Research ArticleModulatory Effect of Citrate Reduced Gold and BiosynthesizedSilver Nanoparticles on 120572-Amylase Activity
Kantrao Saware1 Ravindra Mahadappa Aurade2
P D Kamala Jayanthi2 and Venkataraman Abbaraju13
1Materials Chemistry Laboratory Department of Materials Science Gulbarga University Gulbarga Karnataka 585106 India2Indian Institute of Horticultural Research Hessaraghatta Lake Post Bengaluru Karnataka 5600 89 India3Department of Chemistry Gulbarga University Gulbarga Karnataka 585106 India
Correspondence should be addressed to Venkataraman Abbaraju raman chemrediffmailcom
Received 28 July 2014 Accepted 5 October 2014
Academic Editor Amir Kajbafvala
Copyright copy 2015 Kantrao Saware et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Amylase is one of the important digestive enzymes involved in hydrolysis of starch In this paper we describe a novel approach tostudy the interaction of amylase enzymewith gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) and checked its catalyticfunction AuNPs are synthesized using citrate reduction method and AgNPs were synthesized using biological route employingFicus benghalensis and Ficus religiosa leaf extract as a reducing and stabilizing agent to reduce silver nitrate to silver atoms Amodulatory effect of nanoparticles on amylase activitywas observedGold nanoparticles are excellent biocompatible surfaces for theimmobilization of enzymes Immobilized amylase showed 1- to 2-fold increase of activity compared to free enzymeThe biocatalyticactivity of amylase in the bioconjugate was marginally enhanced relative to the free enzyme in solution The bioconjugate materialalso showed significantly enhanced pH and temperature stability The results indicate that the present study paves way for themodulator degradation of starch by the enzyme with AuNPs and biogenic AgNPs which is a promising application in the medicaland food industry
1 Introduction
Nanoparticles are defined as particulate dispersions with asize in the range of 10ndash100 nm [1] The nanostructures havealso a great potential in biotechnological processes takinginto account that each may be used as carriers for enzymesduring different biocatalytic transformations [2] Differenttypes of biomolecules such as proteins enzymes antibodiesand anticancer agents can be immobilized on these nanopar-ticles
Interaction of metal nanoparticles with biomoleculeshas received much attention in the recent years for thedevelopment of diagnostics for sensors and for targeted drugdelivery Further nanoparticles also have promising medicalapplications for wound healing diagnostics biosensing anddrug delivery [3ndash6] Increased activity of glucose oxidasewas noticed when immobilized onto AgNPs [7] AgNPs apotential nanocatalyst for the rapid degradation of starch
hydrolysis by 120572-amylase were studied by researchers [8]The functionlised AuNPs have been extensively used for thedetection of various enzymes and also for measurement oftheir activity [9ndash11] For example chemical or electrostaticattachment of enzymes to functionlised AuNPs may alter thecatalytic properties either by increasing or by decreasing theaffinity for enzyme-substrate formation [12ndash14] enhancingits stability [12 13] enhancing the rate of product formation(although significant report in this regard is still lacking) [1213] or retention [15] and even having some loss of activity ofthe enzyme [16] Similarly interaction with AuNPs has beenfound to enhance the stability of peptides [17] Their uniqueproperty arises specifically from higher surface to volumeratio and increased percentage of atoms at the grain bound-aries They represent an important class of materials in thedevelopment of novel devices that can be used in variousphysical biological biomedical and pharmaceutical appli-cations [18ndash20] Nanotechnology has a wide application in
Hindawi Publishing CorporationJournal of NanoparticlesVolume 2015 Article ID 829718 9 pageshttpdxdoiorg1011552015829718
2 Journal of Nanoparticles
pest management as nanocapsules for herbicide deliveryvectorpest control and nanosensors for pest detection [21]Synthesized silver or gold nanoparticles also help to producenew insecticides and insect repellants
AuNPs and biointeractions studies are now widelyaccepted as protein corona rapidly forms on AuNPs surfaceswhen introduced into a biological medium [22] The adsorp-tion of proteins to the surface of the AuNPs can signifi-cantly change the surface charge of the AuNPs which hasimportant consequences for nanoparticle fate and transportin biological systems Therefore the composition of theprotein corona largely defines the biological identity of thenanoparticle In addition because of their extremely largesurface area to volume ratios a significant number of proteinscan be adsorbed and ldquotrappedrdquo on AuNPs surfaces whenthey are introduced into biological entities [22ndash27] Goldnanoparticles present an alternate and advantageous syn-thetic scaffold for targeting protein surfaces [28] and havebeen demonstrated to bind biomacromolecules [29] facilitateDNA transfection [30] and reversibly inhibit enzymes [31]The binding of albumin protein on the surfaces of silver andgold nanoparticles has been studied for surface adhesion byresearchers to understand the effect on its structural changes[32 33] Biointeraction studies with AgNPs and protein andits SPR effect surface charge effect was studied for variousapplications in biological sciences by various researchers [3435]
Enzymes are biological catalysts that speed up reactionsin the presence or absence of cofactors without any changein their activity 120572-Amylase is one of the chief digestiveenzymes present in all living organisms and amylases insalivathe pancreas of animals break down the long-chainpolysaccharide starch intomaltose and glucose Amylases arethe most important in food industry and medicinal applica-tions
So in this study we used silver and gold nanoparticles forthe interaction and catalytic action of 120572-amylaseThe presentstudy aims to investigate enzyme-catalyzed starch hydrolysisin the presence of AuNPs and green AgNPs This forms abasis for nanoparticle-bimolecular interaction that may bepotentially applied in the medical and food industry Thuswe report here the modulation of enzymatic activity in thepresence of AuNPs and AgNPs Absorbance fluorescencespectroscopic measurements and other chemical tests weredone to establish that the enzymes were attached to the NPs
2 Materials and Methods
21 Chemicals and Reagents 120572-Amylase (ex-porcine extra-pure) soluble starch AgNO
3 HAuCl
4 and ammonium
molybdate purchased from Himedia Bangalore India andall other chemicals used were of high purity and analyticalgrade For all the experiments ultrafiltered Milli-Q water(Millipore USA) was used
22 Synthesis of Gold Nanoparticles Gold nanoparticles of10ndash20 nm diameter were prepared by the citrate reductionroute [36] Briefly the method involves reducing 5 times 10minus4M
Au+3 by 12 times 10minus3M citrate in water by refluxing for 30minThe cluster solution was red wine for gold [37]
23 Biosynthesis of Silver Nanoparticles Biosynthesis of silvernanoparticles is according to our previous report [38] TheFicus benghalensis (FB) and Ficus religiosa (FR) leaf extractsare used for synthesis of silver nanoparticles Fresh healthyand green leaves from FR and FB plants were collected fromthe Gulbarga University campus Gulbarga India In thisexperiment 5mL of leaf extract was added to 95mL of a10minus3M aqueous AgNO
3(silver nitrate) solution and exposed
for 3min inmicrowave oven Periodically aliquots of the reac-tion solution were removed and subjected to UV-Vis spec-troscopy measurements for surface Plasmon resonance studyof AgNPs synthesis Controls containing leaf extract (withoutsilver nitrate) as positive controls and pure silver nitratesolution (without leaf extract) as negative controls werealso run simultaneously along with the experimental flaskin three replicates
24 UV-Visible Spectral Studies The measurement of max-imum absorbance peak of AuNPs and AgNPs was carriedout using U-3010 spectrophotometer UV-visible absorbancespectrum was measured between 200 and 800 nm in 1 cmquartz cuvette containing 2mL of nanoparticle solution atroom temperature Interactions of silver and gold nanopar-ticles with amylase were measured spectrophotometricallyUV-visible absorbance spectrum was measured between200 and 800 nm in 1 cm quartz cuvette containing protein(50 120583gmL) in 50mM Tris-HCl buffer with pH 74 at roomtemperature Amylase in Tris-HCl buffer is titrated withincreasing concentrations (0 5 10 20 30 and 50120583L) of silverand gold nanoparticles
25 Tryptophan Fluorescence Emission Spectra The inter-action of binding of silver and gold nanoparticles to pure120572-amylase was determined by Trp fluorescence quench-ing titrations as described previously [39] Briefly amylase(50 120583g2mL) was titrated in 50mM Tris-HCl buffer with pH74 with increasing concentrations of silver and gold nanopar-ticles (0 5 10 20 30 40 and 50 120583L) respectively Howeverquenching of Trp fluorescence emission of amylase wasmon-itored at 335 nm following excitation at 280 nm (slit widthfor both 5 nm) using Varian spectrofluorometer
