Thin Solid Films 518 (2009) 614–620

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Metal oxide–chitosan based nanocomposite for cholesterol biosensor

Bansi D. Malhotra ⁎, Ajeet KaushikDepartment of Science and Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India

⁎ Corresponding author. Tel.: +91 11 45609152; fax:E-mail address: bansi.malhotra@gmail.com (B.D. Ma

0040-6090/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.tsf.2009.07.036

a b s t r a c t

a r t i c l e i n f o

Available online 10 July 2009

Keywords:CeO2 nanoparticlesChitosanNanocompositeCholesterol biosensor

Metal oxide [cerium oxide (NanoCeO2)]–chitosan (CH)nanocompositefilmhas been fabricated onto indium-tin-oxide (ITO) coated glass plate to immobilize cholesterol oxidase (ChOx) via physiosorption for cholesteroldetection. Electrochemical studies reveal that the presence of NanoCeO2 in CH–CeO2 nanocomposite results inincreased electroactive surface area for ChOx loading resulting in enhanced electron transport between ChOx andelectrode. The ChOx/CH–NanoCeO2/ITO bioelectrode exhibits interesting characteristics such as detection rangeof 10–400 mg/dL, detection limit of 5 mg/dL, response time of 10 s, low Km value of 3.5 mg/dL and value ofregression coefficient of 0.994.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Estimation of cholesterol has recently aroused much interest sinceit is closely associated with the diagnosis of coronary heart diseases[1]. Due to simple design, specificity and high sensitivity, developmentof cholesterol biosensor has recently been considered very important.The immobilization of ChOx onto an electrode surface is one of themost critical steps to obtain desired biosensing characteristics. Thefuture progress for development in biosensor design will inevitablyfocus upon the technology of newmaterials that offer promise to solvethe biocompatibility and selectivity problems [1–3].

Metal oxide nanoparticles such as zinc oxide [3–5], cerium oxide(CeO2) [2,6–9], iron oxide [10–12], tin oxide [13], titanium oxide[14,15] and zirconium oxide [16] have been found to exhibit inte-resting properties such as large surface-to-volume ratio, high surfacereaction activity, high catalytic efficiency, and strong adsorptionability that make them potential candidate materials for the fab-rication of a biosensor. The large surface area of nanomaterialsis likely to provide a better matrix for the immobilization of desiredenzyme, leading to increased enzyme loading per unit mass ofparticles. Moreover, the multipoint attachment of enzyme mole-cules to nanomaterials surfaces reduces protein unfolding resultingin enhanced stability of enzyme attached to the nanoparticlessurface. The enzyme-attached nanoparticles facilitate enzymes toact as free enzymes in solution that in turn provide enhanced enzyme–substrate interaction by minimizing potential aggregation of the freeenzyme [17].

+91 11 45609310.lhotra).

ll rights reserved.

Among the metal oxide nanomaterials, CeO2 nanostructure isknown to have several advantages such as low temperature processing,tunability in physical parameters, optical transparency, chemicalinertness, thermal stability and negligible swelling in aqueous andnon-aqueous solutions for the immobilization of enzymes. Besides this,CeO2 nanoparticles have attracted much interest owing to their uniqueproperties including highmechanical strength, oxygen ion conductivity,high isoelectric point (IEP), biocompatibility and high adsorptioncapability and oxygen storage capacity for development of desiredbiosensors. The high isoelectric point (IEP~9.2) of CeO2 can behelpful toimmobilize a desired enzyme of low IEP via electrostatic interactions.Furthermore, non-toxicity, high chemical stability and high electrontransfer capability have recentlymadeCeO2 a promisingmaterial for theimmobilization of desired biomolecules for development of implantablebiosensors [2,6]. Choi et al. [7] have fabricated amperometric biosensoremploying CeO2 nanoparticles as insoluble oxidant to minimize theeffect of interferents [7].Mehta et al. [8] have reported novelmultivalentCeO2 based hydrogenperoxide biosensor for application as a 3-terminalamperometric sensor [8]. Anasri et al. have fabricated sol–gel derivednanostructure of CeO2 for application to glucose and cholesteroldetection [2,6]. However, there is a considerable scope to improve thebiosensing characteristics of CeO2 nanoparticles by dispersing in anelectroactive biopolymer e.g. chitosan (CH) to fabricate nanobiocompo-site. Feng et al. [9] have prepared a nanoporous CH–CeO2 compositematrix for the immobilization of single stranded DNA probe fordetection of cancer gene [9].

