InTech-Green Synthesis and Characterization of Biocompatible Gold Nanoparticles Using Solanum Indicum Fruits

7/28/2019 InTech-Green Synthesis and Characterization of Biocompatible Gold Nanoparticles Using Solanum Indicum Fruits

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Nanomaterials and Nanotechnology

Green Synthesis and Characterizationof Biocompatible Gold NanoparticlesUsing Solanum indicumFruitsRegular Paper

Punuri Jayasekhar Babu1,2, Pragya Sharma2, Sibyala Saranya2,3, Ranjan Tamuli2 and Utpal Bora1,2,*

1 Bioengineering Research Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, India2 Biotech Hub, Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, India3 Department of Biotechnology, D.R.W. College, Gudur, SPS Nellore, India* Corresponding author E-mail: [email protected]

Received 24 January 2013; Accepted 10 April 2013

2013 Babu et al.; licensee InTech. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

AbstractThispaperreportsontheecofriendlysynthesis

of gold nanoparticles (AuNPs) using Solanum indicumfruitextract(SFE).Wehaveevaluatedvariousparameters

forsynthesisofAuNPssuchasSFE(0.03%),HAuCl4(0.5

mM) and reaction time (20 seconds). The synthesized

AuNPs were characterized with different physical

techniques such as transmission electron microscopy

(TEM), Fourier transform infrared (FTIR) spectroscopy,

Xray

diffraction

(XRD)

and

energy

dispersive

X

ray

spectroscopy (EDX). TEM experiments showed that

AuNPspresentedananisotropicshapeand sizeranging

from 550nm. FTIR spectroscopy revealed that

biomolecules containing an amine group (NH2), a

carbonyl group, OH groups and other stabilizing

functional groupswere adsorbed on the surface of the

synthesized AuNPs. EDX showed the presence of the

elementsonthesurfaceoftheAuNPs.Thecytotoxicityof

the synthesized AuNPs were tested on two different

human cancer cell lines, HeLa and MCF7 and were

foundtobenontoxic,thusprovidinganopportunitytobe

usedin

biomedical

applications.

Keywords Solanum indicum,Gold nanoparticles,Crystalgrowths,electronmicroscopy,Fouriertransforminfrared

spectroscopy,nanostructure,Xraydiffraction

1.Introduction

Thesynthesisofmetallicnanoparticleshasgainedagreat

significancedue

to

its

remarkable

chemical

and

physical

properties.Inparticular,goldnanoparticles(AuNPs)can

be used in many fields such as biosensors, cosmetics,

electronics,catalysis, semiconductors,drugdeliveryand

tumour imaging, as they possess size and shape

dependent unique properties [1, 2]. Different physical

and chemical methods have been used to synthesize

AuNPsincludingradiation[3],thermaldecomposition[4,

5],vapourdeposition[6],reductioninmicroemulsion[7]

and electro and photo chemical processes [811]. In

theseconventionalmethods,toxicsyntheticchemicalsare

used as reducing and stabilizing agents. Synthetic

reducingagents

are

not

favoured

for

producing

AuNPs

forbiomedical applications, as traces of such chemicals

Punuri Jayasekhar Babu, Pragya Sharma, Sibyala Saranya, Ranjan Tamuli and Utpal Bora:

Green Synthesis and Characterization of Biocompatible Gold Nanoparticles Using Solanum indicum Fruits

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ARTICLE

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leftunreacted in theprocess canbeharmful.Therefore,

there is a considerable interest in developing new

procedures forAuNPsynthesis,whicharesimple,rapid

and environmentally benign. In this context,

microorganisms have been used to synthesize and

stabilizeAuNPs

without

the

involvement

of

synthetic

chemicals [12]. However, a major limitation in using

microorganismsistheintricacyofcontrolovertheshape,

sizeand crystallinityofAuNPs [1314].However,using

plant and fruit extracts for AuNP synthesis has the

potential to address the drawbacks associated with

microorganisms[1517].

