Inhibitory effect of common microfluidic-materials-on-PCR-outcome Sensors & Actuators, B Chemical

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SensorsandActuatorsB:Chemical

journalhomepage:www.elsevier.com/locate/snb

InhibitoryeffectofcommonmicrofluidicmaterialsonPCRoutcome

RimantasKodziusa,b,∗,KangXiaoc,JinboWud,XinYid,XiuqingGongd,IanG.Fouldsb,WeijiaWena,d

a

KAUST-HKUSTMicro/NanofluidicJointLaboratory,TheUniversityofScienceandTechnology,ClearWaterBay,Kowloon,DivisionofPhysicalSciencesandEngineering,4700KingAbdullahUniversityofScienceandTechnology,Thuwal23955-6900,SaudiArabiac

DepartmentofBiology,TheUniversityofScienceandTechnology,ClearWaterBay,Kowloon,d

NanoScienceandNanoTechnologyProgramandPhysicsDepartment,TheUniversityofScienceandTechnology,ClearWaterBay,Kowloon,

b

article

info

abstract

Articlehistory:

Received23July2011

Receivedinrevisedform10October2011Accepted17October2011

Available online 25 October 2011

Keywords:

Bovineserumalbumin(BSA)Microfluidics

PCR(polymerasechainreaction)PCRcompatibilityPolymerase

Surfacepassivation

Inthisstudy,weestablishedasimplemethodforevaluatingthePCRcompatibilityofvariouscommonmaterialsemployedwhenfabricatingmicrofluidicchips,includingsilicon,severalkindsofsiliconoxide,glasses,plastics,wax,andadhesives.Two-temperaturePCRwasperformedwiththesematerialstodeter-minetheirPCR-inhibitoryeffect.Inmostcases,addingbovineserumalbumineffectivelyimprovedthereactionyield.WealsostudiedtheindividualPCRcomponentsfromthestandpointofadsorption.MostofthematerialsdidnotinhibittheDNA,althoughtheynoticeablyinteractedwiththepolymerase.WeprovideasimplemethodofperformingPCR-compatibilitytestingofmaterialsusinginexpensiveinstru-mentationthatiscommoninmolecularbiologylaboratories.Furthermore,ourmethodisdirect,beingperformedunderactualPCRconditionswithhightemperature.Ourresultsprovideanoverviewofmate-rialsthatarePCR-friendlyforfabricatingmicrofluidicdevices.ThePCRreaction,withoutanyadditives,performedbestwithpyrexglass,anditperformedworstwithPMMAoracrylicgluematerials.

© 2011 Elsevier B.V. All rights reserved.

1.Introduction

Microfluidicchipshaveavarietyofapplicationsinthebiologicalsciencesandmedicine.Incontrastwithtraditionalexperimen-talapproaches,microfluidicsentailslowersampleandreagentconsumption,allowsfasterreactionsandenablesefficientsepa-ration.Additionallymicrofluidicsoffersotheradvantagesaccruingfromthefluids’variousdistinctbehaviors,suchasenergydissipa-tion,fluidicresistance,laminarflow,andsurfacetension.Biologicalmoleculessuspendedinfluidandtransportedthroughmicroflu-idicschannelsinteractwiththechannel-wallmaterial[1,2].Thisinteractionisevenstrongerinhighsurface-area-to-volumeratio(SAVR)microfluidicchannels.

Currentlylargenumbersofmaterialsareusedinmicrofluidicchipproductionincludingsilicon,glass,variousplasticsandoth-ers.Adsorptionandinhibitionofbiomoleculesoccurwhenthesematerialscomeincontactwithbiomolecularreactioncomponents.

Abbreviations:RBI,relativebandintensity;SAVR,surface-area-to-volumeratio;WRBI,weightedRBI.

∗Correspondingauthorat:DivisionofPhysicalSciencesandEngineering,4700KingAbdullahUniversityofScienceandTechnology,Thuwal23955-6900,SaudiArabia.Tel.:+96628084556;fax:+96628020140.

E-mailaddresses:rimantas.kodzius@kaust.edu.sa,kodzius@gmail.com

(R.Kodzius),xiaokang@ustc.edu(K.Xiao),ian.foulds@kaust.edu.sa(I.G.Foulds),phwen@ust.hk(W.Wen).

Bothadsorptionandinhibitionareproblemsthatbestbeavoidedbecausethereactionwillnotbesuccessfulevenwhenoneofthecomponentswillbeinhibited[1,3].Polymerasechainreaction(PCR)isathermalcyclingprocedureforamplifyingtargetDNA.ThePCRcompatibilityofsilicon,silicondioxide(SiO2)andothersurfaceshavebeenstudied;howevertheresultsareinconclusive[1,2,4,5].