CH (a linear copolymer of glucosamine and N-acetylglucosamineunits) along with metal oxide nanoparticles has recently been utilizedas a stabilizingagentdue to its excellentfilm-formingability,mechanicalstrength, biocompatibility, non-toxicity, high permeability towardswater, susceptibility to chemical modification and cost-effectivenessetc. for enzyme (ChOx) immobilization. Moreover, amino and hydroxyl

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groups of CH provide a hydrophilic environment compatible with thebiomolecules [1,12].

In this manuscript, we report results of studies relating to theimmobilization of ChOx onto CH–NanoCeO2/ITO electrode for applica-tion to cholesterol detection.

2. Experimental details

2.1. Materials

Chitosan (Mw 2.4×106), cerium ammonium nitrate [(NH4)2Ce(NO3)6], cholesterol oxidase (EC 1.1.36 from Pseudomonas fluorescens)with specific activity of 24U/mg, cholesterol and Triton X-100 werepurchased from Sigma-Aldrich (USA).

2.2. Preparation of CeO2 nanoparticles

Firstly, 1 g of cerium ammonium nitrate [(NH4)2Ce(NO3)6] is dis-solved in distilled 20 mL deionised water. Then a 5 mL (1M) solution ofammoniumhydroxide (NH4OH) is added dropwise in this solutionwithconstant stirring for 4 h at 25 °C to maintain pH 10. A pale yellowprecipitate of Ce(OH)4 thus obtained is washed several times withdeionizedwater until a neutral pH is achieved. Thus obtained Ce(OH)4 isdried at 400 °C for 8 h to obtain nanoparticles of CeO2 [18].

2.3. Preparation of CH–NanoCeO2/ITO nanocomposite electrode

CH (0.50%) solution is preparedbydissolvingCH (50 mg) in 100 mLof acetate buffer (0.05 M, pH 4.2) solution. The calculated amount ofCeO2 nanoparticles is dispersed in the CH solution by stirring at roomtemperature (25 °C) afterwhich it is sonicated. Finally, a highly viscoussolution of CH with uniformly dispersed CeO2 nanoparticles isobtained. The film of nanocomposite is fabricated by dispersing 10 μLsolution of CH–NanoCeO2 nanocomposite onto an ITO (0.25 cm2)surface and allowing it to dry at room temperature for about 12 h incontrolled environment. This CH–NanoCeO2 nanocomposite film iswashed with deionized water to remove any unbound particles.

It has been found that optimized ratio of CH and CeO2 nanoparticlestaken as 10:2 to prepare CH–NanoCeO2 nanocomposite film exhibitsmaximum electrochemical current and that 10 µL of CH–CeO2 nano-composite solution is dispersed onto ITO surface.

2.4. Immobilization of ChOx onto CH–NanoCeO2/ITO electrode

The stock solution of ChOx (1 mg/dL) of 24 U/mL is freshlyprepared in phosphate buffer [PB (50 mM)] at pH 7. The stocksolution of cholesterol is prepared in 10% triton X-100 and is stored at4 °C. 10 µL solution of freshly prepared ChOx (1 mg/dL) is spread ontothe CH-NanoCeO2/ITO electrode. The ChOx/CH–NanoCeO2/ITO bioe-lectrode is kept undisturbed for about 12 h at 4 °C. Finally, the drybioelectrode is immersed in 50 mM PB (pH 7.0) in order to wash outany unbound ChOx from the electrode surface.

It is known that CH is a cationic amine-rich polysaccharide (pH 4.2).The surface charged CeO2 nanoparticles interact with –NH2/OH groupsof CH via electrostatic interactions and hydrogen bonding. Theseelectrostatic interactions between CeO2 nanoparticles and CH havebeen confirmed by FTIR spectra. However, ChOx is negatively chargedbiomolecule at pH 7.0 and it can be easily immobilized onto thepositively charged CH–NanoCeO2 nanocomposite matrix via electro-static interactions.