26 Morphological Studies Using AFM for AgNPs and FESEMfor AuNPs Samples of silver nanoparticles were synthesizedusing leaf extracts of FB and FR Atomic force microscopy(AFM) was prepared by solution casting onto silicon wafersto make thin films These films were analysed in noncontactmode using a Pacific Nanotechnology Nano-R2 instrumentwith SiN probes at a scan rate of 05Hz in the air Themorphology of the gold nanoparticles synthesized by citratereduction method and AuNPs with amylase enzyme wasexamined using Field Emission Scanning ElectronMicroscopy (FESEM FEI Nova nano 600 Netherlands)and for this the images were operated at 15 KV on a 0∘ tiltposition
Journal of Nanoparticles 3
27 Immobilization of Amylase Amylase-silver and goldnanoparticles bioconjugates were prepared according to [40]Briefly the AgNPsAuNPs solution prepared was diluted by afactor of 3 with a glycine buffer (75mM pH 80) Amylase(mgmL) was added with stirring to a portion of the dilutedsolution containing the AgNPsAuNPs (50mL 50mMglycine buffer pH 80) The solution was equilibrated for 1 hbefore being centrifuged at 18000 rpm for 20min to removethe uncoordinated amylase remaining in solution The pre-cipitate obtained was subjected to three repeated wash cyclesinvolving rinsing with 50mM glycine buffer (50mL) andcentrifuging at 18000 rpm for 20min Finally the AgNP-amylase and AuNPs-amylase bioconjugates were suspendedin the 50mM glycine buffer with pH 8 (5mL) for furtherexperiments
28 Amylase Activity by DNS Method The amylase activitywas measured in presence of silver and gold nanoparticles asreported earlier [41] Amylase activity was measured in pres-ence of different concentrations of silver and gold nanoparti-cles by DNSmethodWe also assay the effect of nanoparticleson amylase activity with increasing concentrations of starchBriefly the activity was estimated by DNS method enzyme-AuNPs bionanoconjugates was added to various test tubeseach containing 0 025 5 10 20 30 40 and 50mgmLminus1 ofstarch solution All of these test tubes were then incubatedin a water bath at 37∘C for 20min The UV-Vis spectrumwas recorded at 570 using a U-3010 spectrophotometer(Tokyo Japan) The released sugar in the test sample wasquantified and the enzyme activity was calculated (expressedin 120583gmLmin) We also assayed stabilities of the free andimmobilized 120572-amylase which was studied according to [42]All the experiments were performed at least three times
3 Results and Discussion
Thepresent study reveals themodulatory effect of AuNPs andbiosynthesized AgNPs on enzyme activity during the hydrol-ysis of starch The solution turned pale yellow after 2minand the colour change confirmed the presence of AgNPsTheorganic moiety of soluble leaf extract is assumed to reducesilver nitrate thus acting as both stabilizing and reducingagent [43] The Plasmon peak observed at 435 and 440 nmconfirmed the presence of AgNPs (Figures 1(a) and 1(b))The absorbance peaks that occurred at around 400 nm arethe characteristic SPR signature of AgNPs [44] The HAucl
4
reduced by citrate reduction showing red wine color after10min confirmed the formation of gold nanoparticles and theSPR peak is observed at 510 nm (Figure 2) The absorbancepeak around 526 nm with the peak position remainingpractically constant also indicates the production of goldnanoparticles [45]We observed a faint pink coloration of thesolution after several pulses of the experiment In the solu-tions absorption spectra the surface Plasmon peak (around510ndash530 nm) could be clearly distinguished which was con-sistent with the presence of small 3ndash30 nm particles in thecolloid [46] It was also noted that the resulting AuNPs andAgNPs nanoparticle solutions were found to be stable formore than a month without agglomeration of particles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2000
1525
1050
0575
010020000 40000 60000 80000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
3000
2250
1500
0750
0000
(b)
Figure 1 Absorbance spectra of silver nanoparticles (a) Spectra ofFR nanoparticles (b) spectra of FB silver nanoparticles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
2000
1513
1025
0538
0050
Figure 2 Absorbance spectra of gold nanoparticles
Atomic force microscopy (AFM) was used to probe thesamplersquos surface morphology and roughness Figure 3 showsthe typical three-dimensional (3D) height image of silver
4 Journal of Nanoparticles
2430
3030
30
06
06
12
12
18
1824
24
(120583m)
(120583m)
(120583m)
(120583m) 00
001518
00
(nm
)
(a)
(120583m)
(120583m)
4567
00
0
0
0
(nm
)
10
10
8
8
6
64
42
2
2
(120583m
8
6
4
2
(b)
Figure 3 3D image of AgNPs using atomic force microscopy (a) AgNPs of Ficus benghalensis and (b) Ficus religiosa
(a) (b)
Figure 4 (a) FESEM image of citrate reduced gold nanoparticles 10ndash15 nm (b) Citrate reduced gold nanoparticles with 120572-amylase enzyme
nanoparticles with surface having spherical particles withgrains sized between 20 and 75 nm in diameter with meansize of about 35 nm This magnification is attributed to theconvolution of true particle size with that of the AFM tip(the size and shape of the features observed by AFM can beinfluenced by the effects of tip-sample convolution) and alsoto the preparation of samples for AFM Wider scans cover-ing a few micrometers yielded a root mean square (RMS)roughness of 10 nm with a maximum peak-to-valley distanceof 65 nm
To understand the core-shell morphology of the syn-thesized AuNPs- and AuNPs-amylase bioconjugate FESEMtechnique was employed Figure 4 shows the FESEM imagesof the AuNPs synthesized using citrate reductionmethod Oncareful observation each individualAuNP is seen as sphericalin shape whereas AuNPs and amylase bioconjugate showeda fine capping on each particle and the same may also beresponsible for interparticle binding It is observed from thisimage that the nanoparticles are isolated and are surroundedby a layer of organic matrix at some places which acts as cap-ping Almost all of the AuNPs are spherical in shape and arein the size range of 10ndash15 nm indicating monodispersity
The UV-Vis absorbance spectra were recorded from the120572-amylase solution before (spectrum 1) and after (spectra 2ndash5) addition of AgNPs of FR (Figure 5(a)) and FB (Figure 5(b))
silver colloidal solution (0 5 10 20 30 and 50 120583L) Withthe addition of silver nanoparticles an increase was noticedin absorbance spectra The blue shift of 120572-amylase (2ndash8 nm)was seen for both FB and FR nanoparticles respectively Theresults indicate that these silver nanoparticles interacted withamylase and affect its folding changes toward native structureSimilarly gold nanoparticles also interacted with amylaseand enhanced the absorbance spectra of amylase with theaddition of increasing concentration of AuNPs (Figure 5(c))and a slight conformational change of 120572-amylase with inter-action of AuNPs was observed
Tryptophan fluorescence quenchingmeasurement resultsshowed that AgNPs and AuNPs interacted and bound withhigh affinity to amylase The Trp emission spectra weredecreased with addition of increasing concentrations of FBandFRnanoparticles (Figures 6(a) and 6(b))TheTrpfluores-cence emission of amylase was quenched almost 30ndash60 byFR andFBAgNPs respectivelyHowever there is no emissionshift with addition of AgNPs
Similarly the Trp emission spectra were decreased withaddition of increasing concentrations ofAuNPs (Figure 6(c))AuNPs interacted with high affinity with amylase and a smallblue shift was observed The results indicate that AuNPsinteracted and bound with amylase with high affinity causingconformational changes of amylase Earlier Deka et al [41]reported that gold nanoparticles interacted with 120572-amylase
Journal of Nanoparticles 5
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
3
2
1 1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2
2
1
1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(b)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
30000 40000 50000
0373
0287
0200
0113
002723000
(c)
Figure 5 Measurement of absorbance spectrum of 120572-amylase in presence of nanoparticles Absorbance spectra of 120572-amylase at pH 74Absorbance spectra were increasing with addition of (a) FR silver nanoparticles (b) FB silver nanoparticles (5 10 20 30 40 and 50120583L)respectively and (c) gold nanoparticles
Ernest et al reported that AgNPs enhance amylaseactivity [8] We also observed that FR silver nanoparti-cles stimulate amylase activity at lower concentrations andinhibited it at higher concentration as shown in Figure 7Similarly FB nanoparticles also stimulated amylase activity atlower concentration and inhibited it at higher concentrationSilver nanoparticles demonstrated activator effect on thecholinesterase and monoamine oxidase activities and theseeffects increased with increasing concentrations of the nano-particles [47] The enzyme was attached to the nanoparticlefollowing the degradation of starch from the composite andnot to the reducing sugars although the enzymatic activitywas retained [48] We also found that gold nanoparticlesincreased amylase activity up to 2-fold The rate of reactionwas found to be increased in the presence of gold nanopar-ticles (Figure 7(b)) There is an increase of 2-fold reducingsugar formation suggesting that the gold nanoparticles havea significant role as a nanocatalyst in rapidly degrading thecomplex polysaccharide starch to reducing sugars while athigher concentrations of gold particles amylase activity wasdecreased Deka et al [41] also reported similar modulatoryeffect on amylase where the results have been explained based
