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Colloids and Surfaces B: Biointerfaces 75 (2010) 175–178

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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lue orange light emission from biogenic synthesized silveranoparticles using Trichoderma viride

ohammed Fayaza,∗, C.S. Tiwaryb, P.T. Kalaichelvana, R. Venkatesanc

CAS in Botany, University of Madras, Guindy Campus, Chennai 600 025, IndiaDepartment of Physics, National Institute of Technology, Durgapur, West Bengal, IndiaNational Institute of Ocean Technology, Ministry of Earth Sciences, Chennai, India

r t i c l e i n f o

rticle history:eceived 7 November 2008eceived in revised form 28 July 2009ccepted 17 August 2009vailable online 25 August 2009

a b s t r a c t

Recent advances in nanomaterial have produced a new class of fluorescence labels by conjugating noblemetal with biomolecules. The nanometer size metal conjugates are water soluble, biocompatible andprovide important advantage over the florescence dyes. In this regard we synthesized silver nanoparti-cles at the size of 2–4 nm using biological route and studied fluorescence property of these nanoparticles.

+

eywords:iogenicTIRrichoderma virideilver nanoparticleshotoluminescence

We observe that these silver (Ag ) ions when exposed to filtrate of Trichoderma viride are reduced in solu-tion, thereby leading to the formation of an extremely stable silver hydrosol. These silver nanoparticleswere characterized by means of UV–vis spectrophotometer, FTIR, HrTEM, EDX, XRD and fluorescencespectroscopy. The nanoparticles exhibit maximum absorbance at 405 nm in UV–vis spectrum. The pres-ence of proteins was identified by FTIR. The HrTEM micrograph revealed the formation of monodispersedspherical nanoparticles and the presence of elemental silver was confirmed by EDX analysis and XRD.These monodispersed silver nanoparticles showed emission in the range of 320–520 nm wavelength.

. Introduction

In recent years the nanoparticles of II–VI group are very muchmportant due to its emission properties. These nanoparticles (CdS,nS, CdSe, etc.) are used for wide range of application in the fieldf cathode ray tube, flat-panel display, sensor, laser devices, etc.1–5]. But for biological application these particles are not veryuccessfully applicable. On the other hand, the intense light emis-ion properties of noble metals (gold, silver, etc.) nanoparticles haveaught a lot of attention. These nanoparticles are extensively usedor biological labeling [6], easy to prepare and have a good chemicalnd thermal stability [7].

Silver (Ag) nanoparticles (noble metal) have potential applica-ion in electronics, optoelectronics [8], in heterogeneous catalysis9], and surfaces of heat exchange, gas sensors and as conduc-ive inks [10]. But a limited amount of attention has been giveno the luminescence from Ag nanoparticles due to its very lowfficiency. The absence of band gap makes luminescence exceed-

ngly improbable for these nanoparticles [11]. It was reportedhat Ag nanoparticles emits light in rare gas matrix at cryo-enic temperature under photo activation or electro activation andhis photoluminescence was attributed to sp to sp like transition

∗ Corresponding author. Tel.: +91 09884466583; fax: +91 44 2352494.E-mail address: nanofayaz@gmail.com (M. Fayaz).

927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.08.028

© 2009 Elsevier B.V. All rights reserved.

analogy to inter-band transition in bulk silver [12]. The opticalproperties got enhanced due to lanthanide addition [13]. But forbiological application apart from luminescence synthesis method isalso equally important. There are many methods for synthesis of Agnanoparticles are reported, including both chemical and biologicalmethods. In recent times, scientist has endeavored microorgan-isms as possible eco-friendly nano-factories, for synthesis of silvernanoparticles. The bacterium Pseudomonas stutzeri AG259, isolatedfrom silver mine, when placed in a concentrated aqueous solu-tion of AgNO3, played a major role in the reduction of the Ag+ ionsand it makes silver nanoparticles of well-defined size and distincttopography within the periplasmic space of the bacteria [14].

While intracellular synthesis in principle may accomplish abetter control over the size and shape distributions of the nanopar-ticles, product harvesting, and recovery are more cumbersomeand expensive. The extracellular synthesis by comparison is moreadaptable to the synthesis of a wider range of nanoparticles systems[15]. However, optical properties of the biologically synthesizedsilver nanoparticles have been rarely reported.

