shed light on the surface chemical components and oxidation states of our solvothermalsolvothermal-treated-treated MoSe22 QDs,QDs, XPS XPS was was performed on both pristinepristine bulk MoSe2 powder and MoSe2 QDs.QDs. Figure Figure 44a,ba,b show thethe high-resolutionhigh-resolution MoMo 3d3d andand SeSe 3d3d XPSXPS spectraspectra ofof pristinepristine MoSeMoSe22 powder, respectively. The The two two peaks peaks located located at at 228.1 228.1 eV eV and and 231.2 231.2 eV eVcorrespond correspond to the to theMo Mo3d5/2 3d and5/2 4+ Moand 3dMo3/23d peaks3/2 peaks of the of the Mo Mo4+ statestate in MoSe in MoSe2, which2, which is isin in agreement agreement with with previous previous reports reports [19,41 [19,41].]. Meanwhile, the the peak peak of of Se Se 3d 3d spectrum spectrum can can be deconvoluted be deconvoluted into two into components: two components: the binding the binding energy 2 2 peaksenergy at peaks 53.4 eVat 53.4 and eV 54.2 and eV 54.2 are characteristiceV are characteristic signals signals of Se − of3d Se5/22−and 3d5/2 Se and− 3d Se32/2− ,3d respectively3/2, respectively [42]. 6+ [42].In this In case,this case, a signal a signal from from the the Mo Mostate6+ state was was not not observed, observed, indicating indicating that that therethere isis nono noticeable oxidation in our pristine material. Next, the high high-resolution-resolution Mo 3d and Se 3d spectra of MoSeMoSe22 QDs are presented in Figure 44c,d,c,d, respectively.respectively. TheThe deconvolutiondeconvolution ofof MoMo 3d3d spectralspectral regionregion revealsreveals fourfour contributions. The The two two intense intense peaks peaks at at 227.9 and 231.1 eV belong to the characteristic signals from 4+ 4+ Mo4+ 3d3d5/25/ 2andand Mo Mo4+ 3d3d3/23,/ 2respectively., respectively. Furthermore, Furthermore, the the other other two two minor minor peaks peaks at at binding binding energies of 232.2 and 235.5 eV are ascribed to the Mo(VI) state [[37].37]. This This suggests suggests that a small small portion portion of surface 4+ 6+ Mo4+ waswas oxidized oxidized into into Mo Mo6+ duringduring the the imposed imposed reaction reaction [43,44 [43,44].]. As As shown shown in in Figure Figure 44d,d, thethe twotwo deconvoluted components of the Se 3d doublet appear at 53.5 eV and 54.3 eV, eV, which can be assigned 2 to the SeSe 3d3d55/2/2 and Se 3d33/2/2 orbitalsorbitals of divalent selenide ions (Se2−),), respectively. respectively. It It can can be confirmed conﬁrmed 4+ 2- that the binding energiesenergies ofof MoMo4+ andand Se Se2- orbitalsorbitals of of our our prepared prepared QDs QDs do not deviate noticeably from those those of of the the starting starting material. material. It is reasonable It is reasonable as no heterostructure as no heterostructure formation formation or other interaction or other interactionis involved. is involved.

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FigureFigure 4. 4. High-resolutionHigh-resolution X-ray X-ray photoelectron photoelectron spectrometer spectrometer (XPS) (XPS spectra) spectra showing showi theng binding the binding energy

energyof (a) Mo of 3d(a) and Mo ( b3d) Se and 3d ( electronsb) Se 3d recordedelectrons on recorded bulk MoSe on 2bulkpowder. MoSe High-resolution2 powder. High core-resolution level spectra core levelcorresponding spectra corresponding to (c) Mo 3d andto (c ()d Mo) Se 3d 3d and electrons (d) Se for3d MoSeelectrons2 QDs. for MoSe2 QDs.

3.3.3.3. Optical Optical Property Property Studies Studies ItIt is is known that the dimensionality strongly strongly affects affects the optical properties of nanoscale semiconductors. The optical absorption spectrum of the resultant CTAB-MoSe nanosheets in dispersion semiconductors. The optical absorption spectrum of the resultant CTAB2 -MoSe2 nanosheets in dispersionis displayed is indisplayed Figure5 a.in TheFigure evident 5a. The absorption evident absorption peaks at 805 peaks and at 695 805 nm and can 695 be nm easily can identified be easily identifiedand they areand attributedthey are attributed to the characteristic to the characteristic resonances resonances of A and of B A excitons, and B excitons, respectively respectively [45–47]. [45Their–47 origin]. Their is origin derived is fromderived the from transitions the transitions between thebetween spin-orbit the spin split-orbit valence split bands valence and bands the lowest and theconduction lowest conduction band at K band and K’ at pointsK and K’ of thepoints Brillouin of the Brillouin zone. Moreover, zone. Moreover it is worth, it is of worth noting of that noting the thatdetermined the determined energy separationenergy separation of 244 meVof 244 between meV between the A andthe A B and excitonic B excitonic states states is consistent is consistent with a previous study on the energy splitting of the exciton states in ultrathin MoSe nanosheets [48]. with a previous study on the energy splitting of the exciton states in ultrathin MoSe22 nanosheets [48]. Therefore, it it provides provides a quantitative a quantitative proof proof of pronounced of pronounced quantum quantum confinement confinement effect in oureffect exfoliated in our 2D MoSe nanosheets. The facile and conventional way to address the optical band gap is by means of exfoliated2 2D MoSe2 nanosheets. The facile and conventional way to address the optical band gap is bythe means Tauc plot. of the The Tauc absorption plot. The coe absorptionfficient α of coefficient a direct band-gap α of a direct semiconductor band-gap can semiconductor be related to photoncan be 2 energy hυ by (αhυ) = A (hυ Eg), where2 A is a constant and Eg is the optical band gap. Figure5b plots related to photon energy hυ− by (αhυ) = A (hυ − Eg), where A is a constant and Eg is the optical band α 2 gap.the relationship Figure 5b plots of ( thehυ) relationshipversus hυ, which of (αh demonstratesυ)2 versus hυ, awhich linear demonstrates dependence. Thea linear calculated dependence. optical Thegap forcalculated the apparent optical absorption gap for the is 1.54apparent eV (805 absorption nm), which is exactly1.54 eV coincides(805 nm), with which the exactly A excitonic coincides states and highlights the 2D nature of CTAB-MoSe nanosheets. with the A excitonic states and highlights the2 2D nature of CTAB-MoSe2 nanosheets.

