Elasticity and nanomechanical response of Aspergillus niger spores using atomic force microscopy

Micron43(2012)407–411

ContentslistsavailableatSciVerseScienceDirect

Micron

jo u rn al h om epa g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n

Elasticity

and

nanomechanical

response

of

Aspergillus

niger

spores

using

atomic

force

microscopy

Te-Hua

Fang

,

Shao-Hui

Kang,

Zheng-Han

Hong,

Cheng-Da

Wu

DepartmentofMechanicalEngineering,NationalKaohsiungUniversityofAppliedSciences,415ChienKungRoad,Kaohsiung807,Taiwan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27July2011

Receivedinrevisedform11October2011 Accepted11October2011 Keywords: Spore Nanomechanicalresponse AFM Adhesion Nanoindentation

a

b

s

t

r

a

c

t

TheelasticityandnanomechanicalresponseofAspergillusnigersporesdeterminedusingatomicforce microscopy(AFM)andnanoindentationarediscussed.Theforce-displacementcurveofthesporesurfaces showsthattheaveragesurfaceroughnessofsporeswasapproximately33nmandthattheadhesionforce rangedfrom9to28nN.TheYoung’smodulusoftheA.nigersporesrangedfrom0.1to21.4GPaandthe hardnessrangedfrom0.01to0.17GPa.Thecriticalbucklingloadofthesporemembraneis290␮N.

©2011ElsevierLtd.Allrightsreserved.

1. Introduction

Food-processingequipmentthatcontainsbacterialandfungal sporescancontaminatefood(Clementetal.,1993;Hameretal., 1998; Husmarkand Ronner, 1990).Microorganismsand spores adheretosurfaces.Normalvegetativebacterialcellsaremore eas-ilykilledthansporesbyheatorchemicals(HusmarkandRonner, 1990).Researchonsporeadhesionhasverifiedthatthevirulence factorcanhavesomeimpactonBacilluscereus(Anderssonetal., 1995),andthat highadhesioniscloselyrelated tofood poison-ingsymptoms.Sporeadhesioninthedairyindustryisaproblem, sincethesporesgothroughpipelineswheretheymaymultiplyand resporulate(Anderssonetal.,1995).

Scanning probe microscopy (SPM) has been used for the quantificationand measurementofsurface potentialin physics, chemistry,medicine,andbiology.Atomicforcemicroscopy(AFM) hasbeenused toobtainsingle-molecule resolution (Hanet al., 1997;Jena,2006;Fangetal.,2005; FangandChang,2004)and for imaging membranesand protein crystalsat highresolution (Kienbergeretal.,2004;Tangetal.,2008).Bowenetal.directly quantifiedtheadhesionbetweensingleAspergillusnigersporesand freshlycleavedmicasurfacesusingAFM(Bowenetal.,2000).

Theobjectivesofthisstudyaretounderstandthemechanical propertiesand surface forces ofspores through theAFM-based nanomechanicalmeasurementtechnologies, whichareessential

∗ Correspondingauthor.Tel.:+88673814526x5336. E-mailaddress:[email protected](T.-H.Fang).

knowledge for removing or breaking spores from environ-ments. For biomedical applications, such as a single cell or a macromolecule manipulation, the AFM-based technologies can effectivelyevaluateacriticalclampingforceforavoidingthe plas-ticdeformationandthebreakage.Inthepresentstudy,thesurface andcontactpropertiesofindividualA.nigersporesareinvestigated usingtheelasticityforce,AFMnanoscratching,and nanoindenta-tion.

