Nanoscale

PAPER

Cite this: DOI: 10.1039/c5nr01728a

Received 17th March 2015,Accepted 15th April 2015

DOI: 10.1039/c5nr01728a

www.rsc.org/nanoscale

Insight into the mechanism revealing theperoxidase mimetic catalytic activity of quaternaryCuZnFeS nanocrystals: colorimetric biosensing ofhydrogen peroxide and glucose†

Amit Dalui,a Bapi Pradhan,a Umamahesh Thupakula,a Ali Hossain Khan,a

Gundam Sandeep Kumar,a Tanmay Ghosh,b Biswarup Satpatib andSomobrata Acharya*a

Artificial enzyme mimetics have attracted immense interest recently because natural enzymes undergo

easy denaturation under environmental conditions restricting practical usefulness. We report for the first

time chalcopyrite CuZnFeS (CZIS) alloyed nanocrystals (NCs) as novel biomimetic catalysts with efficient

intrinsic peroxidase-like activity. Novel peroxidase activities of CZIS NCs have been evaluated by catalytic

oxidation of the peroxidase substrate 3,3’,5,5’-tetramethylbenzidine (TMB) in the presence of hydrogen

peroxide (H2O2). CZIS NCs demonstrate the synergistic effect of elemental composition and photoactivity

towards peroxidase-like activity. The quaternary CZIS NCs show enhanced intrinsic peroxidase-like

activity compared to the binary NCs with the same constituent elements. Intrinsic peroxidase-like activity

has been correlated with the energy band position of CZIS NCs extracted using scanning tunneling spec-

troscopy and ultraviolet photoelectron spectroscopy. Kinetic analyses indicate Michaelis–Menten enzyme

kinetic model catalytic behavior describing the rate of the enzymatic reaction by correlating the reaction

rate with substrate concentration. Typical color reactions arising from the catalytic oxidation of TMB over

CZIS NCs with H2O2 have been utilized to establish a simple and sensitive colorimetric assay for detection

of H2O2 and glucose. CZIS NCs are recyclable catalysts showing high efficiency in multiple uses. Our

study may open up the possibility of designing new photoactive multi-component alloyed NCs as

enzyme mimetics in biotechnology applications.

1. Introduction

Activities of natural enzymes are widely investigated owing totheir high substrate specific catalytic efficiency under mildconditions.1,2 However, the catalytic activity of naturalenzymes depends on the presence of inhibitors and peripheralconditions like temperature and pH.3,4 In addition, the wide-spread applications of natural enzymes are limited due to highcosts of preparation, purification and storage.5 Hence, develop-ing and designing novel artificial enzyme mimetics are at thecentre of current research.6,7 Numerous oxidations in naturecan be initiated using peroxidase enzymes by activating hydro-gen peroxide.8,9 Artificial peroxidase enzyme mimetics like

hemin,10 hematin,11 cyclodextrin12 have recently been develo-ped and used for H2O2 detection. Magnetic nanocrystals (NCs)are shown to exhibit intrinsic enzyme mimetic activity similarto that of natural peroxidases such as horseradish peroxidase(HRP), which opens the scope of using nanoscale materials asenzyme mimetics in the biochemical field.13Advantageously,the inorganic NCs are robust and stable in comparison withthe natural enzymes, which makes the NCs promising candi-dates for enzyme mimetics.

A variety of inorganic functional NCs with peroxidase-likeactivity have been reported, including metal-based NPs(gold,14 copper,15 Pt–Pd bimetallic alloy16), oxides (Fe2O3

nanowire,17 ZnFe2O4 NPs,18 TiO2,19 CuO,20 Co3O4 NPs,21 V2O5

nanowires22), sulfides (CuS,23 FeS24), nickel teluride,25 hetero-structures,26,27 carbon nanomaterials (carbon nanodots,28 gra-phene oxide29), composites30 etc. The stability, superioractivity, easy purification, storage at low cost, flexibility inthe structure and compositionally tunable catalytic activitiesmade these NCs superior in comparison with naturalenzymes. Therefore, the artificial peroxidase mimetics hold

†Electronic supplementary information (ESI) available: Fig. S1–S13. See DOI:10.1039/c5nr01728a

aCentre for Advanced Materials, Indian Association for the Cultivation of Science,

Jadavpur, Kolkata 700032, India. E-mail: camsa2@iacs.res.inbSurface Physics and Material Science Division, Saha Institute of Nuclear Physics,

1/AF Bidhannagar, Kolkata 700064, India

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importance in future enzyme related applications. Despite thegreat advances in designing the peroxidase mimicking NCs,the mechanism of the peroxidase-like activity of these NCsseems to be ambiguous. In addition, the response of thephotoactive NCs to the incident light during the peroxidase-like catalytic process remains unexplored. Photoactive BiOBrmicrospheres show high dark and light enhanced peroxidase-like catalytic activity by catalyzing the oxidation of various per-oxidase substrates in the presence of H2O2.

31 Most of the pre-vious studies mainly focused on composition to design theenzyme mimetics but the photoactivity in peroxidase-like cata-lysis was usually neglected. It is therefore still challenging andimperative to clarify the origin of the peroxidase-like catalyticactivity of photoactive nanomaterials.

It is well known that copper and iron ions are commonions, which efficiently catalyze the oxidation of TMB in thepresence of H2O2.

32 The peroxidase-like activity of binaryCuS,23 FeS,24 ZnFeO2 alloyed NCs18 has been reported earlier.However, the enzyme mimetic activity of quaternary NCs con-taining copper, zinc and iron together with photoactive pro-perties remains unexplored. Earth abundant andenvironmentally friendly metal chalcogenide compoundsbelonging to the I–III–VI2 family usually exhibit a chalcopyritecrystal structure with broad band gap distribution, which is anadded advantage for photocatalysis.33–35 The synergistic effectof individual elements in quaternary alloyed NCs as well asphotoactivity can be used simultaneously for the developmentof superior enzyme mimetic materials. Hence, the develop-ment of alternative inexpensive photoactive catalysts composedof multi-component earth abundant elements is highly desir-able in the context of environmental and industrial demand.