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Figure 4. High-resolution X-ray photoelectron spectrometer (XPS) spectra showing the binding

energy of (a) Mo 3d and (b) Se 3d electrons recorded on bulk MoSe2 powder. High-resolution core

level spectra corresponding to (c) Mo 3d and (d) Se 3d electrons for MoSe2 QDs.

3.3. Optical Property Studies It is known that the dimensionality strongly affects the optical properties of nanoscale semiconductors. The optical absorption spectrum of the resultant CTAB-MoSe2 nanosheets in dispersion is displayed in Figure 5a. The evident absorption peaks at 805 and 695 nm can be easily identified and they are attributed to the characteristic resonances of A and B excitons, respectively [45–47]. Their origin is derived from the transitions between the spin-orbit split valence bands and the lowest conduction band at K and K’ points of the Brillouin zone. Moreover, it is worth of noting that the determined energy separation of 244 meV between the A and B excitonic states is consistent with a previous study on the energy splitting of the exciton states in ultrathin MoSe2 nanosheets [48]. Therefore, it provides a quantitative proof of pronounced quantum confinement effect in our exfoliated 2D MoSe2 nanosheets. The facile and conventional way to address the optical band gap is by means of the Tauc plot. The absorption coefficient α of a direct band-gap semiconductor can be related to photon energy hυ by (αhυ)2 = A (hυ − Eg), where A is a constant and Eg is the optical band gap. Figure 5b plots the relationship of (αhυ)2 versus hυ, which demonstrates a linear dependence. NanomaterialsThe calculated2020, 10optical, 2045 gap for the apparent absorption is 1.54 eV (805 nm), which exactly coincides8 of 19 with the A excitonic states and highlights the 2D nature of CTAB-MoSe2 nanosheets.

Figure 5. (a) Ultraviolet–visible (UV–vis) absorption spectrum of 2D CTAB-MoSe2 nanosheets clearly reveals characteristic excitonic structures. (b) The corresponding plot versus (ahv)2 versus hν for

absorption of 2D CTAB-MoSe2, in which the optical band gap energy can be estimated. (c) UV–vis absorption spectrum of MoSe2 QDs. Note the quenching of previous excitonic features in the spectrum.

The optical absorption spectrum of the as-prepared 0D MoSe2 QDs is in sharp contrast with their 2D counterpart, as shown in Figure5c. Here, the A and B excitonic features in the absorption completely disappeared. Instead, the absorption feature comprised two absorption bands. The prominent band is centered at around 275 nm, which is ascribed to the intrinsic excitonic absorption of the QDs [49,50]. Such a significant blue-shift of the excitonic features directly reflects the dominating quantum confinement effect and is in accordance with previous studies on other TMD QDs [32,51]. Furthermore, there exists another mild absorption band at longer wavelengths. It is wide and centered about 325 nm with a tail extending to ~400 nm, which will be commented upon later. Photoluminescence (PL) spectroscopy provides a complimentary optical means to probe the electronic structure of semiconductor materials [52,53]. The distinct optical property of TMD QDs is well-suited to be further evidenced by the PL technique. It is easily found that our MoSe2 QDs dispersed in aqueous solution emit strong blue fluorescence under irradiation with a typical UV lamp. It is due to the weak interlayer coupling and enhanced quantum efficiency of MoSe2 QDs, which is another signature of TMD QDs [54]. To gain a comprehensive view of the emission property, the PL spectra of resultant MoSe2 QD dispersion were further taken with different excitation wavelengths, as shown in Figure6a. It is observed that when the excitation wavelength was increased from 290 to 400 nm, the PL peak position monotonically increased from 390 to 470 nm. Similar excitation-dependent PL behavior has been reported in a few TMD QD reports [32]. In a strong quantum confinement regime, photons with higher energies resonantly excites smaller QDs with wider band gaps, pushing the emission peak to shorter wavelengths. Accordingly, the characteristic excitation-dependent PL behavior derives from the polydispersity of the synthesized QDs. This idiosyncratic variation of PL intensity in response to varying excitation wavelengths can be clearly presented by the 2D color-converted PL contour map as depicted in Figure6b. We found the strongest emission peaked at 418 nm under an excitation wavelength of 340 nm. This specific wavelength falls coincidentally within the observed absorption band around 325 nm. Thus the close correspondence between the absorption and the emission of the synthesized QDs can be revealed by our optical characterizations. Nanomaterials 2020, 10, x FOR PEER REVIEW 8 of 19

absorption of 2D CTAB-MoSe2, in which the optical band gap energy can be estimated. (c) UV–vis

absorption spectrum of MoSe2 QDs. Note the quenching of previous excitonic features in the spectrum.

