Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Extracellular biosynthesis of CdTe quantum dots by the fungusFusarium oxysporum and their anti-bacterial activity

Asad Syed, Absar Ahmad ⇑Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India

h i g h l i g h t s

" First report on biosynthesis of CdTenanoparticles using fungus Fusariumoxysporum.

" Nanoparticles are water dispersible,fluorescent, highly stable andprotein capped.

" CdTe nanoparticles showedantibacterial activity.

1386-1425/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.saa.2013.01.002

⇑ Corresponding author. Tel.: +91 020 25902226; faE-mail address: a.ahmad@ncl.res.in (A. Ahmad).

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 August 2012Received in revised form 2 January 2013Accepted 6 January 2013Available online 12 January 2013

Keywords:BiosynthesisNanoparticlesFungusCdTeAntibacterial activity

a b s t r a c t

The growing demand for semiconductor [quantum dots (Q-dots)] nanoparticles has fuelled significantresearch in developing strategies for their synthesis and characterization. They are extensively investi-gated by the chemical route; on the other hand, use of microbial sources for biosynthesis witnessedthe highly stable, water dispersible nanoparticles formation. Here we report, for the first time, an efficientfungal-mediated synthesis of highly fluorescent CdTe quantum dots at ambient conditions by the fungusFusarium oxysporum when reacted with a mixture of CdCl2 and TeCl4. Characterization of these biosynthe-sized nanoparticles was carried out by different techniques such as Ultraviolet–visible (UV–Vis) spectros-copy, Photoluminescence (PL), X-ray Diffraction (XRD), X-ray Photoelectron spectroscopy (XPS),Transmission Electron Microscopy (TEM) and Fourier Transformed Infrared Spectroscopy (FTIR) analysis.CdTe nanoparticles shows antibacterial activity against Gram positive and Gram negative bacteria. Thefungal based fabrication provides an economical, green chemistry approach for production of highly fluo-rescent CdTe quantum dots.

� 2013 Elsevier B.V. All rights reserved.

Introduction

Semiconductor nanocrystals, also known as quantum dots(QDs), are nanosized particles composed of groups II–VI or III–Vmain group elements. Q-dots have gained a lot of attention fortheir unique electronic and optical properties resulting due toquantum confinement effects [1,2]. Over the past decades, efforts

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have been made to study different functionalities such as size,shape and chemical compositions [3,4]. The semiconductors havebeen applied to various technological areas which include biologi-cal labels [5], optoelectronics and solar cells [6]. Optical propertiescan be tuned by simply changing the size of nanoparticles and thisalso possesses potential application in cell labeling [7], cell track-ing [8], in vivo imaging [9] diagnostics and DNA detection [10].

Cadmium telluride (CdTe), an important group II–VI semicon-ductor material with large exciton Bohr radius (7.3 nm) and nar-row bulk band gap of 1.5 eV has shown significant potential forLED (energy), FRET (electronics), and biomedical applications [11]

42 A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47

due to their size dependent properties. These nanoparticlesprovide excellent photostability, narrow emission and high quan-tum yield in comparison with organic dyes and therefore exploredin live cell bio-imaging [12]. Organometallic method [3,13] andaqueous (water based) method [14,15] with thiols as cappingagent are the two most useful protocols developed for the produc-tion of CdTe quantum dot nanoparticles. Moreover, these processesrequire high temperature and employ highly toxic chemicals suchas tri-octylphosphane or tri-octylphosphane oxide. To address thisconcern, surface of the quantum dots need to be functionalizedwith proteins or biocompatible layers in order to decrease toxicityand then can be utilized in imaging and labeling [16,17] Therefore,a novel, rational, cost effective and reproducible process is neededfor the scalable synthesis of CdTe nanoparticles.