on a model that considered the presence of enzyme boundto NP and that available for enhanced catalysis enzymebound to NP but unavailable due to being buried inside theagglomerate and the free enzyme
The immobilization of 120572-amylase takes places on thesurface of the FR and FB AgNPs or AuNPs Immobilizedamylase activity was increased by 60ndash90 (Figure 8(a))with FR and FB AgNPs respectively when compared tothe free enzyme AgNPs generally have the tendency toagglomerate faster in any biological medium and sedimentat the bottom To our surprise no such agglomeration wasseen during the reaction and thus we confirm that the AgNPshave the chances of being stabilized by the protein moleculethrough the thiol linkages and thus the enzyme molecule isimmobilized [48] A well-known fact is that upon a solidsupport the efficiency of the enzyme is increased comparedto its free form [49] This may possibly act as a nanocatalystin the hydrolysis of starch catalyzed by an amylase Thus thereaction rate is increased in the presence of AgNPs althoughthe exact binding mechanism remains to be explored Sim-ilarly Figure 8(b) shows that immobilized amylase on goldnanoparticles activity was increased by 100when compared
6 Journal of Nanoparticles
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
336
00 2
920
64
Fluo
resc
ence
inte
nsity
(au
)
abcdefg
(a)
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
335
07 3
220
96
Fluo
resc
ence
inte
nsity
(au
) abc
de
(b)
300 350 400 450 5000
200
400
600
800
1000
Wavelength (nm)
339
07 6
628
88
Fluo
resc
ence
inte
nsity
(au
)
ab
cd
ef g
h
(c)
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
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[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
pest management as nanocapsules for herbicide deliveryvectorpest control and nanosensors for pest detection [21]Synthesized silver or gold nanoparticles also help to producenew insecticides and insect repellants
AuNPs and biointeractions studies are now widelyaccepted as protein corona rapidly forms on AuNPs surfaceswhen introduced into a biological medium [22] The adsorp-tion of proteins to the surface of the AuNPs can signifi-cantly change the surface charge of the AuNPs which hasimportant consequences for nanoparticle fate and transportin biological systems Therefore the composition of theprotein corona largely defines the biological identity of thenanoparticle In addition because of their extremely largesurface area to volume ratios a significant number of proteinscan be adsorbed and ldquotrappedrdquo on AuNPs surfaces whenthey are introduced into biological entities [22ndash27] Goldnanoparticles present an alternate and advantageous syn-thetic scaffold for targeting protein surfaces [28] and havebeen demonstrated to bind biomacromolecules [29] facilitateDNA transfection [30] and reversibly inhibit enzymes [31]The binding of albumin protein on the surfaces of silver andgold nanoparticles has been studied for surface adhesion byresearchers to understand the effect on its structural changes[32 33] Biointeraction studies with AgNPs and protein andits SPR effect surface charge effect was studied for variousapplications in biological sciences by various researchers [3435]
Enzymes are biological catalysts that speed up reactionsin the presence or absence of cofactors without any changein their activity 120572-Amylase is one of the chief digestiveenzymes present in all living organisms and amylases insalivathe pancreas of animals break down the long-chainpolysaccharide starch intomaltose and glucose Amylases arethe most important in food industry and medicinal applica-tions
So in this study we used silver and gold nanoparticles forthe interaction and catalytic action of 120572-amylaseThe presentstudy aims to investigate enzyme-catalyzed starch hydrolysisin the presence of AuNPs and green AgNPs This forms abasis for nanoparticle-bimolecular interaction that may bepotentially applied in the medical and food industry Thuswe report here the modulation of enzymatic activity in thepresence of AuNPs and AgNPs Absorbance fluorescencespectroscopic measurements and other chemical tests weredone to establish that the enzymes were attached to the NPs
2 Materials and Methods
21 Chemicals and Reagents 120572-Amylase (ex-porcine extra-pure) soluble starch AgNO
3 HAuCl
4 and ammonium
molybdate purchased from Himedia Bangalore India andall other chemicals used were of high purity and analyticalgrade For all the experiments ultrafiltered Milli-Q water(Millipore USA) was used
22 Synthesis of Gold Nanoparticles Gold nanoparticles of10ndash20 nm diameter were prepared by the citrate reductionroute [36] Briefly the method involves reducing 5 times 10minus4M
Au+3 by 12 times 10minus3M citrate in water by refluxing for 30minThe cluster solution was red wine for gold [37]
23 Biosynthesis of Silver Nanoparticles Biosynthesis of silvernanoparticles is according to our previous report [38] TheFicus benghalensis (FB) and Ficus religiosa (FR) leaf extractsare used for synthesis of silver nanoparticles Fresh healthyand green leaves from FR and FB plants were collected fromthe Gulbarga University campus Gulbarga India In thisexperiment 5mL of leaf extract was added to 95mL of a10minus3M aqueous AgNO
3(silver nitrate) solution and exposed
for 3min inmicrowave oven Periodically aliquots of the reac-tion solution were removed and subjected to UV-Vis spec-troscopy measurements for surface Plasmon resonance studyof AgNPs synthesis Controls containing leaf extract (withoutsilver nitrate) as positive controls and pure silver nitratesolution (without leaf extract) as negative controls werealso run simultaneously along with the experimental flaskin three replicates
24 UV-Visible Spectral Studies The measurement of max-imum absorbance peak of AuNPs and AgNPs was carriedout using U-3010 spectrophotometer UV-visible absorbancespectrum was measured between 200 and 800 nm in 1 cmquartz cuvette containing 2mL of nanoparticle solution atroom temperature Interactions of silver and gold nanopar-ticles with amylase were measured spectrophotometricallyUV-visible absorbance spectrum was measured between200 and 800 nm in 1 cm quartz cuvette containing protein(50 120583gmL) in 50mM Tris-HCl buffer with pH 74 at roomtemperature Amylase in Tris-HCl buffer is titrated withincreasing concentrations (0 5 10 20 30 and 50120583L) of silverand gold nanoparticles
25 Tryptophan Fluorescence Emission Spectra The inter-action of binding of silver and gold nanoparticles to pure120572-amylase was determined by Trp fluorescence quench-ing titrations as described previously [39] Briefly amylase(50 120583g2mL) was titrated in 50mM Tris-HCl buffer with pH74 with increasing concentrations of silver and gold nanopar-ticles (0 5 10 20 30 40 and 50 120583L) respectively Howeverquenching of Trp fluorescence emission of amylase wasmon-itored at 335 nm following excitation at 280 nm (slit widthfor both 5 nm) using Varian spectrofluorometer
26 Morphological Studies Using AFM for AgNPs and FESEMfor AuNPs Samples of silver nanoparticles were synthesizedusing leaf extracts of FB and FR Atomic force microscopy(AFM) was prepared by solution casting onto silicon wafersto make thin films These films were analysed in noncontactmode using a Pacific Nanotechnology Nano-R2 instrumentwith SiN probes at a scan rate of 05Hz in the air Themorphology of the gold nanoparticles synthesized by citratereduction method and AuNPs with amylase enzyme wasexamined using Field Emission Scanning ElectronMicroscopy (FESEM FEI Nova nano 600 Netherlands)and for this the images were operated at 15 KV on a 0∘ tiltposition
Journal of Nanoparticles 3
27 Immobilization of Amylase Amylase-silver and goldnanoparticles bioconjugates were prepared according to [40]Briefly the AgNPsAuNPs solution prepared was diluted by afactor of 3 with a glycine buffer (75mM pH 80) Amylase(mgmL) was added with stirring to a portion of the dilutedsolution containing the AgNPsAuNPs (50mL 50mMglycine buffer pH 80) The solution was equilibrated for 1 hbefore being centrifuged at 18000 rpm for 20min to removethe uncoordinated amylase remaining in solution The pre-cipitate obtained was subjected to three repeated wash cyclesinvolving rinsing with 50mM glycine buffer (50mL) andcentrifuging at 18000 rpm for 20min Finally the AgNP-amylase and AuNPs-amylase bioconjugates were suspendedin the 50mM glycine buffer with pH 8 (5mL) for furtherexperiments