The optical absorption spectrum of the as-prepared 0D MoSe2 QDs is in sharp contrast with their 2D counterpart, as shown in Figure 5c. Here, the A and B excitonic features in the absorption completely disappeared. Instead, the absorption feature comprised two absorption bands. The prominent band is centered at around 275 nm, which is ascribed to the intrinsic excitonic absorption of the QDs [49,50]. Such a significant blue-shift of the excitonic features directly reflects the dominating quantum confinement effect and is in accordance with previous studies on other TMD QDs [32,51]. Furthermore, there exists another mild absorption band at longer wavelengths. It is wide and centered about 325 nm with a tail extending to ~400 nm, which will be commented upon later. Photoluminescence (PL) spectroscopy provides a complimentary optical means to probe the electronic structure of semiconductor materials [52,53]. The distinct optical property of TMD QDs is well-suited to be further evidenced by the PL technique. It is easily found that our MoSe2 QDs dispersed in aqueous solution emit strong blue fluorescence under irradiation with a typical UV lamp. It is due to the weak interlayer coupling and enhanced quantum efficiency of MoSe2 QDs, which is another signature of TMD QDs [54]. To gain a comprehensive view of the emission property, the PL spectra of resultant MoSe2 QD dispersion were further taken with different excitation wavelengths, as shown in Figure 6a. It is observed that when the excitation wavelength was increased from 290 to 400 nm, the PL peak position monotonically increased from 390 to 470 nm. Similar excitation-dependent PL behavior has been reported in a few TMD QD reports [32]. In a strong quantum confinement regime, photons with higher energies resonantly excites smaller QDs with wider band gaps, pushing the emission peak to shorter wavelengths. Accordingly, the characteristic excitation-dependent PL behavior derives from the polydispersity of the synthesized QDs. This idiosyncratic variation of PL intensity in response to varying excitation wavelengths can be clearly presented by the 2D color-converted PL contour map as depicted in Figure 6b. We found the strongest emission peaked at 418 nm under an excitation wavelength of 340 nm. This specific wavelength falls coincidentally within the observed absorption band around 325 nm. Thus the close correspondence betweenNanomaterials the2020 absorption, 10, 2045 and the emission of the synthesized QDs can be revealed by our optical9 of 19 characterizations.

Figure 6. ((a)) Excitation Excitation-wavelength-wavelength dependent photoluminescence ( (PL)PL) spectra of colloidal MoSe2 QDs at room temperature. ( (bb)) The The 2D 2D contour map map acquired from the PL spectra. The characteristic contourcontour is is due to the pronounced quantum confinement conﬁnement eeffect.ﬀect.

NanomaterialsRaman 20 spectroscopy spectroscopy20, 10, x FOR PEER was was REVIEW adopted adopted to to acquire acquire additional additional insight insight into into the the optical optical characteristic characteristics9 ofs of19 of 2D CTAB-MoSe nanosheets. In general, group theory analysis permits bulk TMDs to have four 2D CTAB-MoSe2 nanosheets.2 In general, group theory analysis permits bulk TMDs to have four RamanRaman-activenamely,-active out-of modes. -plane However, AHowever,1g and in only only-plane two two E modes1 modes2g modes. are are accessible Theaccessible inset insk in typicaletch typical in experimental experimentalFigure 7 depicts configuration, conﬁguration, these two 1 namely,principal out-of-plane Raman-active A1g vibrationand in-plane modes E of2g modes. MoSe2. Figure The inset 7 compares sketch in the Figure Raman7 depicts spectra these of twoboth principalMoSe2 bulk Raman-active and 2D CTAB vibration-MoSe modes2 nanosheets. of MoSe For2. FigureMoSe72 bulk,compares the A the1g mode Raman is spectra located of at both 239.4 MoSe cm−21 1 bulkwhile and the 2Din-plane CTAB-MoSe E12g mode2 nanosheets. appears at For ≈285.5 MoSe cm2 −1bulk,, which the match A1g mode nicely is locatedwith literature at 239.4 values cm− while [55]. 1 1 theThe in-plane A1g mode E 2g formode CTAB appears-MoSe at2 nanosheets285.5 cm− , is which red-shifted match nicely to 237 with cm−1 literature, which valuesis attributed [55]. The to A the1g ≈ 1 modesoftening for of CTAB-MoSe the vibrational2 nanosheets mode [56]. is red-shiftedThe reduced to inter 237- cmplanar− , which restoring is attributed force is another to the softeningproof for ofthe the 2D vibrationalfew-layer structure. mode [56 In]. addition, The reduced a new inter-planar peak emerges restoring on the force lower is- anotherfrequency proof side for of the the A 2D1g few-layerpeak. It is structure.ascribed to In Davydov addition, splitting a new peak of the emerges A1g mode on the that lower-frequency is accompanied side by ofthe the suppressed A1g peak. Itinterlayer is ascribed interaction to Davydov as reported splitting in of TMD the A 1gnanosheetsmode that [57,58 is accompanied]. In contrast, by the suppressedE12g mode for interlayer CTAB- 1 interactionMoSe2 shifts as to reported 302 cm in−1. TMD The dielectric nanosheets screening [57,58]. In of contrast, the long the-range E 2g Coulombmode for interaction CTAB-MoSe and2 shifts the 1 tosurface 302 cm effects− . The of dielectric TMD materials screening were of the proposed long-range to Coulomb be responsible interaction for this and theblue surface-shift [59]. eﬀects The of TMDincreased materials energy were splitting proposed between to be the responsible two allowed for this Raman blue-shift peaks [ 59is ].in The accord increased with the energy 2D nature splitting of betweenour CTAB the-MoSe two2allowed [60,61]. Raman peaks is in accord with the 2D nature of our CTAB-MoSe2 [60,61].