Herein, we report highly-crystalline chalcopyrite CuZnFeSNCs (CZIS NCs) as novel biomimetic catalysts. The CZIS NCspossess intrinsic peroxidase-like activity catalyzing the oxi-dation of the peroxidase substrate TMB in the presence ofH2O2 to produce typical color reactions. The CZIS NCs possessa synergistic effect of elemental composition and photoactivityfor peroxidase-like activity. The catalytic reaction by CZIS NCsobeys the typical Michaelis–Menten mechanism. The mechan-ism of enhanced peroxidase-like activity of the CZIS NCs wasrevealed by probing the energy band diagram using scanningtunneling spectroscopy and ultraviolet photoelectron spec-troscopy. The CZIS NCs manifest a high sensitivity for colori-metric detection of H2O2 and glucose making them potentialbiosensors. The CZIS NCs possess robust structural stabilitywith high catalytic activity for multiple uses.

2. Experimental2.1 Materials

Zinc(II) chloride (anhydrous, 99.9%, Aldrich), copper(I) chlor-ide (Cu2Cl2·6H2O, 97%, Loba Chemie), iron(III) acetylacetonate(97%, Aldrich), oleylamine (OLA, technical grade, 70%,Aldrich), sulfur (100 mesh, Aldrich), 1-octadecene (ODE, techni-cal grade, 90%, Aldrich), oleic acid (OA, technical grade, 90%,

Aldrich), dimethysulfoxide (DMSO), hydrogen peroxide (H2O2),sodium acetate, disodium ethylenediaminetetraaceticacid(Na2-EDTA), ammonium oxalate (AO), 3,3′,5,5′-tetramethyl-benzidine (TMB), glucose oxidase (GOx), and isopropanol wereused in the pristine form. Indium tin oxide (ITO) substrates(70–100 Ω per square inch) were purchased from Aldrich.

2.2 Synthesis of nanocrystals

Synthesis of CuZnFeS NCs. Syntheses have been carried outaccording to our recent report with trivial modifications.36 In atypical synthesis, 0.1 mmol copper(I) chloride, 0.1 mmol zinc(II) chloride, 0.1 mmol iron(III) acetylacetonate, 0.5 mL of OLA,0.5 mL of OA and 6 mL of ODE were taken in a three neckedround-bottom flask and degassed with nitrogen at 120 °C for30 minutes to remove the moisture and oxygen. The reactionsystem was then slowly heated to 200 °C and 0.6 mmol sulfurdissolved in 3 mL ODE was quickly injected into the reactionsystem. Upon injection of sulfur, the reaction solutionimmediately turned into magenta black color indicating therapid formation of the CZIS NCs. The reaction was stoppedafter 2 minutes by removing the heating mantle and slowlycooled down to room temperature. The as-prepared NCs werepurified by several cycles of precipitation–dispersion usingchloroform and ethanol. The synthesized CZIS NCs were driedand stored in powder form for further characterization.

Synthesis of copper sulfide NCs. 0.1 mmol copper(I) chlor-ide, 0.5 mL of OLA, 0.5 mL of OA and 6 mL of ODE weredegassed with nitrogen at 120 °C for 30 minutes. At 200 °C,0.6 mmol of sulfur dissolved in 3 mL of ODE was quicklyinjected and annealed for 2 minutes at 200 °C. The reactionsystem was allowed to cool down to room temperature. The as-prepared NCs were purified by several cycles of precipitation–dispersion using chloroform and ethanol.

Synthesis of iron sulfide NCs. We have followed a similarsynthesis procedure as described above in the case of coppersulfide NCs. For iron sulfide NC preparation, 0.1 mmol iron(III)acetylacetonate was used as the iron precursor.

Synthesis of zinc sulfide NCs. Zinc sulfide nanocrystalswere prepared using a single source precursor, zinc-ethyl-xanthate in a mixture of ligands hexadecylamine (HDA) andtrioctylphosphine (TOP) at 200 °C according to the literaturemethod.37,38 In a typical procedure, a mixture of HDA and TOP(1 : 2 millimolar ratio) was taken in a three necked round-bottom flask and heated to 200 °C. At this temperature,0.64 mmol zinc-ethylxanthate dissolved in 9 mmol TOP wasquickly injected in the reaction round-bottom flask andannealed for 60 minutes at 200 °C. Then, the reaction wascooled down to room temperature. The as-prepared NCs werepurified by several cycles of precipitation–dispersion usingchloroform and ethanol.

Synthesis of copper–zinc-sulfide (CZS) and copper–iron-sulfide (CIS) NCs. For the synthesis of CZS NCs, 0.1 mmol ofcopper(I) chloride, 0.1 mmol of zinc(II) chloride, 0.5 mL ofOLA, 0.5 mL of OA and 6 mL of ODE were degassed with nitro-gen at 120 °C for 30 minutes. The reaction system was thenslowly heated to 200 °C. Then 0.6 mmol of sulfur dissolved in

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3 mL ODE was quickly injected at 200 °C and annealed furtherfor 2 minutes. The reaction system was allowed to cool downto room temperature. CIS NCs are prepared in the samemanner by adding 0.1 mmol iron(III) acetylacetonate instead of0.1 mmol zinc(II) chloride. The as-prepared NCs were purifiedby several cycles of the precipitation–dispersion method usingchloroform and ethanol.