In our current investigation, non-pathogenic fast growing fun-gus Trichoderma viride, which habited in dead organic materials,

was used for extracellular biosynthesis of silver nanoparticles.The biologically synthesized silver nanoparticles were charac-terized using UV–vis spectrophotometer (Cary 300 Conc UV–visspectrophotometer) and fluorescence spectroscopy (PerkinElmer).The size and morphology were characterized using HrTEM (high

176 M. Fayaz et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 175–178

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solution.XRD pattern taken using Cu K� target in the range 20–90 of

the Ag nanoparticles is shown in Fig. 3. The peaks matches withJCPDF Card No-087-0720. There is no new peak because proteinis found in this range. The peak listing is shown in Table 1. The

Table 1d-Spacing standard and calculated from XRD with the plane.

dJCPDF dcalculated (h k l)

Fig. 1. UV–vis spectrum for silver nanoparticles with different time interval.

esolution transmission electron microscope, Technai 300) withDS (energy dispersed spectroscopy), XRD (X-ray diffraction,an Analytical), and Fourier transformation infrared spectroscopyPerkinElmer). The PL emission is found to be 320–520 nm withlue color emission along with very narrow range of particle dis-ribution which is stabilized by protein molecules.

. Experimental details

The fungus T. viride was obtained from Culture Collection Center,AS in Botany, University of Madras, India and maintained in Potatoextrose Agar slant at 27 ◦C.

To prepare the biomass for biosynthesis studies the fungi wererown aerobically in liquid broth containing (g/L) KH2PO4 – 7;2HPO4 – 2; MgSO4·7H2O – 0.1; (NH2)SO4 – 1; yeast extract – 0.6;lucose – 10. The culture flask was incubated in an orbital shakert 27 ◦C and agitated at 150 rpm and the biomass was harvested,fter 72 h of growth by sieving through plastic sieve followed byxtensive washing with sterile double distilled water to removeny medium components from the biomass.

Typically 20 g (wet weight) brought in contact with 100 mL ster-le double distilled water for 48 h at 27 ◦C in an Erlen Meyer flasknd agitated as described earlier, after incubation the cell filtrateas obtained by passing it through Whatman filter paper No. 1.

n 100 mL cell filtrate, carefully weighed quantity of silver nitrateas added to the Erlen Meyer flask to yield an over all Ag+ ions

oncentration of 10−3 M in cell filtrate solution and the reaction isarried out in dark condition at 40 ◦C. The synthesized nanoparticlesere dissolved in water and used for measuring optical proper-

ies. A thin film is coated on glass plate for measuring the XRD.he water dispersed nanoparticles are seen in HrTEM using carbonoated grid.

. Results and discussion

The optical properties of silver nanoparticles are related to thexcitation of plasma resonance or inter-band transition, particu-arly on the size effect. The UV–vis absorbance spectrum of colloidalilver can be calculated from the wavelength dependence of opticalonstant of the particle relative to the surrounding medium usingMIE’ theory. According to ‘MIE’ theory silver nanoparticles less thaniameter 52 nm (electron mean free path of silver) result in a broad-

ning of the plasma absorbance bands, meanwhile the height of thebsorbance peak also decreases [16].

Fig. 1 shows the UV–vis spectrum obtained from biologicallyynthesized silver nano solution. It is observed from the spectrahat the silver surface plasmon band occurs at 405 nm in addition

Fig. 2. FTIR spectrum of silver nanoparticles.

to prominent band at around 260 nm. These peaks are charac-teristic plasmon band for silver nanoparticles [17]. At the initialstage of the reaction, a characteristic absorbance band centered atapproximately 405 nm, as reduction proceeds the intensity of theband significantly increased and the full-width at half-maximum(FWHM) of the peak position changed slightly. As there is anincrease in the intensity of the absorbance, the concentration ofsilver particle increases with reaction time. Furthermore there isno significantly different wavelength shift in the absorption spec-tra at different reaction time interval. The absorbance band at lowerwavelength with a good symmetry indicate that the mean diameterof silver nanoparticles is very small with a uniform size distribution.In addition to a prominent band at above 260 nm, it is attributedto electronic excitation in tryptophan and tyrosine residues in pro-tein which indicates the presence of protein molecule in colloidalsolution. It is interesting to note from our study that NADPH depen-dent reductase enzymes, not only specify to Fusarium oxysporum assuggested by other [18], but also involves in the reduction of Ag+

to Ag0. In case of fungus T. viride under experimental condition thepossible mechanism suggests that the reduction of Ag+ to Ag0 ismainly due to conjugate between the electron shutter with NADPHdependent reductase participation [18].