Figure 7. 7. RamanRaman spectra spectra of the of thesource source MoSe MoSe2 bulk2 bulkpowder powder (brown (brown line) and line) as and-produced as-produced 2D CTAB 2D-

CTAB-MoSeMoSe2 nanosheets2 nanosheets (blue (blueline). line).The Raman The Raman shifts shifts are aredenoted denoted by bythe the dashed dashed lines. lines. The The inset inset sketch depicts the atomic displacements of the two vibrational modes leading to the primary RamanRaman peaks.peaks.

3.4. Peroxidase-Like Activities and Steady-State Kinetic Assay

We evaluated the peroxidase-like activity of MoSe2 QDs by using the catalytic oxidation of TMB in the presence of H2O2, as shown in Figure 8a. The absorption spectrum of the TMB solution showed it is colorless. When only H2O2 was incubated with TMB, the TMB–H2O2 systems showed rather weak absorbance at 652 nm. Yet as TMB coexisted with H2O2 and the MoSe2 QDs, a prominent absorption peak of the oxidation products of TMB at 652 nm was observed. Moreover, the color contrast of these system is presented in the inset of Figure 8a. It can be seen that the bare TMB and the TMB–H2O2 systems are virtually colorless to the naked eye, while TMB–H2O2–MoSe2 QDs system showed an apparent color variation. Figure 8b display time-dependent absorbance changes at 652 nm of these systems. It clearly shows the absorbance at 652 nm increased as the time increased for TMB–H2O2– MoSe2 QDs system. This means that the prepared MoSe2 QDs possess the peroxidase-like catalysis capability, which effectively catalyze the oxidation of TMB by H2O2. On the contrary, rather insignificant and slow oxidation of TMB by the presence of H2O2 was found for the reference TMB– H2O2 system. Our results thus demonstrated that the MoSe2 QDs can facilitate the oxidation of TMB to oxTMB in the presence of H2O2 to generate observable color changes. An identical result was also found for our 2D CTAB-MoSe2 nanosheets (not shown here).

Nanomaterials 2020, 10, 2045 10 of 19

3.4. Peroxidase-Like Activities and Steady-State Kinetic Assay

We evaluated the peroxidase-like activity of MoSe2 QDs by using the catalytic oxidation of TMB in the presence of H2O2, as shown in Figure8a. The absorption spectrum of the TMB solution showed it is colorless. When only H2O2 was incubated with TMB, the TMB–H2O2 systems showed rather weak absorbance at 652 nm. Yet as TMB coexisted with H2O2 and the MoSe2 QDs, a prominent absorption peak of the oxidation products of TMB at 652 nm was observed. Moreover, the color contrast of these system is presented in the inset of Figure8a. It can be seen that the bare TMB and the TMB–H2O2 systems are virtually colorless to the naked eye, while TMB–H2O2–MoSe2 QDs system showed an apparent color variation. Figure8b display time-dependent absorbance changes at 652 nm of these systems. It clearly shows the absorbance at 652 nm increased as the time increased for TMB–H2O2–MoSe2 QDs system. This means that the prepared MoSe2 QDs possess the peroxidase-like catalysis capability, which eﬀectively catalyze the oxidation of TMB by H2O2. On the contrary, rather insigniﬁcant and slow oxidation of TMB by the presence of H2O2 was found for the reference TMB–H2O2 system. Our results thus demonstrated that the MoSe2 QDs can facilitate the oxidation of TMB to oxTMB in the presence of H2O2 to generate observable color changes. An identical result was Nanomaterials 2020, 10, x FOR PEER REVIEW 10 of 19 also found for our 2D CTAB-MoSe2 nanosheets (not shown here).

Figure 8. 8. (a()a UV-visible) UV-visible absorption absorption spectra spectra of (1) of 3,3,5,5-tetramethylbenzidine (1) 3,3,5,5-tetramethylbenzidine (TMB) solution (TMB) (brown);solution

(2)(brown); TMB–H (2)2O 2 TMBsystem–H2O (green);2 system (3) TMB–H(green); 2O (3)2–MoSe TMB–2HQDs2O2– systemMoSe2 (blue).QDs system Inset: the (blue). corresponding Inset: the photographscorresponding of photographs these reaction of these systems. reaction (b) Thesystems. time-dependent (b) The time- absorbancedependent absorbance changes at changes 652 nm ofat these652 nm systems. of these systems.