Biological routes have been employed to synthesize quantumdots with controlled sizes, shapes and other functionalities [17].Many organisms are reported for the synthesis of semiconductornanoparticles, such as ZnS nanoparticles by sulfate-reducing bacte-rium Rhodobacter sphaeroides [18], Fusarium oxysporum [19],Schizosaccharomyces pombe [20], Rhodopseudomonas palustris [21]and Escherichia coli [22] have been developed for the synthesis ofCdS nanoparticles. PbS nanoparticles have been synthesized byTorulopsis sp. [23]. Holmes and co-workers have demonstrated thatthe exposure of Cd2+ ions to the bacterium Klebsiella aerogenes re-sults in the intracellular formation of CdS nanoparticles in the sizerange of 20–200 nm [24,25]. Dameron and co-workers reportedthat the yeasts such as S. pombe and Candida glabrata produceintracellular CdS nanoparticles when challenged with aqueoussolution of cadmium salt [26]. These biosynthesis processes occurat ambient conditions and are considered as environment friendlymethods. Recently, CdSe nanoparticles have been synthesizedintracellularly by using yeast. For the isolation of intracellularnanoparticles, complicated procedures are required which includecell washing, cell disruption and removal of cell fragments for get-ting CdSe nanoparticles [27]. In order to avoid the above complica-tions, scientists started the screening of extracellular inorganicnanomaterials using biological methods. Extracellular CdSe nano-particles synthesis has been reported by the fungus F. oxysporum[28]. Very recently, lead sulfide nanoparticles were biosynthesizedby lead resistant marine yeast Rhodosporidium diobovatum [29].

Present work emphasizes on the use of fungus F. oxysporum tosynthesize highly fluorescent extracellular CdTe (quantum dot)nanoparticles. The process utilizes Cd and Te precursors in a verydilute form and allows bottom-up, one-step preparation of nano-particles. Different techniques were employed for their character-ization such as SAED and XRD which confirmed the crystallinenature of biosynthesized nanoparticles. These biosynthesizednanoparticles are capped by proteins secreted by the fungus inthe reaction mixture, which makes them water dispersible andprovides stability in solution by preventing their agglomeration.These nanoparticles also showed antibacterial activity againstGram positive and Gram negative bacteria. Fungus based approachprovides a novel, rational and environment friendly synthesis pro-tocol for nanomaterials synthesis. To the best of our knowledge,this is the first demonstration of a fungal-mediated approach forthe synthesis of CdTe QDs.

Experimental detail

Materials

Cadmium chloride (CdCl2) and Tellurium tetrachloride (TeCl4)were obtained from Sigma Aldrich. Malt extract, Yeast extract,Glucose and Peptone were obtained from HiMedia and usedas-received.

Fungal growth and maintenance

The fungus F. oxysporum was maintained on Potato DextroseAgar [PDA (potato 25% w/v, dextrose 2% w/v, and agar agar 2%w/v)] slants at 25 �C. Stock cultures were maintained by subcultur-ing at monthly intervals. The fungus was grown at pH 7.0 and 25 �Cfor 5 days, the slants were preserved at 15 �C. From an activelygrowing stock culture, subcultures were made on fresh slantsand after 5 days of incubation at pH 7.0 and 25 �C, were used asthe starting material for synthesis of CdTe nanoparticles.

Extracellular biosynthesis of Cadmium telluride (CdTe) nanoparticles

For biosynthesis of nanoparticles, the fungus was grown in500 mL Erlenmeyer flasks each containing MGYP [malt extract(0.3%), glucose (1%), yeast extract (0.3%), and peptone (0.5%)] med-ia (100 mL) at 25–27 �C under shaking condition at 200 rpm for96 h. After 96 h of fermentation, mycelia were separated fromthe culture broth by centrifugation (5000 rpm) at 10 �C for20 min and then the mycelia were washed thrice with sterile dis-tilled water under sterile conditions. The harvested mycelial mass(20 g of wet mycelia) was then resuspended in 100 mL of aqueoussolution of 1 mM CdCl2 and 1 mM TeCl4 solutions in 500 mL Erlen-meyer flasks and the same was put onto a shaker at 25–27 �C(200 rpm). The reaction was carried out for a period of 96 h andfungal biomass was separated by filter paper to collect biomassand filtrate in sterile conditions. Periodically, aliquots of the reac-tion solution were removed and subjected to UV–Vis spectroscopyand fluorescence spectroscopy to check the formation of nanopar-ticles extracellularly.