2.3 Ligand exchange procedure

To explore the peroxidase-like catalytic activity of the NCs inaqueous medium, as synthesized OLA and OA capped CZISNCs are exchanged with 3-mercaptopropanoic acid (MPA) toget M-CZIS NCs. In a typical procedure, 50 mg of hydrophobicNCs (OLA and OA capped) were dissolved in 3 mL chloroform.Then the aqueous solution of MPA (pH of the solutionadjusted to greater than ten by adding sodium hydroxide ortetramethylammonium hydroxide) was added drop-wise undervigorous stirring. Stirring was performed for 30 minutes. Aseparation of two distinct layers of chloroform and NCs wasobserved, after stopping the stirring. The NCs were separatedout by centrifugation and washed two times with chloroformto remove un-exchanged OA and OLA. The NCs were furtherdispersed in water and precipitated out with acetone to removeexcess MPA. Finally the resulting NCs were dried undervacuum and stored for further characterization.

2.4 Characterization

Transmission electron microscopy (TEM) and high resolutionTEM (HRTEM) investigations were carried out using a JEOLJEM-2010 microscope operating at 200 kV. The sample was pre-pared by drop casting the NCs from chloroform/ethanol solu-tion onto a lacey carbon-coated gold grid (300 meshes) prior tomeasurements. Elemental analysis was carried out using X-rayphoto-electron spectroscopy (XPS) measurements with anOmicron X-ray photoelectron spectrometer. High-angleannular dark-field scanning transmission electron microscopy(STEM-HAADF) measurements were carried out using a FEI,TF30-ST microscope operating at 300 kV equipped with a scan-ning unit and an HAADF detector from Fischione (model3000). TF30 is also equipped for electron energy loss spec-troscopy with a post-column Gatan Quantum SE (model 963).Energy filtered TEM (EFTEM) images were acquired usingGatan Imaging Filter. Chemical maps from Cu M (74 eV),Zn M (87 eV), Fe M (54 eV) and S L (165 eV) edges wereobtained using the jump ratio method by acquiring twoimages (one post-edge and one pre-edge), respectively. Compo-sitional analysis was performed by energy dispersive X-rayspectroscopy (EDX) attached to TF30. Powder X-ray diffraction(XRD) patterns were recorded with a Bruker D8 Advanced diffr-actometer using Cu-Kα radiation (λ = 1.5405 Å). UV-vis-NIRabsorption spectra were recorded with a Varian-cary-5000spectrophotometer. Photoluminescence (PL) spectra wererecorded using a Jobin Yvon nanolog spectrophotometer bydispersing the NCs in tetrachloroethylene/water.

2.5 Peroxidase-like activity of M-CZIS NCs

Peroxidase-like activity of the M-CZIS NCs was evaluated by thecatalytic oxidation of the peroxidase substrate TMB in the pres-ence of H2O2. The measurements were carried out by monitor-ing the absorbance change at 652 nm. In a typical experiment,50 μL of M-CZIS NC dispersion (2 mg mL−1) was mixed in3 mL of sodium acetate buffer solution (pH = 4.2) in a quartzcuvette, followed by addition of 20 μL of TMB solution(15 mM, DMSO solution). Then, 10 μL of H2O2 (1.5 M) wasadded into the mixture and stirred with a tiny magnetic barfor 20–30 minutes for the generation of blue color solution.After stopping the stirring M-CZIS NCs settle down at thebottom of the cuvette and the upper clear blue solution istaken for the absorption measurement. For comparison, thecontrol experiments were also conducted under the same con-ditions. All the catalytic experiments were performed underday light, unless otherwise stated.

2.6 Kinetic assay

Kinetic measurements were carried out in a time course modeby monitoring the absorbance change at 652 nm. To investi-gate the mechanism, assays were carried out by varying con-centrations of TMB at a fixed concentration of H2O2 or viceversa. Experiments were performed using 50 μL of M-CZIS NCdispersion (2 mg mL−1) mixed in 3 mL of sodium acetatebuffer solution (pH 4.2), TMB solution (15 mM, DMSO solu-tion) or 15 mM H2O2 as the substrate unless otherwise stated.The apparent kinetic parameters were calculated using Line-weaver–Burk (L–B) plots of the double reciprocal of theMichaelis–Menten equation.13

2.7 Colorimetric detection of hydrogen peroxide and glucose

Glucose detection was carried out as follows: firstly, 10 μL ofGOx aqueous solution (1 mg mL−1) and 100 μL of D-glucosewith various concentrations in NaH2PO4 buffer (0.5 mM, pH7.0) were mixed and incubated at 37 °C for 1 hour. Then 20 μLof TMB (15 mM, DMSO solution), 50 μL of the M-CZIS NC dis-persion (2 mg mL−1) and 3 mL of sodium acetate buffer (0.1M, pH 4.2) were successively added to the glucose reactionsolution. Finally, the mixed solution was incubated at 40 °Cfor 30 minutes for spectroscopic measurement. Limit of detec-tion (LOD) has been quantitatively calculated from the slope ofa calibration curve using the formula LOD = 3.3 × SD of theintercept/slope at a signal to noise ratio (S/N) equal to 3.

2.8 Test for hydroxyl radicals (•OH) by the PL method

The formation of hydroxyl radicals (•OH) was detected by PLspectroscopy using terephthalic acid as a probe molecule. Tere-phthalic acid readily reacts with •OH to produce a highly fluo-rescent product, namely 2-hydroxyterephthalic acid. The PLintensity of 2-hydroxyterephthalic acid is proportional to theamount of •OH radicals produced in the reaction medium. Theexperiment was carried out by mixing 10 μL of H2O2 (1.5 M)and 100 μL of 2 mg mL−1 of M-CZIS NCs into the 3 mL 5 ×10−4 M aqueous solution of terephthalic acid in 2 × 10−3 M

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NaOH in a cuvette. In a regular time interval, we measured thePL intensity with an excitation wavelength of 315 nm using ananolog spectrofluorometer.