2. Experiments

A. niger spores were collected aseptically from 2-week-old colonies,grownonmaltextractagarslopesat25◦C.Thesespores werethenanchoredonaglassslidebya smallamountofglue. Acommerciallyavailablescanningprobemicroscopesystem (NT-MDTSFC050L)wasused.Adiagramoftheexperimentalsetupis showninFig.1(a).TheAFMmeasurementswereobtainedusing a conductiveprobe withatipradiusof 20nm. Aconstant nor-malforceof5nNwasmaintainedbetweenthetipandthesample surfaceinthecontactmode.Aplotofcantileverdeflectionasa functionoftheinteractionwiththesamplesurfacealongthez-axis wasobtainedfromthisprocess.TherelationshipinHooke’smodel betweentheforce,F,andthecantileverdeflectioncanbeexpressed as:

F=Ktip·z (1)

wherespringconstantKtipisthecantileverstiffness,andzisthe verticaldeflection.The adhesionproducedbetweenthetipand A.nigersporesduringtipretractionwasmeasured.Asshownin

0968-4328/$–seefrontmatter©2011ElsevierLtd.Allrightsreserved. doi:10.1016/j.micron.2011.10.011

410 T.-H.Fangetal./Micron43(2012)407–411

Fig.5. (a)AFMimagesofthesporesurfaceand(b)3Dimage.(c)AFMforceversus distancecurvesatvariouslocationsofspores.

load.Thebehaviorofanidealsporecompressedbyanaxialload PisthatifPishigherthanthecriticalload,Pcr,thespore mem-branebeginstobuckleunderaloadingof290␮N(P).Themaximum strengthofasporeis965␮N.

ThehardnessandYoung’smodulusasfunctionsofthe displace-mentoftheindenterweremeasuredfromtheloading–unloading curve(Fangetal.,2011).Thehardnessofamaterialisdefinedas itsresistancetoplasticdeformation.Indepth-sensing nanoinden-tation,thecompositemodulusE*_{iscalculatedas:}

E∗= S 2ˇ



(7)

whereSisthemeasuredstiffnessandˇisashapeconstant(1.034 fortheBerkovichtip).Fig.6(b)showstheYoung’smodulusandthe hardnessoftheA.nigersporesmeasuredundervariousindentation loads.TheYoung’smodulusandthehardnessoftheA.nigerspores wereaveragedafterthemeasurementsthreetimes.TheYoung’s modulusoftheA.nigersporesrangedfrom0.1to21.4GPa and thehardnessrangedfrom0.01to0.17GPa.Theirregularsurface morphologyofspores(Sealeetal.,2008)causesalargeYoung’s modulusandhardnessrangesobtained.ThehardnessandYoung’s modulusslightlyincreasedatadepthof470nmofthesporelayer. Thestructureofthesporecanrecoverbeforetheindenterreaches adepthof470nm.Whentheindentationdepthwaslargerthan 470nm,thestructuralsurfaceunderwentplasticdeformation.The effectofelasticityofmicroorganismoccursbytwomechanisms,to beginwithbygenericphysicochemicalforce,thenbyinteractions betweenstructuresofcell,forexample,piliandfimbriae.

Fig.6. (a)Indentationcycleload–unloadversuspenetrationdepthcurveofspores. (b)Young’smodulusandthehardnessofsporestructure.

4. Conclusion

In this study, theadhesion properties of individual A. niger sporeswereinvestigatedusingelasticityforce,AFM nanoscratch-ing,andnanoindentation.Thenanoscratchtechniquewasusedto studythemechanicalpropertiesofthesurfaceofA.nigerspores. Thescratch depthincreasedwithincreasingloading;the adhe-sionbetweenthesurfaceandthetipinduceshysteresisbehaviorof tip–surfaceinteraction,whichincreasesresistanceabilityofspores totheAFMtip.Theaveragesurfaceroughnessofsporeswas approx-imately33nmandtheadhesionforcerangedfrom9to28nN.The Young’smodulusoftheA.nigersporesrangedfrom0.1to21.4GPa andthehardnessrangedfrom0.01to0.17GPa.Thestructureof thesporebeganplasticdeformationafteranindentationdepthof 470nmundera loadingof290␮N.Themaximumstrength ofa sporeis965␮N.

Acknowledgment

ThisworkwaspartiallysupportedbytheNationalScience Coun-cilofTaiwanundergrantNSC099-2811-E-151-002.