The kinetic kinetic parameters parameters of ofthe the peroxidase peroxidase-like-like reaction reaction were wereharvested harvested by employing by employing the steady the- steady-statestate kinetics kinetics analysis. analysis. With H With2O2 and H2 OTMB2 and as TMB substrates, as substrates, the measurements the measurements were carried were carriedout by outchanging by changing the concentration the concentration of one substrate of one substrate while keeping while keepingthe other thesubstrate other substrateconcentration concentration constant. constant.This generates This generatesthe typical the Michaelis typical Michaelis–Menten–Menten curves, as curves, shown asin shownFigure in9a,b Figure for our9a,b MoSe for our2 QDs. MoSe For2 QDs.2D CTAB For- 2DMoSe CTAB-MoSe2 nanosheets,2 nanosheets, these curves these are curvesplotted arein Figure plotted 10a,b. in Figure The relevant 10a,b. The kinetic relevant parameters kinetic K V parameterslike the Michaelis like the–Menten Michaelis–Menten constant (Km constant) and the ( maximalm) and the reaction maximal velocity reaction (Vmax velocity) can be ( extractedmax) can be extracted from the Lineweaver–Burk plot according to the relation: 1/v = (Km/Vmax) (1/[S]) + from the Lineweaver–Burk plot according to the relation: 1/v = (Km/Vmax) × (1/[S]) + 1/Vmax×, where v 1stands/Vmax ,for where the vinitialstands velocity for the and initial [S velocity] signifies and the [S concentration] signifies the concentrationof the substrate of the[62,63 substrate]. Figure [62 9c,d,63]. Figuredisplay9 c,dthe display L-B plot the for L-B our plot 0D for MoSe our2 0D QDs MoSe while2 QDs Figure while 10c,d Figure illustrate 10c,d illustratethe L-B plot the L-Bfor plot2D CTAB for 2D- CTAB-MoSeMoSe2 nanosheets.2 nanosheets. The calculated The calculated results results are listed are listed in Table in Table S1. The S1. TheKm valueKm value is regarded is regarded as an as animportant important index index that that measures measures thethe binding binding affinity affinity of enzyme of enzyme to the to the substrates. substrates. A smaller A smaller value value of ofKmK usuallym usually indicates indicates a higher a higher affinity affinity between between the theenzyme enzyme and and the thesubstrate. substrate. It is It found is found that that the theKm Kvaluem value of 2D of 2DCTAB CTAB-MoSe-MoSe2 for2 forH2O H22 isO 2loweris lower than than that that of MoSe of MoSe2 QDs,2 QDs, suggesting suggesting a higher a higher affinity affinity of of2D 2DCTAB CTAB-MoSe-MoSe2 to2 Hto2O H22 thanO2 than MoSe MoSe2 QDs.2 QDs. Meanwhile, Meanwhile, the lower the lower Km valueKm value of MoSe of MoSe2 QDs2 toQDs TMB to TMBrepresents represents its higher its higher affinity affi innity this inrespect. this respect. In addition, In addition, the parallel the parallelslope ofslope the lines of the in the lines double in thereciprocal plots of initial velocity versus different concentrations of one substrate reveals a ping-pong mechanism in the catalytic reaction [64–66]. This indicates that both of our MoSe2–based enzymes bound and reacted with the first substrate and the first product was subsequently released before the reaction with the second substrate.

Nanomaterials 2020, 10, 2045 11 of 19 double-reciprocal plots of initial velocity versus different concentrations of one substrate reveals a ping-pong mechanism in the catalytic reaction [64–66]. This indicates that both of our MoSe2–based enzymes bound and reacted with the first substrate and the first product was subsequently released before theNanomaterials reaction 2020 with, 10, x theFOR PEER second REVIEW substrate. 11 of 19 Nanomaterials 2020, 10, x FOR PEER REVIEW 11 of 19

Figure 9.FigureSteady-state 9. Steady-state kinetic kinetic analysis analysis for for MoSe MoSe2 QDs.2 QDs. The reaction The reaction velocity (v) velocity was measured (v) was when measured Figure 9. Steady-state kinetic analysis for MoSe2 QDs. The reaction velocity (v) was measured when (a) the H2O2 concentration was varied while the concentration of TMB was 5 mM and (b) the TMB when (a) the H2O2 concentration was varied while the concentration of TMB was 5 mM and (b) the (a) the H2O2 concentration was varied while the concentration of TMB was 5 mM and (b) the TMB concentration was varied while the concentration of H2O2 was 0.75 mM. The corresponding double- TMB concentration was varied while the concentration of H2O2 was 0.75 mM. The corresponding reciprocalconcentration plots was with varied a fixed while concentration the concentration of one ofsubstrate H2O2 was relative 0.75 mM. to varying The corresponding the concentration double of- double-reciprocal plots with a ﬁxed concentration of one substrate relative to varying the concentration thereciprocal other substrate plots with are a displayed fixed concentration in (c,d). of one substrate relative to varying the concentration of of the otherthe other substrate substrate are are displayed displayed inin (c,,dd).).

Figure 10. Steady-state kinetic assay of 2D CTAB-MoSe2 nanosheets. (a) Varying the concentrations Figure 10. Steady-state kinetic assay of 2D CTAB-MoSe2 nanosheets. (a) Varying the concentrations Figure 10.of HSteady-state2O2 while the concentration kinetic assay of of TMB 2D was CTAB-MoSe 5 mM. (b) 2Varyingnanosheets. the concentrations (a) Varying of theTMB concentrations while the of of H2O2 while the concentration of TMB was 5 mM. (b) Varying the concentrations of TMB while the H2O2 whileconcentration the concentration of H2O2 was 0.75 of TMBmM. The was double 5 mM.-reciprocal (b) Varying plots for the the concentrations concentration of ( ofc) H TMB2O2 and while the concentration of H2O2 was 0.75 mM. The double-reciprocal plots for the concentration of (c) H2O2 and concentration(d) TMB. of H O was 0.75 mM. The double-reciprocal plots for the concentration of (c)H O and (d) TMB. 2 2 2 2 (d) TMB. 3.5. Actives Species Trapping Tests and Peroxidase-Like Catalytic Mechanism 3.5. Actives Species Trapping Tests and Peroxidase-Like Catalytic Mechanism 3.5. Actives SpeciesTo confirm Trapping the prime Tests species and Peroxidase-Likeresponsible for the Catalytic peroxidase Mechanism-mimetic catalytic activities of our To confirm the prime species responsible for the peroxidase-mimetic catalytic activities of our artificial MoSe2–based enzymes, scavenger tests were employed. It is known that reaction systems Toartificial conﬁrm MoSe the2 prime–based speciesenzymes, responsible scavenger tests for were the employed. peroxidase-mimetic It is known that catalytic reaction activities systems of our involving hydrogen peroxide usually abound with reactive radicals such as ȮH and Ȯ2−. Then IPA artiﬁcialinvolving MoSe –based hydrogen enzymes, peroxide scavengerusually abound tests with were reactive employed. radicals Itsuch is knownas ȮH and that Ȯ2− reaction. Then IPA systems 2