3 Results and discussion3.1 Characterization of CZIS NCs

The transmission electron microscope (TEM) image (Fig. 1a)shows mostly faceted spherical NCs with an average size of12 ± 3 nm along with some triangular shaped larger NCs. Thehigh resolution TEM (HRTEM) image reveals an inter-planarspacing of 0.32 ± 0.01 nm (Fig. 1b). CuFeS2 NCs are taken asreference crystals to assign the crystallographic phase basedon the close elemental composition,39 since JCPDS data fileis not available for the direct assignment of the crystallo-graphic phase of newly designed NCs. A comparison of latticespacing of our NCs with CuFeS2 corresponds to (112)planes (d112 of CuFeS2 = 0.303 nm) of the chalcopyrite phase(JCPDS # 370471).

X-ray photoelectron spectroscopy (XPS) confirms (Fig. S1,ESI†) the presence of Cu1+, Zn2+, Fe3+ and S2− constituentelements within the NCs.40,41 Fig. 1c reveals the STEM-HAADF

image of CZIS NCs and elemental composition was estimatedusing the EDX technique. EDX spectra were collected on tendifferent particles and two different areas using a nano probe(∼1 nm). The average atomic percentage of Cu : Zn : Fe : S∼ 29 : 06 : 29 : 36 is shown in the inset of the STEM-HAADFimage. Elemental mapping of NCs using the EFTEM techniquefurther confirmed the presence of the constituent elements(Fig. 1d–g).42 The OA and OLA capped CZIS NCs have poorwater dispersibility due to the presence of long hydrocarbonchains. To explore the catalytic activity in aqueous medium,3-mercaptopropanoic acid (MPA) was used to exchange OA andOLA capping ligands producing MPA capped CZIS (M-CZIS)NCs.43 The X-ray diffraction (XRD) pattern of the M-CZIS NCsshows Braggs peaks corresponding to (112), (220), (204), (312)and (116) reflections which are identical to OA, OLA cappedCZIS NCs (Fig. 2a) of the chalcopyrite phase (JCPDS#370471).39 The XRD pattern is in-line with the HRTEM obser-vation suggesting that the chalcopyrite crystallographic phaseof CZIS NCs is identical to CuFeS2. M-CZIS NCs have a broadrange of absorption in the UV-visible-NIR region and absorp-tion properties remain unchanged in comparison with as-synthesized CZIS NCs (Fig. 2b).This indicates that M-CZIS NCshave identical optical and structural properties like OA, OLA

Fig. 1 (a) Low magnification TEM image of CZIS NCs capped with OA and OLA, (b) high-resolution TEM image of a single CZIS NC with wellresolved lattice planes. (c) STEM-HAADF image of CZIS NCs: inset shows the atomic percentage of the constituent elements estimated using EDX;here different locations are indicated by red from where EDX are taken. (d–g) Chemical maps of the constituent elements Cu, Zn, Fe and S acquiredusing the EFTEM technique.

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capped CZIS NCs. The broad range of absorption with a highabsorption coefficient indicates applicability of M-CZIS NCs inthe field of photocatalysis.

3.2 Peroxidase-like catalytic activity of M-CZIS NCs

To investigate the peroxidase-like catalytic activity of M-CZISNCs, we have taken TMB as a typical chromogenic substrate,44

because it is a benign and non-carcinogenic color reagent. Thepartially oxidized TMB (oxTMB) has characteristic absorptionpeaks at 370 and 652 nm originating from its distinctive bluecharge-transfer complex of di-amine and di-imine (Fig. S2,ESI† for reaction schematic).45

Fig. 3 reveals that the TMB and H2O2 systems withoutM-CZIS NCs show negligible color variation while M-CZIS NCsand the TMB system are colorless under experimental con-ditions. However, M-CZIS NCs along with TMB and H2O2

systems produce a deep blue color (Fig. 3, blue curve). Likeenzymatic peroxidase activity, such as observed for the com-monly used enzyme horseradish peroxidase (HRP),45 this colorreaction was quenched by adding H2SO4. Acidification withH2SO4 converts the charge transfer complex into a fully oxi-dized di-imine showing a yellow color with λmax = 450 nm(Fig. 3, red curve). These results support the fact that theM-CZIS NCs act like peroxidase toward the typical peroxidasesubstrate TMB to produce a blue charge-transfer complex(chromogen) similar to that of natural peroxidase enzyme.44,45

The absorbance at 652 nm of oxTMB provides a way tomonitor the catalytic reaction. Acetate buffer was used as thereaction medium. A series of buffers with varying pH wereinvestigated to optimize the reaction conditions and themaximum relative catalytic activity was obtained at pH 4.2

(Fig. S3, ESI†). However, there are possibilities that the leachedout copper or iron may catalyze the breakdown of H2O2. Inorder to rule out the possibility of TMB oxidation by leachedout iron or copper, CZIS NCs were incubated at pH 4.2 for1 hour and then the supernatants were used for catalytic reac-tion. A comparison of absorbance and color of the reactionsolution (Fig. S4, ESI†) further demonstrated that the catalytic

Fig. 2 (a) Comparison of XRD patterns of the OA, OLA capped and MPA capped CZIS NCs. XRD patterns have been compared with the standardJCPDS data file (vertical red line) and diffraction planes are assigned to the peaks accordingly. (b) Comparison of the UV-vis-NIR absorption spec-trum of the CZIS NCs capped with OA, OLA and MPA.

Fig. 3 UV-vis absorption spectra of the oxidized TMB in acetate bufferfor different systems; (a) TMB + M-CZIS NC system, (b) TMB + H2O2

system, (c) TMB + M-CZIS NCs + H2O2 system and (d) TMB + M-CZISNCs + H2O2 system after addition of conc. H2SO4. The inset photo-graphs show the color of the corresponding systems.