FTIR spectrum of silver nanoparticles is shown in Fig. 2.This spectrum shows the presence of bands at 1650, 1540,1423 and 1060 cm−1. The bands at 1650 correspond to pri-mary amine NH band, similarly, 1540 and 1060 corresponds tosecondary amine NH band and primary amine CN stretch vibra-tions of the proteins, respectively [19]. The positions of thesebands were close to that reported for native proteins [20]. TheFTIR result indicates that the secondary structures of proteinswere not affected as a consequence of reaction with Ag+ ionsor binding with silver nanoparticles. The band at 1425 cm−1 isassigned to methylene scissoring vibration from the protein in the

2.354 2.321 (1 1 1)2.0386 2.032 (2 0 0)1.4415 1.443 (2 2 0)1.2293 1.231 (3 1 1)1.177 1.181 (2 2 2)

M. Fayaz et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 175–178 177

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Fig. 4. (a) HrTEM microgram showing the size and shape of biologically syn-

Fig. 3. XRD pattern of silver nanoparticles with lattice planes.

btained pattern is for fcc cubic crystal structure. The peak planeatches with the card. The crystalline size is calculated from the

ull-width at half-maximum (FWHM) of the diffraction peaks usinghe Debye–Sherrer formula

= 0.89 �

ˇ cos �(1)

here D is the mean grain size, � is the X-ray wavelength for Cuarget, ˇ is the FWHM of diffraction peak and � is the diffractionngle. In order to measure the size of nanoparticles accuratelyach peak is Gaussian fitted and also the instrumental broaden-ng is subtracted using Si standard sample broadening. The sizef nanoparticles from all the peaks is in the range of 2–4 nm. Tobserve the effect of protein molecule on crystal structure theattice parameter is calculated using d spacing of each peak. Thealculated lattice parameter is found to be 4.084 Å. It confirms thatiologically synthesized silver nanoparticles lattice is unaffected byecreted protein molecules by T. viride.

The HrTEM technique was used to visualize the size and shapef the silver nanoparticles formed. Fig. 4a shows the typical brighteld HrTEM micrograph of the synthesized silver nanoparticles.he morphology of nanoparticles is spherical which is observed inicrograph. The HrTEM micrograph suggests that the size of par-

icles is around 2–4 nm. In EDX analysis, Fig. 4b inset shows theeak in silver region confirming the presence of elemental silver.he peak is observed approximately at 3 KeV, which is typical forhe absorption of metallic silver nanocrystalline [21] due to sur-ace plasmon resonance. From this we confirmed the presence ofanocrystalline elemental silver.

The photoluminescence (PL) of silver metal and that of nobleetal is generally attributed to electronic transition between the

pper d band and conduction sp band [22]. Luminescence of silveran be induced by irradiating the metal surface or film with electron23], photon [24] or laser beam [25], with reported emission peakositions distributed over a wide range of 320–520 nm. However,ost of the reports are silver metal, little has been reported about

uminescence from silver nanoparticles.The PL shown in Fig. 5 shows emission in a range of 320–520 nm.

he three peaks at 364 nm, 410 nm and 460 nm are observed in the

L emission. The peak at 410 nm is so weak that can be neglectedn whole photoluminescence spectra; meanwhile another twotrong bands staying apart at both side of 410 nm were observed.he strong emission band that approximately occurred at 364 nmhould be assigned to Ag–Ag interaction. Xu et al. have shown vis-

thesized nanoparticles; (b) EDS spectrum of silver nanoparticles; different X-rayemission peaks showing strong signals from the atoms in the silver nanoparticlesare observed, whereas signals from Cu atoms are also visible.

ible luminescence at 448 nm on excitation of 309 nm, which is dueto metal–ligand charge transfer absorption [26].

Theoretical work demonstrates that photoluminescence ofsilver metals can be viewed as excitation of electrons from occu-pied d bands into states above the Fermi Level. Subsequentlyelectron–phonon and hole–phonon scattering process leads toenergy loss and finally photoluminescence recombination of anelectron from an occupied Sp band with the hole [22,26]. This mech-anism of the emission is shown in Fig. 5, where the band structurefor a typical noble metal is represented by a sample model whichincludes on s–p conduction bond and two sets of d band in K-space.Excitation occurs from states in upper d bands to level and above theFermi energy. Because of the small photon momentum the inter-

band transition are assumed to be direct. The emission arises fromdirect recombination of conduction band electron below the Fermienergy with holes in the d band that have scattered to momentumstate less than the Fermi momentum KF [27].

178 M. Fayaz et al. / Colloids and Surfaces B:

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ig. 5. PL emission of silver nanoparticles with 250 and 270 excitation inset showshe important peak and change in color due to reaction.

. Conclusion

Silver nanoparticles were synthesized by a novel route using. viride. The UV–vis studies showed Plasma Resonance behaviors.he size of monodisperse silver nanoparticles is 2–4 nm which isonfirmed by HrTEM and XRD. The FTIR studies show the presence

f protein molecules. The PL most interestingly shows an emissionn the range of 320–520 nm, which fall in blue orange region. Theiocompatible silver nanoparticles that are synthesized by the bio-

ogical method in future can be used for biosensor and bioimagingpplication.

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Biointerfaces 75 (2010) 175–178

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