Nanomaterials 2020, 10, 2045 12 of 19

Nanomaterials 2020, 10, x FOR PEER REVIEW 12 of 19 involving hydrogen peroxide usually abound with reactive radicals such as OH˙ and O˙ 2−. Then IPA − and benzoquinonebenzoquinone (BQ) werewere taken taken to to be be the the scavengers scavengers in in the the reaction reaction system system for forOH ˙ȮH and andO ˙Ȯ22− radicals, respectively. respectively. Figure Figure 11 11 shows shows the the results results of active of active species species trapping trapping tests of tests our of reaction our reaction system. Wesystem. found We that found the suppressionthat the suppression of characteristic of characteristic absorption absorption and color and fading color were fading not were evident not withevident the withaddition the addition of IPA. On of theIPA. other On the hand, other significant hand, significant decrease decrease in absorption in absorption and color and contrast color contrast can be seen can − whenbe seen BQ when was added.BQ was This added. indicates This indicates that O˙ 2− radicalthat Ȯ2 plays radical the play majors the role major to oxidize role to TMB oxidize to produce TMB to a TMBproduce oxide a TMB and generateoxide and color generate contrast. color Based contrast. on our Based finding on our and finding several and previous several reports previous [67 ,reports68], we propose[67,68], we the propose peroxidase-like the peroxidase catalytic-like mechanism catalytic mechanism of our MoSe of2 –basedour MoSe enzymes,2–based which enzymes, is illustrated which is illustratedin Figure 12 in. Figure In the 12. reaction In the process,reaction process, TMB molecules TMB molecules are absorbed are absorbed on the surfaceon the surface of MoSe of2 MoSe–based2– basednanomaterials nanomaterials and act and as act the as chromogenic the chromogenic electron electron donors. donors. These These molecules molecules transfer transfer their their lone-pair lone- pairelectrons electrons to MoSe to MoSe2 from2 thefrom amino the amino groups, groups, leading leading to the enhancementto the enhancement of electron of el densityectron anddensity mobility and mobilityon the surface on the of surface MoSe2-based of MoSe catalyst.2-based In catalyst. turn, it accelerates In turn, it theaccelerates electron migrationthe electron from migration MoSe2–based from MoSecatalyst2–based to hydrogen catalyst peroxide. to hydrogen The peroxide. one-electron The transfer one-electron reaction transfer generate reaction a large generate amount a of largeO˙ 2− − amountradicals that of Ȯ oxidize2 radicals TMB that and oxidize form blue-green TMB and product. form blue Briefly,-green the product. MoSe2-based Briefly, catalyst the MoSe promote2-based the catalystelectron promote transfer from the electron TMB to transferH2O2, resulting from TMB in the to H oxidation2O2, resulting of TMB in and the oxidationreduction of hydrogen TMB and peroxide.reduction Theof hydrogen production peroxide. of colored The oxTMB production and water of colored in this oxTMB system canandbe water expressed in this by system the equation can be expressedH O + TMB by the2H equationO + O H+2OoxTMB.2 + TMB → 2H2O + O2 + oxTMB. 2 2 → 2 2

Figure 11. The absorbance at 652 nm of reaction solutions in the absence or presence of scavengers Isopropyl alcohol (IPA) and benzoquinone (BQ) (BQ).. The inset shows the images of color changes for differentdiﬀerent reaction systems.

Nanomaterials 2020, 10, 2045 13 of 19 Nanomaterials 2020, 10, x FOR PEER REVIEW 13 of 19

FigureFigure 12. 12. TheThe schematic schematic diagram diagram depicts depicts the mechanism for colorimetric detection of H22O2 byby using using 2D2D CTAB CTAB-MoSe-MoSe2 NSsNSs and and MoSe MoSe22 QDsQDs as as peroxidase peroxidase mimetics. mimetics.