References

Andersson,A.,Ronner,U.,Granum,P.E.,1995.Whatproblemsdoesthefoodindustry havewiththespore-formingpathogensBacilluscereusandClostridium perfrin-gens?Int.J.FoodMicrobiol.28,145–155.

T.-H.Fangetal./Micron43(2012)407–411 411 Bowen,W.R.,Lovitt,R.W.,2000.DirectquantificationofAspergillusnigerspore

adhe-siontomicainairusinganatomicforcemicroscope.ColloidsSurf.173,205–210. Bowen,W.R.,Lovitt,R.W.,Wright,C.J.,2000.DirectquantificationofAspergillusniger sporeadhesioninliquidusinganatomicforcemicroscope.J.ColloidsInterface Sci.228,428–433.

Clement,J.A.,Martin,S.G.,Porter,R.,Butt,T.M.,Beckett,A.,1993.Characterization oftheextracellularmatrixproducedinvitrobyurediniosporesandsporelings ofUromycesviciae-fabae.Mycol.Res.97,594–602.

Derjaguin,B.V.,Muller,V.M.,Toporov,Y.P.,1975.Effectofcontactdeformationson theadhesionofparticles.J.ColloidsInterfaceSci.53,314–325.

Fang,T.H.,Chang,W.J.,2004.Nanolithographyandnanoindentationof tantalum-oxidenanowiresandnanodotsusingscanningprobemicroscopy.PhysicaB352, 190–199.

Fang,T.H.,Chang,W.J.,Tsai,L.,2005.Nanomechanicalcharacterizationofpolymer usingatomicforcemicroscopyandnanoindentation.Microelectron.J.36,55–59. Fang,T.H.,Wu,C.D.,Kang,S.H.,2011.Thermomechanicalpropertiesofpolymer

nanolithographyusingatomicforcemicroscopy.Micron42,492–497. Hamer,J.E.,Howard,R.J.,Chumley,F.G.,Valent,B.,1998.Amechanismforsurface

attachmentinsporesofaplantpathogenicfungus.Science239,288–290. Han,W.,Lindsay,S.,Dlakic,M.,Harrington,R.,1997.KinkedDNA.Nature386,

563–564.

Hertz,H.,1882.UberdieBeruhrungfesterelasticherKorperJ.Reine.Angew.Math. 92,156–171.

Husmark,U.,Ronner,U.,1990.ForcesinvolvedinadhesionofBacilluscereusspores tosolidsurfacesunderdifferentenvironmentalconditions.J.Appl.Bacteriol.69, 557–562.

Jena,B.P.,2006.Cellsecretionmachinery:studiesusingtheAFM.Ultramicroscopy 106,663–669.

Kienberger, F.,Mueller, H., Pastushenko, V., Hinterdorfer, P., 2004. Following singleantibodybindingto purplemembranes inreal time.EMBORep.5, 579–583.

Samson,R.A.,Houbraken,J.,Summerbell,R.C.,Flannigan,B.,Miller,J.D.,2001. Com-monandimportantspeciesoffungiandactinomycetesinindoorenvironments. In:MicroogranismsinHomeandIndoorWorkEnvironments.Taylor&Francis, NewYork,pp.287–292.

Seale,R.B.,Flint,S.H.,McQuillan,A.J.,Bremer,P.J.,2008.Recoveryofsporesfrom ther-mophilicdairybacilliandeffectsoftheirsurfacecharacteristicsonattachment todifferentsurfaces.Appl.Environ.Microbiol.74,731–737.

Tang,J.L.,Ebner,A.,Badelt-Lichtblau,H.,Vollenkle,C.,Rankl,C.,Kraxberger,B., Leit-ner,M.,Wildling,L.,Gruber,H.J.,Sleytr,U.B.,Ilk,N.,Hinterdorfer,P.,2008. Recognitionimagingandhighlyorderedmoleculartemplatingofbacterial S-layernanoarrayscontainingaffinity-tags.NanoLett.8,4312–4319.