Characterization of biosynthesized CdTe nanoparticles

Various aliquots of the biosynthesized nanoparticles solutionwere collected during the course of reaction, by separating thefungal mycelia from the aqueous component by simple filtration.UV–Vis spectroscopy measurements performed on a Shimadzudual-beam spectrophotometer (model UV-1601 PC) operated at aresolution of 1 nm. Fluorescence measurements were carried outusing Perkin–Elmer LS 50B luminescence spectrophotometer. Thediluted biosynthesized nanoparticles solution was drop cast on acarbon coated copper grid and analyzed using Transmission Elec-tron Microscope-FEI Technai G2 system operated at an acceleratingvoltage of 80 kV at room temperature. The Selected Area ElectronDiffraction (SAED) analysis was carried on the same grid. Thin filmsof the nanoparticles were drop casted on glass substrates and thensubjected to X-ray diffraction analysis and data was recorded onPanalytical ‘X’ Pert PRO system, while Energy Dispersive Analysisof X-ray (EDAX) was carried out on a Leica Stereoscan-440 SEMequipped with a Phoenix EDX attachment. EDX spectrum wasrecorded in the spot-profile mode by focusing the electron beamonto a region on the surface coated with nanoparticles. X-ray pho-toelectron spectrum was recorded on VG MicroTech ESCA 3000instrument. FTIR spectrum of biosynthesized nanoparticles solutionwas recorded on a Perkin Elmer Spectrum One B in diffuse reflec-tance (DRS) mode at a resolution of 2 cm�1. Thermogravimetricanalysis of nanoparticles was carried-out using the Q5000 V2.4Build 223 instrument by TA Instruments at the scan rate10 �C min�1 in N2 environment.

Antibacterial activity

The bacterial cultures Staphylococcus aureus NCIM 2079, Bacillussubtilis NCIM 2063, E. coli NCIM 2065 and Pseudomonas aeruginosaNCIM 2200 used for the antibacterial activity were from our in-house culture collection unit, the National Collection of Industrial

Fig. 1. (a) UV–Vis spectrum of extracellular CdTe nanoparticles, the inset shows glass vial containing CdCl2 and TeCl4 solution before (glass vial on the left side) and afterreaction with the fungal biomass (glass vial on the right side), (b) taucs plot for the CdTe quantum dot nanoparticles, (c) fluorescence emission spectrum of extracellular CdTenanoparticles. The inset shows glass vial containing CdCl2 and TeCl4 solution before (glass vial on the left) and after reaction with the fungal biomass (glass vial on the right),(d) fluorescence spectrum in relation with energy shows peak centered at 2.61 eV.

1 For interpretation of color in Fig. 1, the reader is referred to the web version ofthis article.

A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47 43

Microorganisms (NCIM), Pune, India. Evaluation of antibacterialactivity of CdTe nanoparticles was carried out by filter paper bioas-say [30]. For antibacterial activity, bacterial culture were inocu-lated in nutrient broth and incubated at 37 �C for 24 h. From theactively growing bacterial culture broth, 100 lL (0.1 mL) of bacte-rial suspension (with a concentration of 105 CFU/mL) was mixedwith half strength nutrient broth (0.9 mL) and was immediatelyoverlaid on the surface of the sterile nutrient agar plates (90 mmdiameter) and incubated at 37 �C for some time for initial growth.Sterile filter paper discs (Whatman No. 3: 10 mm square) wereplaced on agar plates and then loaded with 50 lL suspension ofnanoparticles of different concentrations (100 lg, 500 lg and1000 lg). These plates were incubated for 24 h. and visually mon-itored for the zone of inhibition. Filter paper disc on nutrient agarplate without nanoparticles suspension was used as a control. Afterincubation, the zone of inhibition was measured in millimeteracross the filter paper.

Results and discussions

The fungus, F. oxysporum when reacted with aqueous solution ofCdCl2 and TeCl4 at room temperature for 96 h under shaking condi-tion on a rotary shaker (200 rpm) resulted in the formation ofhighly stable, water dispersible and fluorescent CdTe nanoparticles.

The UV–Vis spectrum recorded for the biosynthesized CdTenanoparticles solution, after the reaction with precursor solutionis shown in Fig. 1a, this showed notable feature around400–450 nm [31]. The Tauc plot (Fig. 1b) obtained from this datashows that the band gap is 2.61 eV. The inset of Fig. 1a shows glass

vials of the CdCl2 and TeCl4 reaction solution at the beginning (timet = 0, glass vial on the left side)1 and after 96 h of reaction (glassvial on the right side) with the fungus F. oxysporum. The changein the color of the solution from colorless to dark yellow after reac-tion is clearly indicates the formation of CdTe nanoparticles.Absorption between 270 and 280 nm suggests the presence of pro-teins in the reaction mixture (Fig. 1a). These secreted proteinscapped the nanoparticles surface and made them water dispersibleand stable.