28 Amylase Activity by DNS Method The amylase activitywas measured in presence of silver and gold nanoparticles asreported earlier [41] Amylase activity was measured in pres-ence of different concentrations of silver and gold nanoparti-cles by DNSmethodWe also assay the effect of nanoparticleson amylase activity with increasing concentrations of starchBriefly the activity was estimated by DNS method enzyme-AuNPs bionanoconjugates was added to various test tubeseach containing 0 025 5 10 20 30 40 and 50mgmLminus1 ofstarch solution All of these test tubes were then incubatedin a water bath at 37∘C for 20min The UV-Vis spectrumwas recorded at 570 using a U-3010 spectrophotometer(Tokyo Japan) The released sugar in the test sample wasquantified and the enzyme activity was calculated (expressedin 120583gmLmin) We also assayed stabilities of the free andimmobilized 120572-amylase which was studied according to [42]All the experiments were performed at least three times
3 Results and Discussion
Thepresent study reveals themodulatory effect of AuNPs andbiosynthesized AgNPs on enzyme activity during the hydrol-ysis of starch The solution turned pale yellow after 2minand the colour change confirmed the presence of AgNPsTheorganic moiety of soluble leaf extract is assumed to reducesilver nitrate thus acting as both stabilizing and reducingagent [43] The Plasmon peak observed at 435 and 440 nmconfirmed the presence of AgNPs (Figures 1(a) and 1(b))The absorbance peaks that occurred at around 400 nm arethe characteristic SPR signature of AgNPs [44] The HAucl
4
reduced by citrate reduction showing red wine color after10min confirmed the formation of gold nanoparticles and theSPR peak is observed at 510 nm (Figure 2) The absorbancepeak around 526 nm with the peak position remainingpractically constant also indicates the production of goldnanoparticles [45]We observed a faint pink coloration of thesolution after several pulses of the experiment In the solu-tions absorption spectra the surface Plasmon peak (around510ndash530 nm) could be clearly distinguished which was con-sistent with the presence of small 3ndash30 nm particles in thecolloid [46] It was also noted that the resulting AuNPs andAgNPs nanoparticle solutions were found to be stable formore than a month without agglomeration of particles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2000
1525
1050
0575
010020000 40000 60000 80000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
3000
2250
1500
0750
0000
(b)
Figure 1 Absorbance spectra of silver nanoparticles (a) Spectra ofFR nanoparticles (b) spectra of FB silver nanoparticles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
2000
1513
1025
0538
0050
Figure 2 Absorbance spectra of gold nanoparticles
Atomic force microscopy (AFM) was used to probe thesamplersquos surface morphology and roughness Figure 3 showsthe typical three-dimensional (3D) height image of silver
4 Journal of Nanoparticles
2430
3030
30
06
06
12
12
18
1824
24
(120583m)
(120583m)
(120583m)
(120583m) 00
001518
00
(nm
)
(a)
(120583m)
(120583m)
4567
00
0
0
0
(nm
)
10
10
8
8
6
64
42
2
2
(120583m
8
6
4
2
(b)
Figure 3 3D image of AgNPs using atomic force microscopy (a) AgNPs of Ficus benghalensis and (b) Ficus religiosa
(a) (b)
Figure 4 (a) FESEM image of citrate reduced gold nanoparticles 10ndash15 nm (b) Citrate reduced gold nanoparticles with 120572-amylase enzyme
nanoparticles with surface having spherical particles withgrains sized between 20 and 75 nm in diameter with meansize of about 35 nm This magnification is attributed to theconvolution of true particle size with that of the AFM tip(the size and shape of the features observed by AFM can beinfluenced by the effects of tip-sample convolution) and alsoto the preparation of samples for AFM Wider scans cover-ing a few micrometers yielded a root mean square (RMS)roughness of 10 nm with a maximum peak-to-valley distanceof 65 nm
To understand the core-shell morphology of the syn-thesized AuNPs- and AuNPs-amylase bioconjugate FESEMtechnique was employed Figure 4 shows the FESEM imagesof the AuNPs synthesized using citrate reductionmethod Oncareful observation each individualAuNP is seen as sphericalin shape whereas AuNPs and amylase bioconjugate showeda fine capping on each particle and the same may also beresponsible for interparticle binding It is observed from thisimage that the nanoparticles are isolated and are surroundedby a layer of organic matrix at some places which acts as cap-ping Almost all of the AuNPs are spherical in shape and arein the size range of 10ndash15 nm indicating monodispersity
The UV-Vis absorbance spectra were recorded from the120572-amylase solution before (spectrum 1) and after (spectra 2ndash5) addition of AgNPs of FR (Figure 5(a)) and FB (Figure 5(b))
silver colloidal solution (0 5 10 20 30 and 50 120583L) Withthe addition of silver nanoparticles an increase was noticedin absorbance spectra The blue shift of 120572-amylase (2ndash8 nm)was seen for both FB and FR nanoparticles respectively Theresults indicate that these silver nanoparticles interacted withamylase and affect its folding changes toward native structureSimilarly gold nanoparticles also interacted with amylaseand enhanced the absorbance spectra of amylase with theaddition of increasing concentration of AuNPs (Figure 5(c))and a slight conformational change of 120572-amylase with inter-action of AuNPs was observed
Tryptophan fluorescence quenchingmeasurement resultsshowed that AgNPs and AuNPs interacted and bound withhigh affinity to amylase The Trp emission spectra weredecreased with addition of increasing concentrations of FBandFRnanoparticles (Figures 6(a) and 6(b))TheTrpfluores-cence emission of amylase was quenched almost 30ndash60 byFR andFBAgNPs respectivelyHowever there is no emissionshift with addition of AgNPs
Similarly the Trp emission spectra were decreased withaddition of increasing concentrations ofAuNPs (Figure 6(c))AuNPs interacted with high affinity with amylase and a smallblue shift was observed The results indicate that AuNPsinteracted and bound with amylase with high affinity causingconformational changes of amylase Earlier Deka et al [41]reported that gold nanoparticles interacted with 120572-amylase
Journal of Nanoparticles 5
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
3
2
1 1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2
2
1
1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(b)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
30000 40000 50000
0373
0287
0200
0113
002723000
(c)
Figure 5 Measurement of absorbance spectrum of 120572-amylase in presence of nanoparticles Absorbance spectra of 120572-amylase at pH 74Absorbance spectra were increasing with addition of (a) FR silver nanoparticles (b) FB silver nanoparticles (5 10 20 30 40 and 50120583L)respectively and (c) gold nanoparticles
Ernest et al reported that AgNPs enhance amylaseactivity [8] We also observed that FR silver nanoparti-cles stimulate amylase activity at lower concentrations andinhibited it at higher concentration as shown in Figure 7Similarly FB nanoparticles also stimulated amylase activity atlower concentration and inhibited it at higher concentrationSilver nanoparticles demonstrated activator effect on thecholinesterase and monoamine oxidase activities and theseeffects increased with increasing concentrations of the nano-particles [47] The enzyme was attached to the nanoparticlefollowing the degradation of starch from the composite andnot to the reducing sugars although the enzymatic activitywas retained [48] We also found that gold nanoparticlesincreased amylase activity up to 2-fold The rate of reactionwas found to be increased in the presence of gold nanopar-ticles (Figure 7(b)) There is an increase of 2-fold reducingsugar formation suggesting that the gold nanoparticles havea significant role as a nanocatalyst in rapidly degrading thecomplex polysaccharide starch to reducing sugars while athigher concentrations of gold particles amylase activity wasdecreased Deka et al [41] also reported similar modulatoryeffect on amylase where the results have been explained based
on a model that considered the presence of enzyme boundto NP and that available for enhanced catalysis enzymebound to NP but unavailable due to being buried inside theagglomerate and the free enzyme
The immobilization of 120572-amylase takes places on thesurface of the FR and FB AgNPs or AuNPs Immobilizedamylase activity was increased by 60ndash90 (Figure 8(a))with FR and FB AgNPs respectively when compared tothe free enzyme AgNPs generally have the tendency toagglomerate faster in any biological medium and sedimentat the bottom To our surprise no such agglomeration wasseen during the reaction and thus we confirm that the AgNPshave the chances of being stabilized by the protein moleculethrough the thiol linkages and thus the enzyme molecule isimmobilized [48] A well-known fact is that upon a solidsupport the efficiency of the enzyme is increased comparedto its free form [49] This may possibly act as a nanocatalystin the hydrolysis of starch catalyzed by an amylase Thus thereaction rate is increased in the presence of AgNPs althoughthe exact binding mechanism remains to be explored Sim-ilarly Figure 8(b) shows that immobilized amylase on goldnanoparticles activity was increased by 100when compared