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effect was not from the ions leached at pH 4.2, since theamount of copper or iron ions leached was lower than the con-centration required for the Fenton reaction.13 This further con-firms that the observed reaction cannot be attributed toleaching of ions into solution, but occurs on the surface of theM-CZIS NCs. Similar to natural enzymes, the catalytic activityof the M-CZIS NCs is also dependent on reaction temperature.Since the catalytic activity using M-CZIS is very fast, we havestudied the catalytic activity at a temperature of 32 °C in orderto capture the reaction dynamics (Fig. 4a). The optimal temp-erature and pH were similar to those observed with othernanomaterial based enzyme mimetics and natural enzymehorseradish peroxidase (HRP).13 The catalytic activity ofM-CZIS NCs is also dependent on the concentration of TMB,

H2O2 and the amount of NCs (Fig. 4b–d). The maximum cata-lytic activity of the NCs was obtained using the optimal of con-ditions: pH 4.2, 32 °C, 50 μg mL−1 of NCs, 600 μM TMB and150 μM H2O2.

We have investigated the effect of elemental composition ofthe NCs on the peroxidase-like catalytic activity. We havefreshly designed copper sulfide, iron sulfide, zinc sulfide,copper–zinc-sulfide and copper–iron-sulfide NCs (Fig. S5,ESI†) and ligand exchanged with MPA to investigate their per-oxidase-like catalytic activities. Compared to the binarycounterpart, ternary copper–iron-sulfide and quaternarycopper–zinc–iron-sulfide show higher absorbance at 652 nmhence the superior peroxidase-like catalytic activities (Fig. S6,ESI†).

Fig. 4 Dependence of relative activity of peroxidase-like catalytic reactions by M-CZIS NCs on the (a) temperature where inset photographs areshowing the color of the reaction solutions obtained at different reaction temperatures, (b) TMB concentration, (c) H2O2 concentration and (d)M-CZIS NC amounts respectively. Activity of 100% is set where absorption is highest and the relative activities for other absorptions are calculatedaccordingly.

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3.3 Kinetic assay

To investigate the kinetic parameters, the catalytic activity ofM-CZIS NCs was studied by enzyme kinetics theory andmethods with H2O2 and TMB as substrates (Fig. 5).46 Theabsorbance of oxTMB (at 652 nm) increases gradually withH2O2 concentration (from 0.02 mM to 0.5 mM) and saturationis reached at higher H2O2 concentrations (Fig. 5a). Similarly,the absorbance of oxTMB (at 652 nm) increases gradually withTMB concentration (from 0.1 mM to 1.2 mM) until amaximum is reached at higher concentrations of TMB indicat-ing that the catalytic reaction is substrate concentration depen-dent (Fig. 5b). A series of experiments are performed bychanging the concentration of one substrate and keeping theconcentration of the other constant. Typical Michaelis–Mentencurves could be obtained in a certain range of TMB concen-tration (Fig. 5). The Michaelis–Menten constant (Km), whichindicates the affinity of an enzyme for its substrate, is obtained

by the use of a Lineweaver–Burk (L–B) plot:

1v¼ Km

vmax

1½S� þ

1vmax

where v, vmax, [S] and Km are initial velocity, maximum reactionvelocity, concentration of substrate, and Michaelis constantrespectively. The double reciprocal plots of velocity against oneof the substrate concentrations are also acquired when theother substrate is fixed at three concentration levels (Fig. 5cand d). Km and vmax for photoactive M-CZIS NCs with TMB asthe substrate are found to be 2.2 mM and 390 nM s−1, respect-ively. The Km and vmax values for H2O2 as the substrate were0.07 mM and 5.6 nM s−1 , respectively (Table 1). Generally, thesmaller the value of Km the stronger is the affinity between theenzyme and the substrate. The Km (0.07 mM) for the M-CZISNCs is smaller than 0.16 mM of HRP immobilized on theNafion–cysteine modified gold electrode,47 indicating a higher

Fig. 5 Velocity of the reaction is evaluated by changing the concentration of the substrate such as (a) H2O2 and (b) TMB. The results have beenfitted with a non-linear function of the Michaelis–Menten equation. (c, d) Double reciprocal L–B plot by keeping one substrate fixed and varying theconcentration of the other substrate; for plot (c) TMB concentrations are fixed and H2O2 concentrations are varied whereas for plot (d) H2O2 con-centrations are fixed and TMB concentrations are varied.

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affinity of the M-CZIS NCs to H2O2. This is in agreement withthe fact that a lower H2O2 concentration is required to obtainmaximum activity for M-CZIS NCs. In comparison with othernanoparticles exhibiting peroxidase-like activity (with H2O2 asan oxidant) in previously published reports, M-CZIS NCs haveremarkable advantages as indicated by Km (Table 1).

3.4 Mechanism of peroxidase-like activity

There are two main reaction pathways for catalyzing H2O2 intoa •OH radical in the presence of a suitable catalyst which thenreacts with TMB to form oxTMB. The first one is the reactionof H2O2 with Fe(III) to produce the •OH radical through theFenton reaction50 and the other one is the capturing ofphoton-generated electrons by H2O2 from the conductionband of the catalyst to generate •OH radicals.51 The presenceof Fe(III) on the surface of M-CZIS NCs can initiate reactionswhich produce the •OH radical by the Fenton reaction as

described in the following reactions:51

FeIII þH2O2 ! FeII þ •OOHþHþ;

FeII þH2O2 ! FeIII þ •OHþ OH�:

The peroxidase like activity of Cu ions in copper sulfideNCs has previously been reported in the presence of H2O2.

23,32

Our small sized M-CZIS NCs with a high surface area mayallow the exposure of more Fe(III) and Cu(I) for reactions andenhance the catalytic efficiency. Under visible light irradiation,electrons are excited from the valence band to the conductionband in M-CZIS NCs. Furthermore, the conduction band elec-tron may transfer to H2O2, which is as an effective electron sca-venger, to form •OH or photo excited holes can alsodecompose H2O2 into •OH radicals. As a result, more carriersare available for reactions and more •OH radicals are producedfor the TMB oxidation of peroxidase mimicking catalyticactivity. Indeed M-CZIS NCs show higher absorption at 652 nmwith a faster rate in light in comparison with that under darkconditions (Fig. 6a). In the dark, the absorption shows a linearincrease with time. In the presence of light, the 652 nmabsorption saturates just after 20 minutes and decreases for alonger exposure time. M-CZIS NCs show photo-response uponlight illumination (Fig. 6b). Current density (0.01 mA cm−2)measured in the dark is found to increase to 0.2 mA cm−2

upon illumination of light at a bias voltage of 1 V (Fig. 6b).These results indicate that the carrier mobility is enhanced inthe presence of light, which is beneficial for peroxidase likecatalytic activity.