2.5. Characterization

The FTIR spectra have been recorded on Perkin Elmer, SpectrumBX II spectrophotometer. The morphological studies are performedwith scanning electron microscope (LEO-440) and phase identifica-

tion in the CeO2 nanoparticles is performed with an X-ray diffract-ometer (Rigaku, miniflux 2). Autolab Potentiostat/Galvanostat (EcoChemie, Netherlands) is used for electrochemical measurements aswell as electrochemical impedance spectroscopy measurements(0.01–105Hz). These measurements are carried out using a three-electrode cell with ChOx/CH–NanoCeO2/ITO bioelectrode as theworking electrode, platinum (Pt) wire as the counter electrode, andsaturated Ag/AgCl electrode as a reference electrode in phosphatebuffer saline [PBS (50 mM, pH 7.0, 0.9% NaCl)] containing Fe(CN)63−/4−

(5 mM).

3. Results and discussions

3.1. Optical properties

The FTIR spectra (Fig. 1A) of pure CH (curve a) exhibit bands at3200–3400 cm−1 due to the stretching vibration mode of OH andNH2 groups. The band at 1650 cm−1 is due to amide I group (C–Ostretching along with N–H deformation mode), 1570 cm−1 peak isattributed to NH2 group due to N–H deformation, 1425 cm−1 peak isdue to C–N axial deformation (amine group band), 1350 cm−1 peak isdue to COO− group in carboxylic acid salt, 1145 cm−1 is assigned tothe special broad peak of β (1–4) glucosidic band in polysaccharideunit, 1096 cm−1 is attributed to the stretching vibration mode of thehydroxyl group, 1028 cm−1 stretching vibration of C–O–C in glucosecircle and 1080–1020 cm−1 bands correspond to CH–OH in cycliccompounds.

The FTIR spectra of the CH–NanoCeO2 nanocomposite film (curve b)exhibits characteristic IR bands of the functional group corresponding topure CH [12] and the NanoCeO2 nanoparticles [2]. The IR bands of CHcorresponding to the –NH and OH stretching modes are shifted to thelower wave number in the IR spectra of the CH–CeO2 nanocompositefilm (curve b), corresponding to pure CH. This indicates that NH2/OHgroupof CH is involved in theassemblyof CeO2nanoparticles. In general,CeO2 nanoparticles show IR band at ~465 cm−1 assigned to Ce–Ostretching vibration mode indicating the formation of NanoCeO2 [2].However, IR band at 505 cm−1 in CH–NanoCeO2 nanocomposite isattributed to CeO2 nanoparticles. This absorption band is at higherwavenumber than that of pure CeO2 nanoparticle due to the interactionbetween CH and CeO2 in the nanocomposite. The presence of CH in CH–NanoCeO2 composite facilitates immobilization of biomolecules viaamine and hydroxyl group. However, the shapes of the absorptionpeaksbecome broader due to the overlap of functional groups of ChOxcorresponding to amide I (1650 cm−1) and amide II (1545 cm−1) withCH–NanoCeO2 nanocomposite film indicating the immobilization ofChOx on nanocomposite matrix.

SEM(Fig.1B) studies reveal the globular porousmorphologyof CH–NanoCeO2/ITO electrode indicating the formation of CH–NanoCeO2

hybrid nanobiocomposite. This may be attributed to electrostaticinteractions between cationic CH and surface charged CeO2 nanopar-ticles. However, surface morphology of CH–NanoCeO2/ITO electrodeschanges after the immobilization of ChOx (image b) revealingimmobilization of ChOx onto bionanocomposite film.

3.2. Electrochemical studies

The stepwise fabrication of biosensor has been characterizedusing electrochemical impedance spectroscopy (EIS) studies. Fig. 2Ashows EIS spectra of CH/ITO electrode (curve a), CH–NanoCeO2/ITOelectrode (curve b) and ChOx/CH–NanoCeO2/ITO bioelectrodes (curvec) investigated in PBS in the frequency range of 0.01–105Hz. In the EIS,the semicircle part corresponds to electron transfer limited processand its diameter is equal to the electron transfer resistance, RCT thatcontrols electron transfer kinetics of the redox probe at the electrodeinterface. It can be seen from the Nyquist plots (Fig. 2A) thatsemicircle of CH/ITO electrode (RCT=1.27 KΩ curve a), characteristic

Fig. 2. A) Electrochemical impedance spectroscopy (EIS) studies of CH/ITO electrode(curve a), CH–NanoCeO2/ITO electrode (curve b) and ChOx/CH–NanoCeO2/ITObioelectrodes (curve c). B) Differential pulse voltammetry (DPV) of the CH/ITOelectrode (curve a) CH–NanoCeO2/ITO electrode (curve b) and ChOx/CH–NanoCeO2/ITO bioelectrode (curve c).