3.6.3.6. Colorimetric Colorimetric Detection Detection of of H H22OO22 byby MoSe MoSe2-2Based-Based As Assaysay System System

InIn view view of of the the intrinsic intrinsic peroxidase-like peroxidase property-like property of as-prepared of as-prepared MoSe2–based MoSe catalysts,2–based a catalysts, colorimetric a colorimetricstrategy for thestrategy detection for the of Hdetection2O2 was of established. H2O2 was established. The absorption The spectraabsorption of TMB–H spectra2O of2 –MoSeTMB–H2 2QDsO2– MoSesystem2 QDs with system different with H2 Odifferent2 concentration H2O2 concentration is presented is in presented Figure S1. in It canFigure be seenS1. It that can thebe seen characteristic that the µ characteristicabsorption of absorption TMB at 652 of nm TMB is dependentat 652 nm is on dependent the concentration on the concentration of H2O2 varied of H from2O2 varied 10 M from to 4 10 M. μMAnalogous to 4 M. resultsAnalogous can beresults found can with be ourfound TMB–H with 2ourO2–2D TMB CTAB-MoSe–H2O2–2D CTAB2 system,-MoSe as shown2 system, in as Figure shown S2. inFigures Figure 13 S2.a and Fig ure14as display13a and the 14a absorbance display the variations absorbance at variations 652 nm of theat 652 oxidized nm of the TMB oxidized in the presence TMB in theofH presence2O2 with of di Hff2erentO2 with concentrations different concentrations for the TMB–H for the2O 2TMB–MoSe–H22OQDs2–MoSe system2 QDs and system TMB–H and2O TMB2–2D– HCTAB-MoSe2O2–2D CTAB2 system,-MoSe2 respectively. system, respectively. Besides, the Besides, corresponding the corresponding image in responseimage in to response the change to the of changeH2O2 is of shown H2O2 is in shown the insets in the of Figuresinsets of 13 Figurea ands 1413aa, and which 14a, shows which the shows color the variation color variation could be could seen beby seen the nakedby the eye.naked For eye. TMB–H For TMB2O2––MoSeH2O2–2MoSeQDs2 system,QDs system, the H the2O 2Hconcentration-response2O2 concentration-response curve curve has hasa linear a linear relationship relationship in thein the range range of of 10 10µM μM to to 100 100µ MμM with with a a detection detection limit limit ofof 44 µμM,M, as illustrated inin Figure Figure 13b. 13b. Figure Figure 14b 14 drawsb draws the thecalibration calibration curve curve for H for2O2 Hwith2O2 awith linear a range linear from range 10 fromto 250 10 μM to for250 theµM TMB for the–H2 TMB–HO2–2D CTAB2O2–2D-MoSe CTAB-MoSe2 system. 2Thesystem. detection The limit detection was limitalso reckoned was also to reckoned be around to be 4 μM.around It shows 4 µM. that It shows the TMB that–H the2O TMB–H2–2D CTAB2O2–2D-MoSe CTAB-MoSe2 system could2 system have could a wider have linear a wider range linear compared range withcompared that with of the that TMB of the–H TMB–H2O2–MoSe2O22–MoSe QDs 2 system.QDs system. Finally, Finally, we we compare compare some some representative representative colorimetriccolorimetric detections of H 2OO22 byby using using novel novel 2D 2D materials materials in in Table Table 11[ [6969–72]. It It can can be be seen seen that that the the colorimetriccolorimetric sensing sensing ability ability of of H 2HO2Obased2 based on the on peroxidase-like the peroxidase property-like property of our MoSeof our2-based MoSe2 catalyst-based catalystis comparable is comparable to other to reported other reported 2D materials-based 2D materials- colorimetricbased colorimetric platforms. platforms. Therefore, Therefore, our work our workprovides provides a facile, a facile, simple, simple, cost-e costffective,-effective, and and alternative alternative 2D 2D materials-based materials-based colorimetriccolorimetric sensing platform for sensitive detection of H 2OO22. .In In principle, principle, this this sensing sensing platform platform can can be be applied applied to to a a few few diversediverse applications, applications, yet yet proper proper selectivity selectivity tests tests should should then then be be imposed to to understand the the specific specific accuracyaccuracy in the measurement [31,73 [31,73–75].

Nanomaterials 2020, 10, 2045 14 of 19 Nanomaterials 2020, 10, x FOR PEER REVIEW 14 of 19 Nanomaterials 2020, 10, x FOR PEER REVIEW 14 of 19

Figure 13. (a) The absorbance changes at 652 nm for MoSe2 QDs in different amount of H2O2 (0.01, Figure 13.13. (a) TheThe absorbanceabsorbance changeschanges atat 652652 nmnm forfor MoSeMoSe22 QDs in didifferentﬀerent amountamount ofof HH22O2 (0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3 and 4 mM). Inset: the images of color contrast for different 0.02, 0.03, 0.04, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3 and 4 mM). Inset: the the images images of of color color contrast contrast for different diﬀerent concentrations of H2O2. (b) The linear calibration plot for H2O2. concentrations of H22O2..( (bb)) The The linear linear calibration calibration plot plot for for H H22OO2.2 .

FigureFigure 14.14. ((a)) TheThe dose-responsedose-response curvecurve forfor HH22O22 detection by usingusing 2D CTAB-MoSeCTAB-MoSe22 NSs as artiﬁcialartificial Figure 14. (a) The dose-response curve for H2O2 detection by using 2D CTAB-MoSe2 NSs as artificial enzyme.enzyme. The inset are thethe photosphotos ofof reactionreaction solutionssolutions afterafter addingadding didifferentﬀerent concentrationsconcentrations ofof HH22OO22. enzyme. The inset are the photos of reaction solutions after adding different concentrations of H2O2. ((bb)) TheThe linearlinear calibrationcalibration plotplot forfor HH22O22 concentration. (b) The linear calibration plot for H2O2 concentration.