In order to reveal the mechanism of peroxidase-like activity,we further carried out scanning tunneling microscopy andspectroscopy (STM and STS) on M-CZIS NCs. STM and STSreveal the valence band (VB) and conduction band (CB)information of a single NC by directly probing the current

Table 1 Michaelis–Menten constant (Km) and maximum velocity (vmax)obtained from the double reciprocal plots which have been comparedwith the natural enzyme HRP and other artificial enzyme mimetic NCs

Catalysts

Km [mM] vmax [nM s−1]

Ref.H2O2 TMB H2O2 TMB

M-CZIS NCs 0.07 2.2 5.6 390 Present workHRP 3.7 0.434 87.1 100 13Fe3O4 154 0.098 97.8 34.4 13ZnFe2O4 MNPs 1.66 0.85 77.4 133.1 18Co3O4 NP 140.07 0.037 121 62.7 21FeS 9.36 0.008 192 87 24BiOBr 0.046 1.61 3.7 26.3 31Fe0.5Co0.5 NPs 0.06 1.79 132 456 48VO2 (B) 1.69 0.146 1770 1310 49

Fig. 6 (a) Comparison of the time dependent absorption change at wavelength 652 nm under dark and visible-light illumination. (b) Current density(J)–voltage (V) characteristic of the device made by M-CZIS NCs under dark and 1 sun illumination; inset shows the schematic of the device struc-ture used in the J–V measurement.

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(I)–voltage (V) characteristics by the resonant tunneling mech-anism.38 Fig. 7a (inset) shows the STM image of an isolatedM-CZIS NC on a HOPG substrate revealing a dimension of∼15 nm in diameter. The size of the NC is observed to beslightly enlarged in comparison with the sizes measured inTEM. A finite radius of STM tip may contribute to the apparentenlargement in the NC diameter. The I–V curve (average of∼50 measurements) collected on an isolated M-CZIS NCreveals a semiconductor nature (Fig. S7a, ESI†). The corres-ponding dI/dV curve calculated by numerical differentiationalso reveals a finite zero conductivity at the zero bias.

In addition, a series of tunneling resonance peaks areclearly observed after exceeding the zero conductivity regionsat both positive and negative bias voltages. It is well knownthat the dI/dV curve of NC in STM represents the local densityof states (LDOS) at a particular position from where the spec-trum has been collected. In addition, the peak like behavioron either side of the zero bias reflects the LDOS of CB and VBat positive and negative bias, respectively. Indeed, similar dI/dV spectral features are observed for our NCs. The corres-ponding onset/threshold positions of CB at positive bias(+0.55 V) and VB at negative bias (−0.5 V) reveal a local tunnel-ing band gap value of ∼1.05 eV (Fig. 7a). We further comparedthe relative VB and CB positions of M-CZIS with that of hydra-zine capped CZIS NCs.36 The corresponding VB position(−0.5 V) is observed to be constant for both the NCs, however,the CB position is modified from +0.25 V of hydrazine cappedCZIS to +0.55 V for M-CZIS NCs demonstrating a larger bandgap for the M-CZIS NCs.

We carried out ultraviolet photoelectron spectroscopy (UPS)measurements on M-CZIS NCs to estimate the relative VB edgeposition with respect to the vacuum level. Generally, the UPSmeasurements reveal the information of the top most valence

band which can be assigned to a specific orbital of a particularelement present in the NCs. The UPS spectrum of M-CZIS NCsis presented in the ESI (Fig. S7b†). It is well known from theliterature that the top most VB contains the contribution of Cu‘3d’ orbitals in CuFeS2 like chalcopyrite compounds.52 Earlier,the structural characterization revealed that the CZIS NCs aresimilar to CuFeS2 chalcopyrite compounds.39 Therefore, wehave assigned the top most orbitals in the UPS VB spectrum toCu ‘3d’ orbitals. The UPS measurements suggest a bindingenergy (BE) of ∼1.95 eV for Cu ‘3d’ orbitals in M-CZIS NCs.A relative shift of ∼0.35 eV in comparison with the BE of Cu‘3d’ orbitals in bulk copper (∼1.6 eV) was found. In addition,the top most Cu ‘3d’ orbitals in bulk (Fermi level) are placed at∼−4.7 eV with respect to the vacuum level.53 Hence, by addingthe relative BE shift of Cu ‘3d’ orbitals (∼0.35 eV) to the Fermienergy of bulk copper (−4.7 eV) we can achieve the VB positionof M-CZIS NCs at −5.05 eV with respect to the vacuum level.Moreover, addition of the band gap value (1.05 eV) extractedfrom the STS measurements to the VB position of M-CZIS NCs(−5.05 eV) reveals the relative CB position at −4.0 eV withrespect to the vacuum. Fig. 7b shows the resulting banddiagram drawn in combination of the STM, STS and UPSmeasurements of M-CZIS NCs. We have correlated the bandposition of NCs extracted using STS and UPS techniques withthe redox potential of oxidative species in order to reveal thecatalysis pathways (Fig. 7c). Formation of oxidative species •OHradicals from the decomposition of H2O2 by the photo-excitedelectrons or holes is favorable since the corresponding redoxpotentials E° H2O2/

•OH = −4.8 eV (−0.33 V with respect tonormal hydrogen electrode scale)54 remain within the bandgap of M-CZIS NCs (Fig. 7c). Photo-excited electrons may reactwith the dissolved oxygen (O2) to form the super oxide radical(O2

•−) since the redox potential of O2 (aq)/O2•− (E° O2(aq)/O2

•− =

Fig. 7 (a) dI/dV curve measured on isolated M-CIZS NCs. Typically ∼50 I–V curves are averaged to improve the signal to noise ratio. The dI/dVmeasurement was performed at a set-voltage of 1 V and a set-current of 0.1 nA. Inset: STM image of M-CIZS nanocrystals on the HOPG substrate.The STM image was obtained in constant current mode by using the set-voltage of 1 V and set-current of 0.1 nA. (b) Energy band diagram forM-CZIS NCs obtained from a combination of STS and UPS measurements with respect to the vacuum level. (c) Standard redox potentials (E°) ofdifferent oxidative species with respect to the vacuum energy level scale.