Fig. 1. A) Fourier transform infrared (FTIR) spectra of CH/ITO electrode (curve a), CH–NanoCeO2/ITO nanocomposite electrode (curve b) and ChOx/CH–NanoCeO2/ITO bioelec-trode (curve c). B) Scanning electron microscopic (SEM) images of CH–NnaoCeO2/ITOelectrode (image a) and ChOx/CH–NanoCeO2/ITO bioelectrode (image b).

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of a diffusion limiting step of the electrochemical process, decreasesfor CH–NanoCeO2/ITO electrode (RCT=1.01 KΩ curve b). These resultssuggest that electron transfer in the CH–NanoCeO2 nanocompositefilm is easier between solution and the electrode i.e., CeO2

nanoparticles not only provide the hydrophilic surface, but alsopromote electron transfer due to permeable structure of CH/ITO.

Fig. 3. A) Cyclic voltammetry (CV) of the CH/ITO electrode (curve a) CH–NanoCeO2/ITOof ChOx/CH–NanoCeO2/ITO bioelectrode as a function of scan rate (10–100 mV/s). C) CV

However, on the immobilization of ChOx onto CH–NanoCeO2/ITO, thesemicircle part of the bioelectrode further decreases (RCT=0.61 KΩ).This suggests that CH–NanoCeO2 nanocomposite provides desiredmicroenvironment for ChOx resulting in enhanced electron transportbetween medium and electrode.

Fig. 2B shows differential pulse voltammogram (DPV) of the CH/ITO electrode (curve a) CH–NanoCeO2/ITO electrode (curve b) andChOx/CH–NanoCeO2/ITO bioelectrode (curve c) in PBS. CH/ITOelectrode shows DPV curve arising due to cationic characteristicsthat accept electrons from the medium and transfer these to theelectrode. The magnitude of the peak current increases for the CH–NanoCeO2/ITO electrode suggesting that CeO2 nanoparticles promoteelectron transfer due to uniform dispersion throughout the CHnetwork at the electrode. Themagnitude of response increases furtherfor the ChOx/CH–NanoCeO2/ITO bioelectrode. This suggests that thepresence of CeO2 nanoparticles results in increased electroactivesurface area of CH and the CH–NanoCeO2 nanocomposite andproviding favourable environment for the immobilization of ChOxresulting in enhanced electron transfer.

Fig. 3A shows cyclic voltammograms (CV) obtained for CH/ITOelectrode, CH–NanoCeO2/ITO electrode and ChOx/CH–NanoCeO2/ITO

electrode (curve b) and ChOx/CH–NanoCeO2/ITO bioelectrode (curve c). B) CV curvecurve of ChOx/CH–NanoCeO2/ITO bioelectrode as a function of pH (6.0–8.0).

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Fig. 4. A) Cyclic voltammograms of ChOx/CH–NanoCeO2/ITO bioelectrode as a function of cholesterol concentration (10–400 mg/dL). Inset: calibration curve betweenelectrochemical response current and ln[cholesterol concentration (mg/dL)]. B) Effect on of interferents on ChOx/CH–NanoCeO2/ITO bioelectrode during cholesterol estimation.