Inacontinuousefforttosearchfordesirableplantmaterial

fortheecofriendlysynthesisofAuNPs,wefoundSolanum

indium fruits, which haveboth reducing and stabilizing

capabilities. S. indicum is an indigenous plant grown

widelyin

India

and

Taiwan

and

has

many

medical

applicationssuchasanantiinflammatory,woundhealing

and analgesic and is used for the treatment of rhinitis,

coughsandbreastcancer.InAssamandThailand,fruitsof

S.indicumareusedasfood[18,19].

Here we report the use of microwaves for the rapid

synthesis of the AuNPs using Solenum indicum Fruit

Extract (SFE). Microwaves are electromagnetic waves,

which can generate enormous amounts of heat by

molecular dipole moment interactions with high

frequencyelectromagneticradiations.Theheatgenerated

by

microwaves

assists

in

rapid

and

uniform

synthesis

of

AuNPs.Incontrasttoconventionalmethods,microwaves

also have the advantages of homogeneous heating that

directly influences the nucleation process of AuNP

synthesis[2022].

2.ExperimentalDetails

2.1Materials

S.indiumwasgrownintheexperimentalfieldandhealthy

leaveswere harvested forAuNP synthesis.All reagents

were purchased from Sisco Research Laboratories

(Mumbai,India)

and

Merck

(Mumbai,

India).

Cell

culture

relatedplasticwareandMTT(3,4,5 dimethylthiazol2yl

25diphenyltetrazoliumbromide)wereobtainedfromthe

SigmaAldrich(Bangalore,India).Celllineswereobtained

fromNationalCentreforCellSciences(Pune,India).

2.2PreparationofaqueousSolanumindicumfruitsextract

S. indicum fruits were washed in deionised water to

remove adsorbed dirt.To 25ml ofdistilledwater, 50g of

choppedfruitwasaddedandgrinded(BajajModelGX11,

India) for 15 minutes (min). The grinding mixture was

filteredwith

Whatman

filter

paper

(50mm)

and

the

filtrate

wasstoredat40Cforvariousexperiments.

2.3Biochemicaltestforflavonoidsandphenoliccompounds

To 5ml of diluted ammonium solution, 2ml of SFEwas

added, followed by several drops of concentrated

sulphuric acid. The appearance of a yellowish colour

indicatedthe

presence

of

flavonoids.

To 2ml of SFE, several drops of 5% ferric chloridewere

added.Theappearanceofadarkgreencolourindicatesthe

presenceofpolyphenoliccompounds.

2.4SynthesesofAuNPs

ThesynthesesofAuNPswerecarriedoutbyvarying the

SFE concentration (0.0050.05%) against 0.5mM HAuCl4

and the final volumewasmade up to 2mlwith double

distilledwater. The desired percentage of the SFE was

obtained by dissolving the dried powder of SFE in

distilledwater

to

make

0.1%

(W/V)

of

the

stock

solution

fromwhichtheworkingsolutionwasobtainedbyfurther

dilutions. To the whole resulting mixture, we applied

microwave irradiation for 15 seconds. To find out the

optimum concentration of HAuCl4, experiments were

carried out by varying the gold solution (0.11mM)

concentrationagainst0.03%of theSFE.30mlof0.1%SFE

wasmixedwith10mlofthe5mMHAuCl4solutionandthe

final volume was made up to 100ml with the distilled

watertoobtaintheconcentrationsof0.03%SFEand0.5mM

HAuCl4 (working solution). To determine the optimum

time for the AuNPs, a fixed amount of the workingsolution

(2ml)

was

taken

and

exposed

to

different

microwave irradiation times from 525 seconds with an

intervalof3seconds.

2.5CharacterizationofAuNPs

2.5.1UVvisiblespectroscopy

UVvisiblespectroscopicmeasurementsofthesynthesised

AuNPs were carried out on a Cary 100 BIO UVVis

spectrophotometer(Varian,CA,USA).

2.5.2TransmissionElectronMicroscope(TEM)

A volume of 10ml ofAuNP solutionwas centrifuged at

20000rpm for 20 minutes. The resulting pellet was

resuspended in 3mlofdistilledwater and centrifuged at

20000rpm for 20 minutes and the process was repeated

threetimes.Theobtainedpelletwasfinallyresuspendedin

1ml of distilled water. Several drops of the redispersed

colloidalsolutionwereplacedoveracarboncoatedcopper

grid and the water was evaporated in a hot air oven

(Daihan Labtech Co. Ltd. model LDO150F) at 65oC for

four hours. TEM measurements were performed on a

TransmissionElectronMicroscope(TEMJEOLmodel2100,

200kV)operated

at

avoltage

of

190kV.