Fluorescence measurement was studied by exciting the reactionmixture at 400 nm. As shown in Fig. 1c, the biosynthesized QDspossess fluorescence emission band centered at 475 nm, which isin good agreement with nanoparticles synthesized by chemicalmethods. The inset of Fig. 1c shows glass vials containing CdCl2

and TeCl4 reaction solution at the beginning (time t = 0, glass vialon the left side) and after 96 h of reaction (glass vial on the rightside). When the solution of nanoparticles was illuminated with a365 nm lamp, an intense green luminescence was seen (the insetof Fig. 1c, glass vial on the right side). Green luminescence is signif-icant for several applications and a subject of intense scientific re-search. The fluorescence studies in relation with energy (Fig. 1d)shows peak centered at 2.61 eV (band gap of nanoparticles).

The TEM analysis of the nanoparticles showed that they are inthe size range of 15–20 nm. The morphology of the nanoparticleswas essentially spherical (Fig. 2a). Fig. 2b shows the Selected Area

Fig. 2. (a) TEM micrograph and (b) SAED pattern recorded from extracellular CdTe nanoparticles shown in (a).

Fig. 3. (a) XRD pattern and (b) EDAX spectrum for extracellular CdTe nanoparticles.

44 A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47

Electron Diffraction (SAED) pattern obtained from nanoparticlesshown in Fig. 2a. The Scherrer ring pattern characteristic of facecentered cubic (fcc), CdTe is clearly observed, showing that thestructures seen in TEM images are nanocrystalline in nature.

X-ray diffraction (XRD) analysis of these biosynthesized CdTenanoparticles was carried out by depositing them as a thin filmon a glass substrate (Fig. 3a) which showed intense peaks corre-sponding to (111), (220) and (311) planes in the 2h range of15h–60h and agree with the standard diffraction pattern [32]. Thebroadening of Bragg’s peaks indicates that the particles formed arein nanoscale dimensions. EDAX spectrum in Fig. 3b shows the spotprofile mode from one of the densely populated regions on the sur-face. Signals are observed from C, O, Cd and Te. The C and O signalsare likely to be due to X-ray emission from proteins/enzymes pres-ent onto the nanoparticles surface.

The presence of CdTe nanoparticles was also confirmed by ana-lyzing the sample by XPS as shown in Fig. 4. The results showed thepresence of Cd, Te, C and O as the prominent elements. In Fig. 4,background corrected XPS was presented, peaks correspond tochemically distinct C 1s core levels (Fig. 4a) originating from thea-carbon and –COOH groups present in the proteins with bindingenergies 285 and 286.5 eV respectively. The peak (in Fig. 4b) corre-sponds to chemically distinct O 1s core level transition with bindingenergy 532 eV of proteins. The Cd 3d spectrum (Fig. 4c) could bedecomposed into two spin–orbit components 1 and 2 (spin–orbitsplitting � 6.6 eV). The Cd 3d5/2 and 3d3/2 peaks occurred at bindingenergies of 406.6 eV and 413.2 eV respectively, which agrees withthe core level binding energies and are characteristic of metallic

Cd. Moreover, the binding energies of 573.19 eV and 585.2 eV forTe 3d peak are comparable to the core level binding energies andare typical for metallic Te (Fig. 4d). The XPS data clearly indicatesthat all the CdTe metal ions are fully reduced by the fungus F. oxy-sporum and are in metallic form. These findings are comparable andin good agreement with the reported literature [33].

FTIR analysis was performed to determine the nature of thepresent protein on the surface of nanoparticles. The presence oftwo bands in the region 1644 and 1530 cm�1 assigned to amidebands I and II respectively of proteins (Fig. 5a). Similar results werereported in our previous reports on the synthesis of CdS nanopar-ticles by F. oxysporum [19]. The data provide some insights on thenature of the capping layer of nanoparticles surface, which may befurther utilized for biofunctionalization for different applications.

As mentioned earlier, biosynthesized nanoparticles are naturallycapped with secreted proteins into the reaction mixture during syn-thesis process. Thermogravimetric analysis (TGA) was performed tofurther probe the thermal property (desorption/decomposition) ofthe synthesized nanoparticles. TGA spectrum was plotted in termsof weight loss against temperature and presented in Fig. 5b, thespectrum shows weight loss (�30%) in the temperature range of200–250 �C. The weight loss for these nanoparticles attributed torelease of water vapor and biomolecules which are bound/cappedon the surface of nanoparticles. These nanoparticles show furtherdegradation (weight loss accounts for decomposition) uponincrease in temperature, but this step stabilizes over a temperaturerange of 500–700 �C when approximately �30% mass of the nano-particles remains as it is.