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bioelectrode recorded in PBS in the potential range of−0.3 to 0.6 V at10 mV/s rate. CH/ITO shows a well-defined redox behaviour (curve a)due to cationic CH that accepts electrons from ferricyanide species(that are negatively charged in PBS) resulting in enhanced redoxcurrent. The magnitude of the current response for CH–NanoCeO2/ITOelectrode (curve b) increases in comparison to that of CH/ITOelectrode. These results suggest that the presence of CeO2 nanopar-ticles results in increased electroactive surface area of CH andenhanced electron transfer. The surface concentrations of the CH/ITO and CH–NanoCeO2/ITO electrodes have been estimated usingEq. (1).

ip = 0:227nFAC04k0 exp

−αnaFRT

ðEp−E′0Þ� �

ð1Þ

where, ip is the anodic peak current, n is the number of electronstransferred (1), F is the Faraday constant (96,485.34°C mol−1), A issurface area (0.25 cm2), R is the gas constant (8.314 J mol−1 K−1), C0* isthe surface concentration of the ionic species of film surface (mol cm−3),Ep is the peak potential and E′0 is the formal potential. −αnaF/RT and k0

(rate constant) correspond to the slope and intercept of ln (ip) versusEp–E′0 curve obtained at different scan rates. Itmay benoted that surfaceconcentration of CH–NanoCeO2/ITO electrode (2.2×10−6mol cm−3) ishigher than that of CH/ITO (1.4×10−6mol cm−3). The higher surfaceconcentration of CH–NanoCeO2 electrode suggests that loading of CeO2

nanoparticles results in increased electroactive surface area that helps inhigh loading of ChOx. However, after the immobilization of ChOx ontoCH–NanoCeO2/ITO electrode, magnitude of the current response in-creases (curve c), revealing strong binding of ChOxwith CH–NanoCeO2/ITO electrode that enhances electron transport.

Table 1Characteristics of ChOx/CH–NanoCeO2/ITO bioelectrode along with those reported in literature.

Bioelectrode Detection range Detection limit Sensitivity Km value Response time Shelf-life Ref

ChOx/NanoZnO 25–400 mg/dL – 80 mg/dL 15 s 75 days [3]ChOx/NanoFe3O4 50–200 mg/dL – – 17 mg/dL 15 days [11]ChOx/Au nanowires 38–250 mg/dL – 2.63 μA/dL−1 cm−2 – 10 days [19]ChOx/Ni/K3Fe(CN)6/CNT 0.2–120 mg/dL – – – b20 s 30 days [20]ChOx/NanoPt/CNT 0.2–380 mg/dL – – – – [21]ChOx/CS/MWCNT 0.2–270 mg/dL – 1.55 µA mM−1 9.1 mg/dL b20 s 50 days [22]ChOx/NanoZnO/CH 5–400 mg/dL – 141 μA mg/dL 8.6 mg/dL 15 s 85 days [1]ChOx/NanoCeO2 10–400 mg/dL 2 μA/mg dL−1 cm−2 76 mg/dL 15 – [2]ChOx/CH–NanoCeO2 10–400mg/dL 5 mg/dL 47 μA/mg dL−1 cm−2 3.5 mg/dL 10 s 60 days Present work

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Fig. 3B shows CV of ChOx/CH–NanoCeO2/ITO bioelectrode in PBS(50 mM, pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4− as afunction of scan rate from 10 to100 mV s−1. It can be seen that bothcathodic (Ip) and anodic (Ic) peak currents of the electrode increaselinearly and are proportional to the scan rate (inset Fig 2B) accordingto Eqs. (2) and (3).

IpðAÞ = 3:26 × 10−4ðAÞ + 6:11 × 10−6A ðs=mVÞ⁎ scan rate ðmV=sÞ with regression coefficient 0:994

ð2Þ

IcðAÞ = −3:52 × 10−4ðAÞ− 4:83 × 10−6A ðs=mVÞ⁎ scan rate ðmV=sÞ with regression coefficient 0:986

ð3Þ

These results reveal surface controlled electrode and reversibleelectron transfer in the CH–NanoCeO2 composite. It has been shownthat ChOx adsorbed onto CH–NanoCeO2/ITO electrode undergoes areversible electron transfer with nanobiocomposite film.

The effect of pH on ChOx/CH–NanoCeO2/ITO bioelectrode has beencarried out using CV technique (Fig. 3C). It is observed that magnitudeof the current increases in the pH range from 6.0 to 7.0. Themagnitudeof current has been found to decrease on further increase in pH from7.0 to 8.0. This suggests that ChOx/CH–NanoCeO2 bioelectrode showsmaximum activity at pH 7 at which ChOx retains its natural structure.