Table 1. Comparison of colorimetric detections of H2O2 in the linear range and detection limit between Table 1. Comparison of colorimetric detections of H2O2 in the linear range and detection limit between ourTable MoSe 1. Comparison2 nanostructures of colorimetric and other detections peroxidase of mimics H2O2 in based the linear on nanoscale range and 2D detection materials. limit between our MoSe2 nanostructures and other peroxidase mimics based on nanoscale 2D materials. our MoSe2 nanostructures and other peroxidase mimics based on nanoscale 2D materials. Catalyst Linear Range (µM) Detection Limit (µM) Ref. Catalyst Linear Range (μM) Detection Limit (μM) Ref. Positively-chargedCatalyst Au nanoparticles Linear Range (μM) Detection Limit (μM) Ref. 2–200 0.5 [15] Positively(NPs)h-BN-charged/ N-MoSAu nanoparticles (NPs) Positively-charged Au nanoparticles2 (NPs) 2–200 0.5 [15] h-BNh-/N-MoSBN/N-MoS2 2 1–10002–200 0.40.5 [[15]69] h-BN/N-MoS2 Few-layered MoSe2 nanosheets (NSs) 10–160 0.4 [27] Few-layered MoSe2 nanosheets (NSs) 1–1000 0.4 [69] Few-layeredMoS MoSe2 NPs2 nanosheets (NSs) 3–1201–1000 1.250.4 [[69]23] SDS–MoSMoS2 2NPs NPs 2–10010–160 0.320.4 [[27]24] MoS2 NPs 10–160 0.4 [27] g-C3N4 5–100 1 [70] SDS–MoS2 NPs 3–120 1.25 [23] MoS2 QDsSDS/g-C–MoS3N24 NPsNSs 2–503–120 0.1551.25 [[23]71] WS2 Nanosheetsg-C3N4 5–2002–100 1.50.32 [[24]72] g-C3N4 2–100 0.32 [24] 2D CTAB-MoSe2 10–250 4 This work MoS2 QDs/g-C3N4 NSs 5–100 1 [70] 0D MoSe2 MoSQDs2quantum QDs/g-C3 dotsN4 NSs (QDs) 10–1005–100 41 This[70] work WS2 Nanosheets 2–50 0.155 [71] WS2 Nanosheets 2–50 0.155 [71] 5–200 1.5 [72] 2D CTAB-MoSe2 5–200 1.5 [72] 2D CTAB-MoSe2 10–250 4 This work 10–250 4 This work 0D MoSe2 QDs quantum dots (QDs) 10–100 4 This work 0D MoSe2 QDs quantum dots (QDs) 10–100 4 This work

Nanomaterials 2020, 10, 2045 15 of 19

4. Conclusions

In summary, we prepared 2D and 0D MoSe2 nanostructures based on systematic and non-toxic top-down strategies. The characteristic excitation-dependent PL of the MoSe2 QDs can be attributed to the polydispersity of the synthesized QDs. The Raman shift of ultrathin MoSe2 nanosheets manifests the 2D nature of its structure. We demonstrated that these MoSe2 nanostructures possess intrinsic peroxidase-like activity in that they can facilitate the oxidation of TMB in the presence of H2O2, generating a visible color reaction. For the catalysis mechanism, kinetic analysis indicates that the catalytic reaction follows the typical Michaelis–Menten theory and a ping–pong mechanism. Moreover, active species study shows that O˙ 2− plays a pivotal role in the peroxidase-like catalytic reaction. Based on the color reaction of TMB catalyzed by our MoSe2 nanomaterials, we have developed a new colorimetric method for H2O2 detection by using 2D and 0D MoSe2 nanostructures as peroxidase mimetics. It is shown that the colorimetric sensing capability of our MoSe2 catalysts is comparable to other 2D materials-based colorimetric platforms. Overall, the synthesis strategy we proposed is environmentally friendly and economic, and it can easily be adapted to construct novel inorganic low-dimensional enzyme-free mimetic with intrinsic catalytic activity. The potential of the presented 2D and 0D MoSe2 nanostructures for use as a catalyst in other oxidation reactions could be explored in the extended study and this could create a new opportunity for this enzyme-mimicking MoSe2 nanostructures in many signiﬁcant ﬁelds, such as environmental protection, food monitoring, medical diagnostics, and photocatalysis.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/10/2045/s1, Figure S1: UV–vis absorption spectra of MoSe2 QDs with different concentrations of H2O2, Figure S2: UV–vis absorption spectra of 2D CTAB-MoSe2 NSs with different amounts of H2O2, Table S1: Michaelis–Menten parameters of the 2D CTAB-MoSe2 NSs and 0D MoSe2 QDs. Author Contributions: Conceptualization, D.-R.H. and Y.-Q.P.; methodology, Y.-Q.P. and K.H.S.; validation, Y.-Q.P., K.H.S. and S.E.I.; formal analysis, Y.-Q.P. and K.H.S.; investigation, Y.-Q.P. and K.H.S.; data curation, D.-R.H. and Y.-Q.P.; writing—original draft preparation, D.-R.H. and Y.-Q.P.; writing—review and editing, D.-R.H. and C.-T.L.; resources, H.-F.W. and M.M.C.C.; supervision, D.-R.H.; project administration, D.-R.H.; funding acquisition, D.-R.H. and C.-T.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Ministry of Science and Technology, Taiwan under grant Nos: MOST 108-2221-E-110-044, MOST 109-2811-M-002-634, and MOST 108-2119-M-002-025-MY3. Acknowledgments: We acknowledge financial support from Center of Crystal Research, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan. We thank Yuan-Kuei Hu for valuable help with the AFM characterization. Conflicts of Interest: The authors declare no conflict of interest.

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