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2.5.3XRD,FTIRandEDXanalysis

30ml of AuNP colloidal solution was centrifuged at

20000rpmandtheresultingpelletwasresuspendedin4ml

ofdoubledistilledwater.Thisprocesswasrepeated three

timestoobtainpurenanoparticles.Thepelletobtainedwas

redispersed in 5ml of double distilled water and freeze

dried in a lyophiliser (Christ Gefriertrocknungsanlagen

GmbHModel14)for16hours.Thefinedriedpowderwas

analysed with the help of an XRD instrument (Bruker

AdvanceD8XRDmachine)withaCusourceat1.5406A

wavelength in thin filmmode.The infrared spectrawere

recorded using a FTIR spectroscope (Spectrum One,

PerkinElmer,MA,USA), from4000/cmto450/cm,witha

resolution of 2cm and five scans/sampleby using aKBr

pelletcontaining1mgofdriedAuNPs.

TheelementalcompositionoftheaspreparedAuNPswasobtainedby using EnergyDispersiveXray Spectroscopy

(LEO 1430 VP) at variable pressures with a scanning

electronmicroscopeequippedwithanINCAOxfordEDX

detector,byusinganaccelerationvoltageof10keV

2.6Cytotoxicitystudies

TheMinimalEssentialMedium(MEM)containing1.0mM

C3H3NaO3, 0.1mM nonessential amino acids, 1.5g/l

NaHCO3,2mMLglutaminesupplementedwith10%FBS

(heat inactivated) and 1% antibioticantimycotic solution

(1000U/mLpenicillinG,10mg/mL streptomycin sulphate,5mg/mL gentamycin and 25g/mL amphotericin B)was

used tomaintain theHeLa (Human cervical cancer) and

MCF7 (Human breast cancer) cells. The cells were

culturedat37Cinahumidifiedincubator(HealForce,HF

160W,China)supplementedwith5%CO2.

TheMTTassaywasusedtotestthecytotoxicityofAuNPs

oncancercells.MonoculturesoftheHeLaandMCF7cells

were incubatedwith increasing filter concentrations (0.2

microns) sterilized the AuNPs for 24 hours. The cell

viabilitywasestimatedbyanMTTdyeconversionassay.

Cellsnotexposed toAuNPswereconsideredasreferring

samples. In a 96well plate (Cell Bind, Corning),

approximately 1 104 cellswere seeded andmaintained

usingMEMmediumcontainingserum.After24hoursof

incubation,themediumwasreplacedwiththeserumfree

mediumcontainingvariousconcentrationsofAuNPs (10

100M). The media were removed after 24 hours of

treatmentandcellswerewashedwithphosphatebuffered

saline (PBS, 0.01M, pH7.2) followedby the addition of

100l of MTT (0.5mg/mL), prepared in a serum free

medium.Themediawerethenincubatedforfourhoursin

an incubator. Subsequently, medium was removed and

100lofdimethylsulphoxide(DMSO)wasaddedtoeach

welltosolubilisetheformazancrystals.Theconcentration

of formazan was determined using a multiwell plate

reader(Teconmicroplatereader,model680,CA,USA)at

470nm absorbance.The cellviabilitywas calculatedwith

thefollowingequation.

Cellviability(%)=(Atreated/Acontrol)x100

whereAtreatedandAcontrolare theabsorbanceof the treated

anduntreatedcells,respectively.

Figure 1.UVVisible absorption spectra ofAuNPs synthesizedby (A) different concentrations of SFE (0.0050.05%) for 15

seconds against 0.5mMHAuCl4 (B) varying concentrations of

HAuCl4(0.1 1mM)againsttothe0.03%SFE.