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−4.34 eV)55 resides below the CB position of M-CZIS (Fig. 7c).The superoxide radical (O2

•−) then undergoes protonation gen-erating the hydroxyl radicals (•OH) required for peroxidase-likecatalytic reaction (Fig. 7c).

In order to investigate reaction pathways involved in the oxi-dation process of TMB catalyzed by M-CZIS NCs, we have per-formed an experiment by adding different quenchers to thecatalytic reaction system that can scavenge the relevant reactivespecies such as hydroxyl radicals (•OH), superoxide anions(O2

•−), and photogenerated holes (h+) etc. Here, 2-propanol(IPA) or methanol (MA) was utilized to scavenge •OH radicalsin solution whereas ethylenediaminetetraacetic acid (EDTA) orammonium oxalates (AO) was used to scavenge the photogene-rated holes (h+) (Fig. S8, ESI†).56 N2 was purged in the reactionsystem to perform TMB oxidation under an oxygen free atmos-phere. The catalytic activity of photoactive M-CZIS NCs for theoxidation of TMB was not largely influenced by the inertatmospheric conditions with nitrogen.56 However, the catalyticactivity of M-CZIS NCs for TMB oxidation decreases in thepresence of a •OH scavenger like IPA or MA, indicating theinvolvement of •OH radicals in the TMB oxidation process.However, the catalytic activity of M-CZIS NCs was hinderedmostly in the presence of a hole scavenger like a disodium saltof EDTA or AO, indicating that photogenerated holes (h+) playvital roles in TMB oxidation (Fig. S8, ESI†). Also, the chelatingagent EDTA or AO may cover the surface of the M-CZIS NCswhich hindered the Fenton reaction between H2O2 and Fe(III)or Cu(I) at the NC surface to form •OH radicals. We have inves-tigated the TMB oxidation with M-CZIS NCs under UV lightexcitation without addition of H2O2 (Fig. S9, ESI†). The photo-generated exciton (electrons or holes) of M-CZIS NCs can cata-lyze the oxidation of TMB generating blue color but requires amuch higher amount of TMB and M-CZIS NCs. In comparison,

much higher activity was obtained using very less concen-tration of TMB or M-CZIS NCs in the presence of 5 mM H2O2.This indicates that generation of •OH radicals from H2O2 byreaction with photogenerated holes and reaction of H2O2 withFe(III) or Cu(I) at the NC surface to form •OH radicals are thereaction pathways involved in the oxidation of TMB.

The presence of •OH radicals was investigated using a spintrapping experiment by electron paramagnetic resonance(EPR) spectroscopy of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)and hydroxyl radical adduct (DMPO-•OH adduct).57 In general,the DMPO-•OH adduct produces four hyper fine peaks withinthe range 3400 to 3500 gauss. Unfortunately, in our study theresulting hyperfine peaks are surpassed by a broad peak in therange of 3405 to 3530 gauss (Fig. S10, ESI†). However, we suc-cessfully investigated the generation of •OH by the fluo-rescence method using terephthalic acid as a probe moleculewhere terephthalic acid reacts with •OH radicals to producehighly fluorescent 2-hydroxy terephthalic acid.58 A gradualincrease in PL intensity at 425 nm was observed with the incre-ment of the M-CZIS NC amount (Fig. S11, ESI†). Noticeably,PL intensity was not detected in the absence of the M-CZISNCs. This observation helps us to conclude that the M-CZISNCs can catalytically activate H2O2 to produce •OH radicalsthat can react with TMB to produce the color reaction. Thusthe activation of H2O2 by the M-CZIS NCs to produce a higheramount of •OH radicals enhances the peroxidase-like catalyticactivity.

3.5 Colorimetric detection of hydrogen peroxide and glucose

The color variation caused by TMB oxidation is dependent onH2O2 concentration, which is monitored by an absorbancechange at 652 nm. The phenomenon could be used as a strat-egy for quantitative detection of H2O2. As the absorbance of

Fig. 8 (a) Absorbance change at 652 nm for TMB oxidation by variation of H2O2 concentration; lower inset shows the evolution of the absorptionat 652 nm with the increment of H2O2 concentration and upper inset photograph of the reaction solution on increasing the H2O2 concentration. (b)Absorbance change at 652 nm for TMB oxidation by variation of glucose concentration; lower inset shows the evolution of the absorption at652 nm with the increment of glucose concentration and upper inset photograph of the reaction solution on increasing the glucose concentration.Error bars (3–7%) are shown for each data point.

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oxTMB at 652 nm is in proportion to H2O2 concentration, it isa facile approach to detect H2O2 using a spectrophotometer.