6 Journal of Nanoparticles
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
336
00 2
920
64
Fluo
resc
ence
inte
nsity
(au
)
abcdefg
(a)
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
335
07 3
220
96
Fluo
resc
ence
inte
nsity
(au
) abc
de
(b)
300 350 400 450 5000
200
400
600
800
1000
Wavelength (nm)
339
07 6
628
88
Fluo
resc
ence
inte
nsity
(au
)
ab
cd
ef g
h
(c)
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
27 Immobilization of Amylase Amylase-silver and goldnanoparticles bioconjugates were prepared according to [40]Briefly the AgNPsAuNPs solution prepared was diluted by afactor of 3 with a glycine buffer (75mM pH 80) Amylase(mgmL) was added with stirring to a portion of the dilutedsolution containing the AgNPsAuNPs (50mL 50mMglycine buffer pH 80) The solution was equilibrated for 1 hbefore being centrifuged at 18000 rpm for 20min to removethe uncoordinated amylase remaining in solution The pre-cipitate obtained was subjected to three repeated wash cyclesinvolving rinsing with 50mM glycine buffer (50mL) andcentrifuging at 18000 rpm for 20min Finally the AgNP-amylase and AuNPs-amylase bioconjugates were suspendedin the 50mM glycine buffer with pH 8 (5mL) for furtherexperiments
28 Amylase Activity by DNS Method The amylase activitywas measured in presence of silver and gold nanoparticles asreported earlier [41] Amylase activity was measured in pres-ence of different concentrations of silver and gold nanoparti-cles by DNSmethodWe also assay the effect of nanoparticleson amylase activity with increasing concentrations of starchBriefly the activity was estimated by DNS method enzyme-AuNPs bionanoconjugates was added to various test tubeseach containing 0 025 5 10 20 30 40 and 50mgmLminus1 ofstarch solution All of these test tubes were then incubatedin a water bath at 37∘C for 20min The UV-Vis spectrumwas recorded at 570 using a U-3010 spectrophotometer(Tokyo Japan) The released sugar in the test sample wasquantified and the enzyme activity was calculated (expressedin 120583gmLmin) We also assayed stabilities of the free andimmobilized 120572-amylase which was studied according to [42]All the experiments were performed at least three times
3 Results and Discussion
Thepresent study reveals themodulatory effect of AuNPs andbiosynthesized AgNPs on enzyme activity during the hydrol-ysis of starch The solution turned pale yellow after 2minand the colour change confirmed the presence of AgNPsTheorganic moiety of soluble leaf extract is assumed to reducesilver nitrate thus acting as both stabilizing and reducingagent [43] The Plasmon peak observed at 435 and 440 nmconfirmed the presence of AgNPs (Figures 1(a) and 1(b))The absorbance peaks that occurred at around 400 nm arethe characteristic SPR signature of AgNPs [44] The HAucl
4
reduced by citrate reduction showing red wine color after10min confirmed the formation of gold nanoparticles and theSPR peak is observed at 510 nm (Figure 2) The absorbancepeak around 526 nm with the peak position remainingpractically constant also indicates the production of goldnanoparticles [45]We observed a faint pink coloration of thesolution after several pulses of the experiment In the solu-tions absorption spectra the surface Plasmon peak (around510ndash530 nm) could be clearly distinguished which was con-sistent with the presence of small 3ndash30 nm particles in thecolloid [46] It was also noted that the resulting AuNPs andAgNPs nanoparticle solutions were found to be stable formore than a month without agglomeration of particles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2000
1525
1050
0575
010020000 40000 60000 80000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
3000
2250
1500
0750
0000
(b)
Figure 1 Absorbance spectra of silver nanoparticles (a) Spectra ofFR nanoparticles (b) spectra of FB silver nanoparticles
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
20000 40000 60000 80000
2000
1513
1025
0538
0050
Figure 2 Absorbance spectra of gold nanoparticles
Atomic force microscopy (AFM) was used to probe thesamplersquos surface morphology and roughness Figure 3 showsthe typical three-dimensional (3D) height image of silver
4 Journal of Nanoparticles
2430
3030
30
06
06
12
12
18
1824
24
(120583m)
(120583m)
(120583m)
(120583m) 00
001518
00
(nm
)
(a)
(120583m)
(120583m)
4567
00
0
0
0
(nm
)
10
10
8
8
6
64
42
2
2
(120583m
8
6
4
2
(b)
Figure 3 3D image of AgNPs using atomic force microscopy (a) AgNPs of Ficus benghalensis and (b) Ficus religiosa
(a) (b)
Figure 4 (a) FESEM image of citrate reduced gold nanoparticles 10ndash15 nm (b) Citrate reduced gold nanoparticles with 120572-amylase enzyme
nanoparticles with surface having spherical particles withgrains sized between 20 and 75 nm in diameter with meansize of about 35 nm This magnification is attributed to theconvolution of true particle size with that of the AFM tip(the size and shape of the features observed by AFM can beinfluenced by the effects of tip-sample convolution) and alsoto the preparation of samples for AFM Wider scans cover-ing a few micrometers yielded a root mean square (RMS)roughness of 10 nm with a maximum peak-to-valley distanceof 65 nm
To understand the core-shell morphology of the syn-thesized AuNPs- and AuNPs-amylase bioconjugate FESEMtechnique was employed Figure 4 shows the FESEM imagesof the AuNPs synthesized using citrate reductionmethod Oncareful observation each individualAuNP is seen as sphericalin shape whereas AuNPs and amylase bioconjugate showeda fine capping on each particle and the same may also beresponsible for interparticle binding It is observed from thisimage that the nanoparticles are isolated and are surroundedby a layer of organic matrix at some places which acts as cap-ping Almost all of the AuNPs are spherical in shape and arein the size range of 10ndash15 nm indicating monodispersity
The UV-Vis absorbance spectra were recorded from the120572-amylase solution before (spectrum 1) and after (spectra 2ndash5) addition of AgNPs of FR (Figure 5(a)) and FB (Figure 5(b))
silver colloidal solution (0 5 10 20 30 and 50 120583L) Withthe addition of silver nanoparticles an increase was noticedin absorbance spectra The blue shift of 120572-amylase (2ndash8 nm)was seen for both FB and FR nanoparticles respectively Theresults indicate that these silver nanoparticles interacted withamylase and affect its folding changes toward native structureSimilarly gold nanoparticles also interacted with amylaseand enhanced the absorbance spectra of amylase with theaddition of increasing concentration of AuNPs (Figure 5(c))and a slight conformational change of 120572-amylase with inter-action of AuNPs was observed
Tryptophan fluorescence quenchingmeasurement resultsshowed that AgNPs and AuNPs interacted and bound withhigh affinity to amylase The Trp emission spectra weredecreased with addition of increasing concentrations of FBandFRnanoparticles (Figures 6(a) and 6(b))TheTrpfluores-cence emission of amylase was quenched almost 30ndash60 byFR andFBAgNPs respectivelyHowever there is no emissionshift with addition of AgNPs
Similarly the Trp emission spectra were decreased withaddition of increasing concentrations ofAuNPs (Figure 6(c))AuNPs interacted with high affinity with amylase and a smallblue shift was observed The results indicate that AuNPsinteracted and bound with amylase with high affinity causingconformational changes of amylase Earlier Deka et al [41]reported that gold nanoparticles interacted with 120572-amylase
Journal of Nanoparticles 5
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
3
2
1 1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2
2
1
1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(b)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
30000 40000 50000
0373
0287
0200
0113
002723000
(c)
Figure 5 Measurement of absorbance spectrum of 120572-amylase in presence of nanoparticles Absorbance spectra of 120572-amylase at pH 74Absorbance spectra were increasing with addition of (a) FR silver nanoparticles (b) FB silver nanoparticles (5 10 20 30 40 and 50120583L)respectively and (c) gold nanoparticles
Ernest et al reported that AgNPs enhance amylaseactivity [8] We also observed that FR silver nanoparti-cles stimulate amylase activity at lower concentrations andinhibited it at higher concentration as shown in Figure 7Similarly FB nanoparticles also stimulated amylase activity atlower concentration and inhibited it at higher concentrationSilver nanoparticles demonstrated activator effect on thecholinesterase and monoamine oxidase activities and theseeffects increased with increasing concentrations of the nano-particles [47] The enzyme was attached to the nanoparticlefollowing the degradation of starch from the composite andnot to the reducing sugars although the enzymatic activitywas retained [48] We also found that gold nanoparticlesincreased amylase activity up to 2-fold The rate of reactionwas found to be increased in the presence of gold nanopar-ticles (Figure 7(b)) There is an increase of 2-fold reducingsugar formation suggesting that the gold nanoparticles havea significant role as a nanocatalyst in rapidly degrading thecomplex polysaccharide starch to reducing sugars while athigher concentrations of gold particles amylase activity wasdecreased Deka et al [41] also reported similar modulatoryeffect on amylase where the results have been explained based