3.Resultsanddiscussion

3.1SFEcatalysedsynthesisyieldsdiverseshaped

AuNPsunderoptimumconditions

The reactionmixtures (0.0050.025%) showed a gradual

increment in the formation of theAuNPs,whereas the

reactionmixtures above 0.03% of SFE showed flattened

peaks.This revealed that0.03%ofSFEwas sufficient to

reduce 0.5mM of HAuCl4. Thus, 0.03% of SFE was

consideredasoptimumforAuNPsynthesis(Fig.1a).UV

Vis spectra ofproducts obtainedby reacting 0.03% SFE

with varying concentrations (0.11mM) displayed an

intensepeakat547nm for0.5mMHAuCl4.However, the

peaksoftheSurfacePlasmonResonance(SPR)displayed



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by 0.1 and 0.3mM HAuCl4 were suboptimal, whereas

thoseof0.7and1mMHAuCl4werebroadened,indicating

an insignificant formation of the AuNPs. Thus, 0.5mM

HAuCl4 provided the optimum conditions for the

synthesisoftheAuNPs(Fig1b).

Several experiments were performed to determine the

optimum time for the synthesis of the AuNPs by

incubating 0.03% SFE and 0.5mM HAuCl4 solution at

different MI time intervals from 525 seconds. We

observedbroadenedbands above 20 seconds indicating

no further significant formation of AuNPs. The peaks

below20secondswereconsideredassuboptimalbecause

of their low intensity.Thisrevealed that20secondswas

sufficient to reduce0.5mMofgold solutionusing0.03%

SFEforAuNPssynthesis(Fig2).

Figure2.MWirradiationtime(525seconds)with0.03%SFEand0.5mMHAuCl4.

3.2TEMandXRDstudiesconfirmedthecrystallinenatureofAuNPsWeemployedaTEMtechniquetodeterminetheshapeand

size of the AuNPs formed. A typical brightfield TEM

image of AuNPs obtained under optimum reaction

conditions showed the formation of anisotropic AuNPs

(Fig.3a).TheAuNPswithdifferentshapesandwerefound

asshown

in

the

insert

of

Fig.

3b,

where

the

pie

chart

presents thedistributionof thedifferent shapes (circular,

hexagonal,triangularandrod).Thesizeofthesynthesized

AuNPswithacircularshaperangedbetween5and50nm,

whiletheaveragesizewasfoundtobe7.4nm(Fig.3b).

The crystalline nature of the synthesized AuNPs was

confirmed from the selected area electron diffraction

(SAED)patternwithbrightcircular ringscorresponding

to the (1 1 1), (2 0 0) and (2 2 0) planes (Fig. 4a). The

HRTEM image showed lattice fringes of 0.22nm,

revealing that the growth of the AuNPs occurred

preferentially

on

the

(1

1

1)

plane

(Fig.

4b).

The

inter

planedistanceof theAu(111)planewas inagreement

withthe(111)dspacingofbulkAu(0.2255nm)[2224].

Figure 3. Transmission Electron Microscope of (a) AuNPssynthesizedwith0.03%and0.5mMHAuCl4 for20seconds (b)

sizedistributionhistogram.

Figure 4. (a) SAED pattern of the Au NPs, the white markerrepresents 51nm1. (b) HRTEM of AuNPs synthesized with

optimizedparameters(0.03%PLE,0.5mMHAuCl4and20seconds).



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Figure 5. XRD pattern of AuNPs synthesizedwith optimizedparameters.

The XRD pattern revealed prominent Bragg reflections,

whichwere indexedon thebasisof the fcc structureofgold. The intensity of the (1 1 1), (2 0 0) and (2 2 0)

diffractionpeaks corresponding to 38.1o, 44.5o and 64.8o

respectively,confirmedthatthesynthesizedAuNPswere

of crystalline nature [20, 22]. The ratio between the

intensityof the (200)and (111)diffractionpeakswas

much lower than theusual value (0.52) suggesting that

the (11 1)planewas thepredominant among the three

diffractionpeaks(Fig.5).