Fig. 8a shows the calibration curve of the absorbancechange at 652 nm against H2O2 concentration. Enhancementof absorbance at 652 nm was observed with the increment ofH2O2 concentration (Fig. 8a, lower inset).The curve was linearin a range from 10 to 55 μM (correlation coefficient R =0.9995). The limit of detection for hydrogen peroxide was 3 μM(S/N = 3), indicating a potentially novel strategy to detect lowH2O2 concentration. Furthermore, the color variation isobvious on visual observation, which offers a convenientapproach to detect H2O2 by the eye with lowest visual concen-tration being 12 μM (Fig. 8a, upper inset photograph). Hydro-gen peroxide is the main product of the glucose oxidase (GOx)catalyzed reaction.59 GOx catalyzes the oxidation of glucose toproduce gluconic acid and hydrogen peroxide in the presence

of oxygen. The as produced hydrogen peroxide was then cata-lyzed by NCs which reacts with TMB, producing a blue color.Hence, color change of the oxTMB can be employed indirectlyto measure the glucose content using M-CZIS NCs instead ofthe traditionally used horseradish peroxidase. Glucose is amajor energy source in cellular metabolism, and has animpact on the natural growth of cells. It has been establishedthat there is a correlation between the breakdown of glucosetransport in the human body and the occurrence of certaindiseases, such as diabetes and some cancers. So, the detectionof glucose has been an active area of research.56 Since GOxwould be denatured in a pH 4.2 buffer solution, the glucosedetection can be performed in two steps: (i) GOx catalyzes oxi-dation of glucose to gluconic acid and H2O2 in a pH 7.0 buffersolution, while in the meantime, the substrate oxygen is con-verted to H2O2,

59 (ii) H2O2 will be decomposed by M-CZIS NCs

Fig. 9 (a) Stability of the M-CZIS NCs towards the relative activity of TMB oxidation after annealing the NCs at different temperatures up to 100 °C;inset: color of the reaction solution of the oxTMB produced by the M-CZIS NCs after annealing at different temperatures. (b) Relative activity of per-oxidase-like activity of the M-CZIS NCs in fourteen repetitive cycles where the inset shows the absorption of the reaction solution in fourteen repeti-tive cycles. (c) XRD patterns of the M-CZIS NCs before catalysis and after reuse of ten cycles where diffraction patterns are compared with JCPDSdata files (red vertical lines). (d) TEM image of M-CZIS NCs before catalysis; inset: HRTEM image of M-CZIS NCs with well resolved lattice planes ofchalcopyrite (112). (e) TEM image of M-CZIS NCs after ten reaction cycles where the inset reveals the corresponding HRTEM image indexed withlattice planes corresponding to the (112) plane of the chalcopyrite crystal structure.

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to produce •OH radicals which react with the TMB co-substrateat pH 4.2, to produce a blue color (detailed procedure in theExperimental section). Fig. 8b shows the standard curve of theabsorption versus glucose concentration. A higher absorptionwas observed at 652 nm with the increase of glucose concen-tration (Fig. 8b, lower inset). The linear range for glucose was16 to 60 μM, with a limit of detection as low as 4.1 μM (S/N =3). The sensitivity of our system is appropriate for bloodglucose detection considering that blood glucose concen-tration in healthy and diabetic persons is about 3–8 mM and9–40 mM, respectively.60 The color variation for glucose wasalso obvious by visual observation, even lower than 20 μM(Fig. 8b, upper inset photograph). Our method is likely to bepractically useful for glucose detection upon further develop-ment in biotechnology and clinical diagnosis of enzymaticmimetics. To explore the selectivity of our method for detec-tion of glucose, responses of the sensor to other saccharidesand potential interferents such as ascorbic acid, maltose,galactose, mannose as control samples were also investigatedunder the same detection conditions. Our observation shows(Fig. S12, ESI†) that the absorbance of these glucose analoguesare not obvious when their concentrations are five timeshigher than that of glucose, indicating that our sensor showshigh selectivity toward glucose. The color difference can alsobe distinguished by the eye.

Surface area measurement by the BET (Brunauer–Emmett–Teller) reveals a specific surface area of 25.2 m2 g−1 for M-CZISNCs (Fig. S13, ESI†) indicating larger ion active sites (Fe andCu) at the surfaces, a feature beneficial for observed enhancedperoxidase-like catalytic properties compared to HRP, whichhas only one iron ion at the active center.13,61 M-CZIS NCsshow high catalytic activity even after annealing under harshreaction conditions like high temperature.They show over 80%realtive activity after being annealed upto a temperature of100 °C (Fig. 9a). In addition, M-CZIS NCs are recyclable cata-lysts showing over 85% relative catalytic activity up to fourteenrepetitive cycles (Fig. 9b) for TMB oxidation suggesting thatM-CZIS can be reused in catalytic processes.The NCs retaintheir crystallographic phase even after the 10th cycle of cata-lysis showing structural robustness (Fig. 9c). TEM and HRTEManalysis for fresh and NCs after the tenth cycle samples revealunchanged structural stability and crystallinity of the NCs,proving their robustness as environmentally benign peroxidasemimetic catalysts (Fig. 9d and e).

4 Conclusions

In conclusion, we have demonstrated that photoactive quatern-ary CZIS NCs possess highly efficient peroxidase-like catalyticactivity towards the oxidation of the peroxidase substrate TMBin the presence of H2O2. We show the effect of elemental com-position by designing NCs of different elemental combinationstowards TMB oxidation, which suggests that the quaternaryCuZnFeS composition possesses superior catalytic activity.The study gives an insight into the mechanism of enhanced

catalytic efficiency as has been revealed by extracting theenergy levels of the CZIS NCs, using a combination of STS andUPS, which suggests favorable catalysis pathways for TMB oxi-dation. In terms of the color reaction, we have successfullyestablished a simple and sensitive colorimetric assay for thedetection of H2O2 and glucose based on CZIS NCs.The recycl-ability and structural robustness promise multiple uses of theNCs which may have potential applications in biotechnologyand clinical diagnosis as enzymatic mimics. The inherent cata-lytic properties of the CZIS NCs may open up the prospect ofdesigning and understanding the peroxidase-like activity ofmulti-component photoactive NCs.

Acknowledgements

The authors acknowledge DST, India for financial support.B. P. acknowledges CSIR, India for research fellowship. G.S.K.acknowledges DST INSPIRE fellowship.

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