on a model that considered the presence of enzyme boundto NP and that available for enhanced catalysis enzymebound to NP but unavailable due to being buried inside theagglomerate and the free enzyme
The immobilization of 120572-amylase takes places on thesurface of the FR and FB AgNPs or AuNPs Immobilizedamylase activity was increased by 60ndash90 (Figure 8(a))with FR and FB AgNPs respectively when compared tothe free enzyme AgNPs generally have the tendency toagglomerate faster in any biological medium and sedimentat the bottom To our surprise no such agglomeration wasseen during the reaction and thus we confirm that the AgNPshave the chances of being stabilized by the protein moleculethrough the thiol linkages and thus the enzyme molecule isimmobilized [48] A well-known fact is that upon a solidsupport the efficiency of the enzyme is increased comparedto its free form [49] This may possibly act as a nanocatalystin the hydrolysis of starch catalyzed by an amylase Thus thereaction rate is increased in the presence of AgNPs althoughthe exact binding mechanism remains to be explored Sim-ilarly Figure 8(b) shows that immobilized amylase on goldnanoparticles activity was increased by 100when compared
6 Journal of Nanoparticles
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
336
00 2
920
64
Fluo
resc
ence
inte
nsity
(au
)
abcdefg
(a)
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
335
07 3
220
96
Fluo
resc
ence
inte
nsity
(au
) abc
de
(b)
300 350 400 450 5000
200
400
600
800
1000
Wavelength (nm)
339
07 6
628
88
Fluo
resc
ence
inte
nsity
(au
)
ab
cd
ef g
h
(c)
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
Figure 3 3D image of AgNPs using atomic force microscopy (a) AgNPs of Ficus benghalensis and (b) Ficus religiosa
(a) (b)
Figure 4 (a) FESEM image of citrate reduced gold nanoparticles 10ndash15 nm (b) Citrate reduced gold nanoparticles with 120572-amylase enzyme
nanoparticles with surface having spherical particles withgrains sized between 20 and 75 nm in diameter with meansize of about 35 nm This magnification is attributed to theconvolution of true particle size with that of the AFM tip(the size and shape of the features observed by AFM can beinfluenced by the effects of tip-sample convolution) and alsoto the preparation of samples for AFM Wider scans cover-ing a few micrometers yielded a root mean square (RMS)roughness of 10 nm with a maximum peak-to-valley distanceof 65 nm
To understand the core-shell morphology of the syn-thesized AuNPs- and AuNPs-amylase bioconjugate FESEMtechnique was employed Figure 4 shows the FESEM imagesof the AuNPs synthesized using citrate reductionmethod Oncareful observation each individualAuNP is seen as sphericalin shape whereas AuNPs and amylase bioconjugate showeda fine capping on each particle and the same may also beresponsible for interparticle binding It is observed from thisimage that the nanoparticles are isolated and are surroundedby a layer of organic matrix at some places which acts as cap-ping Almost all of the AuNPs are spherical in shape and arein the size range of 10ndash15 nm indicating monodispersity
The UV-Vis absorbance spectra were recorded from the120572-amylase solution before (spectrum 1) and after (spectra 2ndash5) addition of AgNPs of FR (Figure 5(a)) and FB (Figure 5(b))
silver colloidal solution (0 5 10 20 30 and 50 120583L) Withthe addition of silver nanoparticles an increase was noticedin absorbance spectra The blue shift of 120572-amylase (2ndash8 nm)was seen for both FB and FR nanoparticles respectively Theresults indicate that these silver nanoparticles interacted withamylase and affect its folding changes toward native structureSimilarly gold nanoparticles also interacted with amylaseand enhanced the absorbance spectra of amylase with theaddition of increasing concentration of AuNPs (Figure 5(c))and a slight conformational change of 120572-amylase with inter-action of AuNPs was observed
Tryptophan fluorescence quenchingmeasurement resultsshowed that AgNPs and AuNPs interacted and bound withhigh affinity to amylase The Trp emission spectra weredecreased with addition of increasing concentrations of FBandFRnanoparticles (Figures 6(a) and 6(b))TheTrpfluores-cence emission of amylase was quenched almost 30ndash60 byFR andFBAgNPs respectivelyHowever there is no emissionshift with addition of AgNPs
Similarly the Trp emission spectra were decreased withaddition of increasing concentrations ofAuNPs (Figure 6(c))AuNPs interacted with high affinity with amylase and a smallblue shift was observed The results indicate that AuNPsinteracted and bound with amylase with high affinity causingconformational changes of amylase Earlier Deka et al [41]reported that gold nanoparticles interacted with 120572-amylase
Journal of Nanoparticles 5
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
3
2
1 1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(a)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
2
2
1
1
2000
1500
1000
0500
000020000 30000 40000 50000 60000
(b)
Scan spectrum curve
Wavelength (nm)
Abso
rban
ce
30000 40000 50000
0373
0287
0200
0113
002723000
(c)
Figure 5 Measurement of absorbance spectrum of 120572-amylase in presence of nanoparticles Absorbance spectra of 120572-amylase at pH 74Absorbance spectra were increasing with addition of (a) FR silver nanoparticles (b) FB silver nanoparticles (5 10 20 30 40 and 50120583L)respectively and (c) gold nanoparticles
Ernest et al reported that AgNPs enhance amylaseactivity [8] We also observed that FR silver nanoparti-cles stimulate amylase activity at lower concentrations andinhibited it at higher concentration as shown in Figure 7Similarly FB nanoparticles also stimulated amylase activity atlower concentration and inhibited it at higher concentrationSilver nanoparticles demonstrated activator effect on thecholinesterase and monoamine oxidase activities and theseeffects increased with increasing concentrations of the nano-particles [47] The enzyme was attached to the nanoparticlefollowing the degradation of starch from the composite andnot to the reducing sugars although the enzymatic activitywas retained [48] We also found that gold nanoparticlesincreased amylase activity up to 2-fold The rate of reactionwas found to be increased in the presence of gold nanopar-ticles (Figure 7(b)) There is an increase of 2-fold reducingsugar formation suggesting that the gold nanoparticles havea significant role as a nanocatalyst in rapidly degrading thecomplex polysaccharide starch to reducing sugars while athigher concentrations of gold particles amylase activity wasdecreased Deka et al [41] also reported similar modulatoryeffect on amylase where the results have been explained based
on a model that considered the presence of enzyme boundto NP and that available for enhanced catalysis enzymebound to NP but unavailable due to being buried inside theagglomerate and the free enzyme
The immobilization of 120572-amylase takes places on thesurface of the FR and FB AgNPs or AuNPs Immobilizedamylase activity was increased by 60ndash90 (Figure 8(a))with FR and FB AgNPs respectively when compared tothe free enzyme AgNPs generally have the tendency toagglomerate faster in any biological medium and sedimentat the bottom To our surprise no such agglomeration wasseen during the reaction and thus we confirm that the AgNPshave the chances of being stabilized by the protein moleculethrough the thiol linkages and thus the enzyme molecule isimmobilized [48] A well-known fact is that upon a solidsupport the efficiency of the enzyme is increased comparedto its free form [49] This may possibly act as a nanocatalystin the hydrolysis of starch catalyzed by an amylase Thus thereaction rate is increased in the presence of AgNPs althoughthe exact binding mechanism remains to be explored Sim-ilarly Figure 8(b) shows that immobilized amylase on goldnanoparticles activity was increased by 100when compared
6 Journal of Nanoparticles
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
336
00 2
920
64
Fluo
resc
ence
inte
nsity
(au
)
abcdefg
(a)
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
335
07 3
220
96
Fluo
resc
ence
inte
nsity
(au
) abc
de
(b)
300 350 400 450 5000
200
400
600
800
1000
Wavelength (nm)
339
07 6
628
88
Fluo
resc
ence
inte
nsity
(au
)
ab
cd
ef g
h
(c)
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
Figure 5 Measurement of absorbance spectrum of 120572-amylase in presence of nanoparticles Absorbance spectra of 120572-amylase at pH 74Absorbance spectra were increasing with addition of (a) FR silver nanoparticles (b) FB silver nanoparticles (5 10 20 30 40 and 50120583L)respectively and (c) gold nanoparticles