Fig. 4. XPS analysis of extracellular CdTe nanoparticles.

Fig. 5. (a) FTIR analysis and (b) TGA analysis of extracellular CdTe nanoparticles.

A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47 45

A range of antibacterial compounds of different chemical clas-ses such as alkaloids, peptides, terpenoids, steroids, flavonoids,quinones, lignans, phenols and lactones, and several other second-ary metabolites have been isolated from plants and their associ-ated microorganisms. As cases of drug resistance in bacteria arealarmingly on a rise, new and improved therapeutic agents andderivatives are required for effective treatments of diseases inhumans, plants and animals. In order to provide a new class ofantibacterial compounds to overcome resistance in bacteria, we

evaluated the antibacterial activity of biologically synthesized,water dispersible, protein capped, highly stable CdTe nanoparticlesagainst few Gram positive and Gram negative bacteria, using filterpaper bioassay method. Filter paper bioassay for anti-bacterialactivity revealed a zone of inhibition developed around the filterpaper disc against the bacterial growth of both Gram-negative (E.coli and P. aeruginosa) and Gram-positive (S. aureus and B. subtilis)bacteria, thus exhibiting the great potential of these CdTe nanopar-ticles to be developed as a broad-spectrum anti-bacterial agent.

Fig. 6. Antibacterial activity of CdTe nanoparticles against Gram positive bacteria (a) Staphylococcus aureus NCIM 2079, (b): Bacillus subtilis NCIM 2063 and Gram negativebacteria, (c) Escherichia coli NCIM 2065 and (d) Pseudomonas aeruginosa NCIM 2200) C: Control, Different concentration (1:100 lg, 2:500 lg and 3:1000 lg).

Table 1Antibacterial activity in terms of zone of inhibition of CdTe nanoparticles againstbacterial cultures was presented.

Organism Concentrations/zone ofinhibition

100 lg 500 lg 1000 lg

Gram positive bacteriaStaphylococcus aureus (NCIM 2079) (mm) 17 30 35Bacillus subtilis (NCIM 2063) (mm) – 22 26

Gram negative bacteriaEscherichia coli (NCIM 2065) (mm) – 29 33Pseudomonas aeruginosa (NCIM 2200) (mm) – 27 30

46 A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47

This is the first report of antibacterial activity exhibited by biolog-ically synthesized CdTe nanoparticles isolated by us from the fun-gus F. oxysporum. These CdTe nanoparticles, apart from havingantibacterial activity, may also be used in paints and other biomed-ical applications such as cell tracking, in vivo imaging, diagnostics,optoelectronics and in solar cells. Our observations are supportedby the fact that CdTe nanoparticles synthesized by various chemi-cal methods also exhibited antibacterial activity against a range ofGram positive and Gram negative bacteria. CdTe nanoparticlesshowed growth inhibition against these bacteria (Fig. 6). The

growth inhibition in terms of zone of inhibition was estimatedand presented in Table 1.

Conclusions

In conclusion, the present investigation for the first time demon-strates a novel, rational, eco-friendly, efficient and most impor-tantly an extracellular biosynthetic process for the synthesis ofCdTe quantum dot nanoparticles. As these nanoparticles are cappedby natural proteins secreted by the fungus, they do not require theaddition of any external stabilizing agents which are usually toxic.These nanoparticles were characterized by different techniquessuch as UV–Visible spectroscopy, fluorescence measurements,XRD, XPS and TEM. Also, these nanoparticles show antibacterialactivity against Gram positive and Gram negative bacteria.

Acknowledgements

A.S. thanks the Council of Scientific and Industrial Research(CSIR), New Delhi for Senior Research Fellowship. A.A. thanks theDepartment of Biotechnology, Govt. of India (New Delhi) for theTata Innovation Fellowship award and financial support throughNWP0035 CSIR, New Delhi. The authors thank the Centre for Mate-rials Characterization (CMC), Pune for assistance regarding TEMmeasurements.

A. Syed, A. Ahmad / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106 (2013) 41–47 47

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