4. Electrochemical response studies

The electrochemical response of the ChOx/CH–NanoCeO2/ITObioelectrode has been investigated as a function of cholesterolconcentration (100 μL of 2.5–400 mg dL−1) using CV technique(Fig. 4A) at a scan rate of 10 mV/s. The magnitude of current responseis found to increase on addition of cholesterol. This can be attributedto the formation of CH–NanoCeO2 bioconjugate that accepts electronsduring re-oxidation of ChOx and transfer these to electrode. ThusChOx/CH–NanoCeO2/ITO bioelectrode surface acts as electron trans-fer-accelerating layer for transfer of electrons.

Inset (Fig. 4A) shows the calibration curve obtained as a function ofcholesterol concentration. The inset in Fig. 4A shows variation ofcurrent response against cholesterol concentration. However, the plotof current response versus logarithmic function of cholesterolconcentration yields a linear behaviour with high regression value of0.994.

IðAÞ = 2:43 × 10−4ðAÞ + 47:2 × 10−6 A ðdL=mgÞ⁎ cholesterol concentration ðmg=dLÞ

ð4Þ

The reproducibility of ChOx/CH–NanoCeO2/ITO bioelectrode hasbeen investigated at 10 mg/dL cholesterol concentration, no signifi-cant change in the current response is observed after using it at least10 times and the results have been found to be reproducible. Theshelf-life of the ChOx/NS–CeO2/ITO bioelectrode measured after aninterval of 1 week has been estimated as 10 weeks. The decrease in thevalue of current has been found to be about 10% up to about 7 weeks

where after the current decreases sharply resulting in about 65% lossin about 10 weeks (data not shown).

Thevalueof theMichaelis–Mentenconstant (Km)hasbeenobtainedas4 mg/dL using Lineweaver–Burke plot. The observed lowKmvalue revealsthe higher affinity of ChOx/CH–NanoCeO2/ITO biosensor and is attributedto favourable orientation of ChOx and higher loading of ChOx provided bythe microenvironment of CH–NanoCeO2 nanocomposite film.

The selectivity of ChOx/CH–NanoCeO2/ITO bioelectrode has beendetermined by comparing magnitude of the current response byadding normal concentration (physiological range) of interferentssuch as glucose, ascorbic acid, uric acid, urea and lactic acid. The role ofinterferents has been examined bymixing desired interferent in equalconcentration with that of cholesterol. It has been found that ChOx/CH–NanoCeO2/ITO bioelectrode is not significantly affected due topresence of these interferents. The value of the electrochemicalcurrent response remains nearly same except for ascorbic acidwherein there is a decrease of about 2% (Fig. 4B).

Table 1 summarizes results of the present studies obtained usingChOx/CH–NanoCeO2/ITO bioelectrode along with those reported inliterature.

5. Conclusions

In summary, CeO2 nanoparticles have been successfully incorpo-rated in CH matrix and ChOx has been immobilized onto CH–NanoCeO2 nanocomposite for cholesterol detection. ChOx/CH–

NanoCeO2/ITO bioelectrode displays excellent catalytic property tocholesterol due to increased active site of nanocomposite byNanoCeO2 and provides friendly microenvironment for high loadingof ChOx resulting in enhanced electron transport between analyte andCH–NanoCeO2/ITO electrode surface. ChOx/CH–NanoCeO2/ITO bioe-lectrode exhibits sensing characteristics such as detection range of 10–400 mg/dL, detection range 5 mg/dL, response time of 10 s, low Km

value of 3.5 mg/dL, high sensitivity of 47 μA/mg dL−1 cm−2 and theregression coefficient of 0.994. Efforts should be made to fabricateother biosensors based on this interesting nanocomposite.

Acknowledgements

We thank Dr Vikram Kumar, Director, NPL, New Delhi, India for thefacilities. We thank Dr. Anees A. Ansari, Dr. Pratima R. Solanki and othermembers of the group for interesting discussions. Financial supportreceived under the DST sponsored projects (DST/CLP 041332, DST/INT/JAP/P-21/17, DST/TST/TE/2007/06 and DST/SEN/SI/03), CSIR funded(Non-Network) and DBT sponsored projects (DBT/PR7667/MEP/14/1057/2006) is gratefully acknowledged.

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