Ernest et al reported that AgNPs enhance amylaseactivity [8] We also observed that FR silver nanoparti-cles stimulate amylase activity at lower concentrations andinhibited it at higher concentration as shown in Figure 7Similarly FB nanoparticles also stimulated amylase activity atlower concentration and inhibited it at higher concentrationSilver nanoparticles demonstrated activator effect on thecholinesterase and monoamine oxidase activities and theseeffects increased with increasing concentrations of the nano-particles [47] The enzyme was attached to the nanoparticlefollowing the degradation of starch from the composite andnot to the reducing sugars although the enzymatic activitywas retained [48] We also found that gold nanoparticlesincreased amylase activity up to 2-fold The rate of reactionwas found to be increased in the presence of gold nanopar-ticles (Figure 7(b)) There is an increase of 2-fold reducingsugar formation suggesting that the gold nanoparticles havea significant role as a nanocatalyst in rapidly degrading thecomplex polysaccharide starch to reducing sugars while athigher concentrations of gold particles amylase activity wasdecreased Deka et al [41] also reported similar modulatoryeffect on amylase where the results have been explained based
on a model that considered the presence of enzyme boundto NP and that available for enhanced catalysis enzymebound to NP but unavailable due to being buried inside theagglomerate and the free enzyme
The immobilization of 120572-amylase takes places on thesurface of the FR and FB AgNPs or AuNPs Immobilizedamylase activity was increased by 60ndash90 (Figure 8(a))with FR and FB AgNPs respectively when compared tothe free enzyme AgNPs generally have the tendency toagglomerate faster in any biological medium and sedimentat the bottom To our surprise no such agglomeration wasseen during the reaction and thus we confirm that the AgNPshave the chances of being stabilized by the protein moleculethrough the thiol linkages and thus the enzyme molecule isimmobilized [48] A well-known fact is that upon a solidsupport the efficiency of the enzyme is increased comparedto its free form [49] This may possibly act as a nanocatalystin the hydrolysis of starch catalyzed by an amylase Thus thereaction rate is increased in the presence of AgNPs althoughthe exact binding mechanism remains to be explored Sim-ilarly Figure 8(b) shows that immobilized amylase on goldnanoparticles activity was increased by 100when compared
6 Journal of Nanoparticles
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
336
00 2
920
64
Fluo
resc
ence
inte
nsity
(au
)
abcdefg
(a)
300 350 400 450 5000
100
200
300
400
Wavelength (nm)
335
07 3
220
96
Fluo
resc
ence
inte
nsity
(au
) abc
de
(b)
300 350 400 450 5000
200
400
600
800
1000
Wavelength (nm)
339
07 6
628
88
Fluo
resc
ence
inte
nsity
(au
)
ab
cd
ef g
h
(c)
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
Figure 6 Tryptophan fluorescence quenching spectra of120572-amylase with nanoparticles Line (a) corresponds to amylase alone Line (b-g) withnanoparticles (a) Amylase + FB AgNPs (b) amylase + FR AgNPs (c) amylase + gold nanoparticles Decreasing of Trp fluorescence spectrawith increasing (5 10 15 20 and 30120583L) concentration of silver and gold nanoparticles Amylase titrated with increasing concentration ofnanoparticles excitation at 280 nm followed emission at 335 nm (slit width for both 5 nm)
with free enzymes In our present study biosynthesizedAgNPs and citrate reduced AuNPs upon interaction with120572-amylase were capable of breaking down the starch com-plex with the attachment of the enzyme over its surfacethereby being immobilized and degrading starch much fasterthan when compared to free enzyme Because the collisionfrequency between the soluble (free) enzyme the substratemolecule and their steric orientations form the basis ofthe enzyme activity and the constraint is overcome by theimmobilized enzyme with the support of a solid nanoparticlewhereas it does not occur in the case of free starchThereforethe reaction velocity is high and the breakdown of starchto smaller molecules as monosaccharides and disaccharidesis faster Amylase activity was increased with increasing
concentration of starch in presence of nanoparticles as shownin Figure 9 It indicates that nanoparticles enhance the basalamylase activity
Pepsin immobilized on AuNPs surface was more stablewhen compared with the free enzyme [50] Similarly weobserved that AgNPs andAuNPswere increasing the stabilityof amylase activity Free amylase and amylase mixed withAuNPs or AgNPs solutions were kept at room temperaturefor 5 days and the comparison of the activities was deter-mined every 12 hours The results showed a slight enzymeactivity retained in all tests this kind of stability increaseis time dependent (Figure 10) This means that the longerthe enzymes are kept at room temperature the more activityincrease can be detected in the sample of amylase mixed with
Journal of Nanoparticles 7
0
20
40
60
80
100
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Silver nanoparticles (120583L)
FR AgNPsFB AgNPs
(a)
0
20
40
60
80
100
120
0 50 100 150 200 250
Am
ylas
e act
ivity
(mg
mL
min
)
Gold nanoparticles (120583L)
FR AgNPsFB AgNPs
(b)
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
Figure 7 Modulatory effect of silver and gold nanoparticles on 120572-amylase activity (a) (998771) effect of FBAgNPs on amylase (998779) effect ofFR AgNPs on amylase (b) effect of gold nanoparticles Data pointsrepresent the mean of at least three determinations
AuNPs or AgNPs solutions This result clearly demonstratedthat the AuNPs or AgNPs can stabilize the activity of amylasewhen compared with the free enzyme
4 Conclusion
In summary biosynthesized AgNPs and AuNPs showed anincreased rate of reaction with 120572-amylase The degradationof starch digestion kinetics in the presence of AgNPsAuNPsrapidly produced larger amounts of reducing sugars Thisstudy showed that the nanoparticle may have a significanteffect in the field of nanocatalysis promising their potentialuse in industries for rapid degradation of the complex
0
50
100
150
200
Amylase FR AgNPs-amylase FB AgNPs-amylase
Am
ylas
e act
ivity
()
(a)
0
50
100
150
200
250
Amylase AuNPs-amylase
Am
ylas
e act
ivity
()
(b)
Figure 8 Assay of immobilized amylase activity (a) Amylase +silver nanoparticles (b) amylase + gold nanoparticles Data pointsrepresent the mean of at least three determinations
0
20
40
60
80
100
120
0 10 20 30 40 50 60Starch (mgmL)
Am
ylas
e act
ivity
(mg
mL
min
)
AmylaseAgNPs-amylaseAuNPs-amylase
Figure 9 Effect of substrate concentrations on 120572-amylase activity inthe presence of nanoparticles as compared to the control (enzyme inabsence of NPs) Legend represents the respective concentration ofstarch at which the experiments were carried outThe concentrationofAgNPsAuNPswas same for all of the aboveData points representthe mean of at least three determinations
8 Journal of Nanoparticles
0
20
40
60
80
100
120
0 1 2 3 4 5 6Time (days)
Am
ylas
e act
ivity
()
AmylaseAmylase-AgNPsAmylase-AuNPs
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
Figure 10 Comparison of storage stabilities of the free and immo-bilized 120572-amylase at room temperature Data points represent themean of at least three determinations
molecule to simpler ones by immobilizing the enzymes ontothe surface of nanoparticlesThey could also be possibly usedin assay kits as they are less time consuming and for bio-medical applications such as drug delivery and sensing
Conflict of Interests
The authors report no conflict of interests The authors aloneare responsible for the content and writing of the paper
Authorsrsquo Contribution
Kantrao Saware and Ravindra Mahadappa Aurade con-tributed equally to the paper
Acknowledgments
The author Kantrao Saware thanks Professor G U Kulkarnifor fruitful guidance and Selvi Rajan JNCASR Bangalorefor FESEM measurements Venkataraman Abbaraju thanksUGC NewDelhi Government of India for financial supportin the form of project Kantrao Saware thanks Dr Shiv-ashankar KS IIHR Bangalore for providing fluorescencespectrophotometer
References
[1] S P Gubin Y A Koksharov G B Khomutov and G YYurkov ldquoMagnetic nanoparticles preparation structure andpropertiesrdquo Russian Chemical Reviews vol 74 no 6 pp 489ndash520 2005
[2] K J Dussan O H Giraldo and C A Cardona ldquoApplica-tion of magnetic nanostructures in biotechnological processesbiodiesel production using lipase immobilized on magneticcarriersrdquo in Proceedings of the European Congress of ChemicalEngineering Copenhagen Denmark September 2007
[3] D Roe B Karandikar N Bonn-Savage B Gibbins and J-B Roullet ldquoAntimicrobial surface functionalization of plasticcatheters by silver nanoparticlesrdquo Journal of AntimicrobialChemotherapy vol 61 no 4 pp 869ndash876 2008
[4] C-F Chau S-H Wu and G-C Yen ldquoThe development of reg-ulations for food nanotechnologyrdquo Trends in Food Science andTechnology vol 18 no 5 pp 269ndash280 2007
[5] F Furno K S Morley B Wong et al ldquoSilver nanoparticles andpolymeric medical devices a new approach to prevention ofinfectionrdquo Journal of Antimicrobial Chemotherapy vol 54 no6 pp 1019ndash1024 2004
[6] M Das C Mohanty and S K Sahoo ldquoLigand-based targetedtherapy for cancer tissuerdquo Expert Opinion on Drug Delivery vol6 no 3 pp 285ndash304 2009