3.3FTIRandEDXrevealedadsorptionofbiomoleculesontheAuNPsThe FTIR spectra of AuNPs synthesized under

optimized conditions indicated the presence of the

biomolecules on the AuNPs. FTIR spectra of SFE

showed strong signals at 3454, 2951, 1649, 1431 and

1082cm1, corresponding to thehydroxylgroup arising

from alcohols and phenolic compounds, secondary

amine, amide I bond of proteins, asymmetric

deformation of CH3 from alkenes and CN stretching

vibrationsoftheamidebond,respectively(Fig.6a).The

FTIR spectra of AuNPs showed strong signals at

3440cm1 (hydroxyl group of alcohols or phenols),

2949cm1(secondaryamine),1668cm1(amideIbondof

proteins), 1425cm1 (deformation of the CH3 from

alkanes) and 1082cm1 (stretching vibrations of CN

from amide bond) (Fig. 6b) [25]. This confirmed the

presence of bioactive molecules in the SFE and their

adsorption ontoAuNPs. The adsorption pattern (200

290nm) of the SFE after biochemical tests shows the

presence of phenolic compounds and flavonoids [26].

These bioactive molecules (phenolic compounds and

flavonoids)reduced theAu3+ ion intoAuonanoparticles

by oxidizing hydroxyl (ROH) to carbonyl groups (R

C=O) or transfer their electrons. During the

microwave irradiation process thebioactive molecules

were adsorbed onto the AuNPs and stabilized the

synthesizedAuNPs.

Figure 6. FTIR spectra of lyophilized SFE (spectrum a) andAuNPs(spectrumb)

TheEDXprofileofAuNPspresentsastrongsignalforgold

and copper along with very strong carbon and oxygenpeaks (Fig 7).The signal for copperwasdue to the grid

used for the TEM observations. We found molecular

carbonandoxygen,whichmayoriginate from flavonoids

orphenolicororganicbiomoleculesboundtothesurfaceof

the AuNPs. The EDX suggested the presence of

biomoleculesonthesurfaceofthesynthesizedAuNPs.

Figure 7.EDX pattern ofAuNPs synthesizedwith 0.03% FLE,0.5mMHAuCl4and20secondsofMWirradiationtime.

3.4SFEprovidedanontoxiccoatingonthesurfaceofAuNPsThe AuNPs synthesizedwere tested on the HeLa and

MCF7 cells andwere found tobebiocompatible. The

cellularmorphologiesofthetwocelllineswereunaltered

after treatment with AuNPs suggesting that treatment

withAuNPsdidnot induceanycytotoxiceffectcausing

significant damage or death of the treated cells. Both

HeLaandMCF7cellslinesshowedexcellentviabilityup

to 100mol of AuNPs/L of MEM after 24 hours post

treatment (Fig.8).HeLacellsshowedmore resistance to

the synthesized AuNPs, compared to theMCF7 cells.

This indicated that theSFEprovidedanontoxiccoating

on the surface of theAuNPs. Therefore, the rapid and

ecofriendly method presented in this work for the

synthesis of AuNPs provides an effective solution for

applicationsindrugdeliveryandmolecularimaging.



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Figure 8.Cytotoxicity assay:CellviabilityofHeLa andMCF7cells exposed todifferent concentrationsofAuNPs (10100M)

overaperiodof24hourstreatment.

4.Conclusion

Wehave successfully synthesizedAuNPsusing an eco

friendly method using Solanum indicum fruit extract

underoptimizedconditions (0.03%SFE,0.5mMHAuCl4

and 16 seconds). TEM observations showed that the

synthesizedAuNPswereanisotropic.HRTEMandXRD

analysisconfirmedthecrystallinenatureofAuNPs.Also,

FTIR and EDX studies revealed the adsorption of

bioactivemoleculeson thesurfaceofAuNPs.Moreover,

cytotoxic studies showed that the synthesized AuNPs

were non toxic on human cancer cell lines, indicatingtheir biocompatibility and thus their potential use in

severalbiomedicalapplications.Themethodpresentedin

thiswork has the advantages of ecofriendliness, rapid

synthesis, cost effectiveness and most importantly

producingbiocompatibleAuNPs.

5.Acknowledgments

TheDepartmentofBiotechnology(DBT)Govt.ofIndiais

acknowledged for providing funds through the project

Establishment of IBT Hubs by DBT under special

programme forNorthEasternStatesof India (Sanction

No BT/04/NE/2009). PJB thanks MHRD for providing

researchfellowship.SSandPSthankBiotechHubforthe

traineeship.

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InTech-Green Synthesis and Characterization of Biocompatible Gold Nanoparticles Using Solanum Indicum Fruits