[7] X Ren X Meng D Chen F Tang and J Jiao ldquoUsing silvernanoparticle to enhance current response of biosensorrdquo Bio-sensors and Bioelectronics vol 21 no 3 pp 433ndash437 2005
[8] V Ernest P J Shiny A Mukherjee and N ChandrasekaranldquoSilver nanoparticles a potential nanocatalyst for the rapiddegradation of starch hydrolysis by 120572-amylaserdquo CarbohydrateResearch vol 352 pp 60ndash64 2012
[9] Z Wang R Levy D G Fernig and M Brust ldquoKinase-catalyzedmodification of gold nanoparticles a new approach tocolorimetric kinase activity screeningrdquo Journal of the AmericanChemical Society vol 128 no 7 pp 2214ndash2215 2006
[10] M R Choi K J Stanton-Maxey J K Stanley et al ldquoA cellulartrojan horse for delivery of therapeutic nanoparticles intotumorsrdquo Nano Letters vol 7 no 12 pp 3759ndash3765 2007
[11] R Bonomi A Cazzolaro A Sansone P Scrimin and L J PrinsldquoDetection of enzyme activity through catalytic signal ampli-fication with functionalized gold nanoparticlesrdquo AngewandteChemie International Edition vol 50 no 10 pp 2307ndash23122011
[12] P Pandey S P Singh S K Arya et al ldquoApplication of thiolatedgold nanoparticles for the enhancement of glucose oxidaseactivityrdquo Langmuir vol 23 no 6 pp 3333ndash3337 2007
[13] K G Kouassi J Irudayaraj and G McCarthy ldquoExaminationof cholesterol oxidase immobilization onto magnetic nanopar-ticlesrdquo BioMagnetic Research and Technology vol 3 article 12005
[14] B J Jordan R Hong G Han S Rana and V M RotelloldquoModulation of enzyme-substrate selectivity using tetraethy-lene glycol functionalized gold nanoparticlesrdquo Nanotechnologyvol 20 no 43 Article ID 434004 2009
[15] G K Ahirwal and C K Mitra ldquoDirect electrochemistry ofhorseradish peroxidase-gold nanoparticles conjugaterdquo Sensorsvol 9 no 2 pp 881ndash894 2009
[16] LM Rossi A D Quach and Z Rosenzweig ldquoGlucose oxidase-magnetite nanoparticle bioconjugate for glucose sensingrdquo Ana-lytical and Bioanalytical Chemistry vol 380 no 4 pp 606ndash6132004
[17] M M Varma D D Nolte H D Inerowicz and F E RegnierldquoSpinning-disk self-referencing interferometry of antigen-anti-body recognitionrdquo Optics Letters vol 29 no 9 pp 950ndash9522004
[18] J-M Nam C S Thaxton and C A Mirkin ldquoNanoparticle-based bio-bar codes for the ultrasensitive detection of proteinsrdquoScience vol 301 no 5641 pp 1884ndash1886 2003
[19] A G Tkachenko H Xie Y Liu et al ldquoCellular trajectoriesof peptide-modified gold particle complexes comparison ofnuclear localization signals and peptide transduction domainsrdquoBioconjugate Chemistry vol 15 no 3 pp 482ndash490 2004
Journal of Nanoparticles 9
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
[20] L R Hirsch R J Stafford J A Bankson et al ldquoNanoshell-mediated near-infrared thermal therapy of tumors under mag-netic resonance guidancerdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 23 pp13549ndash13554 2003
[21] G Scrinis and K Lyons ldquoThe emerging nano-corporateparadigm nanotechnology and the transformation of naturefood and agri-food systemsrdquo International Journal of Sociologyof Agriculture and Food vol 15 no 2 pp 22ndash44 2007
[22] I Lynch andK A Dawson ldquoProtein-nanoparticle interactionsrdquoNano Today vol 3 no 1-2 pp 40ndash47 2008
[23] I Lynch T Cedervall M Lundqvist C Cabaleiro-Lago SLinse and K A Dawson ldquoThe nanoparticle-protein complexas a biological entity a complex fluids and surface science chal-lenge for the 21st centuryrdquoAdvances in Colloid and Interface Sci-ence vol 134-135 pp 167ndash174 2007
[24] P Ghosh G Han M De C K Kim and V M Rotello ldquoGoldnanoparticles in delivery applicationsrdquoAdvanced Drug DeliveryReviews vol 60 no 11 pp 1307ndash1315 2008
[25] A E Nel L Madler D Velegol et al ldquoUnderstanding bio-physicochemical interactions at the nano-bio interfacerdquo NatureMaterials vol 8 no 7 pp 543ndash557 2009
[26] A Lesniak F FenaroliM PMonopoli C Aberg KADawsonand A Salvati ldquoEffects of the presence or absence of a proteincorona on silica nanoparticle uptake and impact on cellsrdquo ACSNano vol 6 no 7 pp 5845ndash5857 2012
[27] L Lartigue CWilhelm J Servais et al ldquoNanomagnetic sensingof blood plasma protein interactions with iron oxide nano-particles impact on macrophage uptakerdquo ACS Nano vol 6no 3 pp 2665ndash2678 2012
[28] A Verma and V M Rotello ldquoSurface recognition of biomacro-molecules using nanoparticle receptorsrdquoChemical Communica-tions no 3 pp 303ndash312 2005
[29] C M McIntosh E A Esposito III A K Boal J M SimardC T Martin and V M Rotello ldquoInhibition of DNA transcrip-tion using cationic mixed monolayer protected gold clustersrdquoJournal of the American Chemical Society vol 123 no 31 pp7626ndash7629 2001
[30] MThomas and A M Klibanov ldquoConjugation to gold nanopar-ticles enhances polyethyleniminersquos transfer of plasmid dna intomammalian cellsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 16 pp 9138ndash9143 2003
[31] N O Fischer A Verma C M Goodman J M Simardand V M Rotello ldquoReversible ldquoirreversiblerdquo inhibition of chy-motrypsin using nanoparticle receptorsrdquo Journal of the Amer-ican Chemical Society vol 125 no 44 pp 13387ndash13391 2003
[32] P Kumari and P Majewski ldquoAdsorption of albumin on silicasurfaces modified by silver and copper nanoparticlesrdquo Journalof Nanomaterials vol 2013 Article ID 839016 7 pages 2013
[33] S P Boulos T A Davis J A Yang et al ldquoNanoparticle-protein interactions a thermodynamic and kinetic study ofthe adsorption of bovine serum albumin to gold nanoparticlesurfacesrdquo Langmuir vol 29 no 48 pp 14984ndash14996 2013
[34] V Banerjee and K P Das ldquoInteraction of silver nanoparticleswith proteins a characteristic protein concentration dependentprofile of SPR signalrdquo Colloids and Surfaces B Biointerfaces vol111 pp 71ndash79 2013
[35] L Shang L Yang J Seiter et al ldquoNanoparticles interacting withproteins and cells a systematic study of protein surface chargeeffectsrdquo Advanced Materials Interfaces vol 1 no 2 Article ID1300079 2014
[36] J Turkevich P C Stevenson and J Hillier ldquoA study of thenucleation and growth processes in the synthesis of colloidalgoldrdquo Discussions of the Faraday Society vol 11 pp 55ndash75 1951
[37] P V Kamat Nanoparticles and Nanostructured Films JohnWiley amp Sons New York NY USA 1998
[38] K Saware and A Venkataraman ldquoBiosynthesis and character-ization of stable silver nanoparticles using Ficus religiosa leafextract a mechanism perspectiverdquo Journal of Cluster Sciencevol 25 no 4 pp 1157ndash1171 2014
[39] R Liu A Siemiarczuk and F J Sharom ldquoIntrinsic fluores-cence of the P-glycoprotein multidrug transporter sensitivityof tryptophan residues to binding of drugs and nucleotidesrdquoBiochemistry vol 39 no 48 pp 14927ndash14938 2000
[40] S-H Huang M-H Liao and D-H Chen ldquoDirect binding andcharacterization of lipase onto magnetic nanoparticlesrdquo Bio-technology Progress vol 19 no 3 pp 1095ndash1100 2003
[41] J Deka A Paul and A Chattopadhyay ldquoModulating enzymaticactivity in the presence of gold nanoparticlesrdquo RSC Advancesvol 2 no 11 pp 4736ndash4745 2012
[42] A Rangnekar T K Sarma A K Singh J Deka A Rameshand A Chattopadhyay ldquoRetention of enzymatic activity of120572-amylase in the reductive synthesis of gold nanoparticlesrdquoLangmuir vol 23 no 10 pp 5700ndash5706 2007
[43] D Raghunandan M D Bedre S Basavaraja B Sawle S YManjunath andA Venkataraman ldquoRapid biosynthesis of irreg-ular shaped gold nanoparticles from macerated aqueous extra-cellular dried clove buds (Syzygium aromaticum) solutionrdquoColloids and Surfaces B Biointerfaces vol 79 no 1 pp 235ndash2402010
[44] X P Zhu T Suzuki T Nakayama H Suematsu W Jiang andK Niihara ldquoUnderwater laser ablation approach to fabricatingmonodisperse metallic nanoparticlesrdquoChemical Physics Lettersvol 427 no 1ndash3 pp 127ndash131 2006
[45] NV Tarasenko A V Butsen E ANevar andNA SavastenkoldquoSynthesis of nanosized particles during laser ablation of gold inwaterrdquo Applied Surface Science vol 252 no 13 pp 4439ndash44442006
[46] FMafune J-Y Kohno Y Takeda andT Kondow ldquoFull physicalpreparation of size-selected gold nanoparticles in solution laserablation and laser-induced size controlrdquo Journal of PhysicalChemistry B vol 106 no 31 pp 7575ndash7577 2002
[47] S A R Abbas ldquoThe effects of gold and silver nanoparticleson choline estrase andMonoamino oxidase enzymes activitiesrdquoInternational Journal of Chemistry vol 3 no 4 pp 61ndash68 2011
[48] J Deka A Paul A Ramesh and A Chattopadhyay ldquoProbingAu nanoparticle uptake by enzyme following the digestion ofa starch-Au-nanoparticle compositerdquo Langmuir vol 24 no 18pp 9945ndash9951 2008
[49] X Jiang J Jiang Y Jin E Wang and S Dong ldquoEffect ofcolloidal gold size on the conformational changes of adsorbedcytochrome 119888 probing by circular dichroism UV-visible andinfrared spectroscopyrdquo Biomacromolecules vol 6 no 1 pp 46ndash53 2005
[50] AGole CDashC Soman S R SainkarMRao andM SastryldquoOn the preparation characterization and enzymatic activityof fungal protease-gold colloid bioconjugatesrdquo BioconjugateChemistry vol 12 no 5 pp 684ndash690 2001