Usuallyforprotein–surfaceinteractionmeasurements,bulkyandexpensiveequipmentisused,suchasatomicforcemicroscopy(AFM),scanningortransmissionelectronmicroscopy(SEM,TEM),spectrophotometricproteinconcentrationmeasurement,Fouriertransforminfraredspectroscopy(FTIR)orX-rayphotoelectronspectroscopy(XPS).Further,testslikeSEM,TEMandXPSmustbeperformedinvacuum,wherewaterisremovedfromthesample[6–9].AlthoughAFMworksatambienttemperature,eveninliquidthesurfaceisvisualizedonlyoveraverysmallarea[7,8,10–12].FTIRissuitableonlyformicro-samples,allowingthemolecularbondandgroupingvibrationstobededucted[7,13–16].

Wedesignedasimple,relativelyquickmeasurementthatonlyrequiresaPCRcycler;thusitmimicsactualconditionsinPCRcycling.Inourstudy,weevaluatedtheinhibitoryaffectofdiffer-entmaterialsonPCR,whichisoneofthemostfrequentlyusedenzymaticreactionsinmicrofluidics[7,17–21].ThePCRreactioncomponentsincludetheDNAtemplate,primers,DNApolymerase(polymerase),dNTPs,abuffer,divalentions(MgCl2),andKCl.Themaincomponentinthisreactionisheat-stablepolymerase.PCRconsistsof20–40ofrepeatedcycles,withtemperaturetransitions

0925-4005/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2011.10.044

350R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

between∼55◦Cand∼95◦C.Currentdevelopmentsallowfortemperaturecyclingbetween∼71◦Cand∼91◦Corevenlowertemperatures[22].LowerTd,enablesawiderchoiceofmaterialsforthePCR.

Initially,mostPCRmicrofluidicdeviceswerefabricatedfromsilicon,aseffectivetechnologieshadbeenderiveddirectlyfromsemiconductorfabrication.Morerecently,owingtodemandsforspecificopticalcharacteristics,bio-orchemicalcompatibility,lowerproductioncosts,andfasterprototypingmicrofluidics,glass,polymersandothermaterialshavebeenutilizedinstead.

AdsorptiontosilicasurfacesiscausedbytheselectiveactionofSiOx–surfacesilanol(Si–OH)groupsonpolarmolecules,whichisitselfaresultofacombinationofionicandhydrogenbondingeffects.Assuch,polarmoleculessuchasDNAandthepolymerasecanbeadsorbedbychipmaterial.Asearlyas1996,Shoffneretal.notedsomesurfaceinteractioninPCR[1].Taylorsuggestedaddingcarrier-proteinbovineserumalbumin(BSA)tothePCRmixtocom-petewithTaqpolymeraseforadsorptionatthechipwalls[2].BSAisthoughttocompetewiththepolymeraseforadsorptionatthechipwallsand,thus,toimprovePCRyields[2].TheadsorptionmechanismofTaqpolymerase[11]andBSAprotein[9,23]hasbeenextensivelystudied.BSAalsoactsasapolymerasecompetitorintheinhibitorchelation[24].Additionally,BSAfacilitatesprimeranneal-ing,stabilizesboththeDNAandthepolymerase,and,insodoing,actsasanosmo-protectant.Insubsequentyears,manymoreadju-vants,alongwithpassiveandactivecoatingstrategies,havebeeninvestigated[21].Jeyachandranetal.showedthatBSAmoleculescanbeadsorbedtobothhydrophobicandhydrophilicsurfaces[23]withdifferingadsorptionmechanismsandrates[9].Prakashetal.systematicallystudiedpolymeraseadsorptionon13materials[11],butthisstudydidnotuseactualPCRconditions,andthustheresultsarenotnecessarilytranslatabletoPCRcompatibilityduetothetemperaturedependenceofadsorption.AndwhileavarietyofmaterialshavebeentestedforPCRcompatibility[11],ourstudyisthefirsttoinvestigateawiderangeofmaterials.Somesolutionsthatavoidtheproblemofinhibitoryeffectsofcertainmaterials’inenzymaticreactions,suchasPCR,havebeenreported[21,25].Forexample,materialsurfacepassivationcanbeachievedbypassivecoating(“staticpassivation”)orbyactivecoating(“dynamicpassi-vation”).Intheformer,chemicalorbiologicalmoleculesareappliedtothemicrochannelsurfacepriortothePCRreaction;inthelatter,additivesareincludedinthereactionmix.PassivationtechniquesarewelldescribedinreviewsofZhangetal.[21,25].

Toaccessthematerialinhibitoryproperties,wedecidedtoperformonlydynamicpassivationusingBSA,themostcommonadjuvantinmicrofluidicPCR.AdsorptionofBSAtobothchargedandhydrophobicsurfaceshasbeenassessedwidely[23].Otherchip-surface-treatmentmaterialsactinasimilarway–limitingtheaccessofreactioncomponentstothesurface.AllofourPCRreactionsolutionscontainedadjuvantbetaine,acommonadditiveinmostPCRcommercialoptimizationkits[26].Betaineactsasanosmoprotectantforpolymerase,therebyincreasingitsresistancetodenaturation.

PCRreactionoptimizationthroughchoiceofsurfacematerialsisoftheupmostimportance,asitenablesandimprovesenzymaticreactioninmicrofluidics.OurassessmentofthePCRcompatibilityofvariousmaterialscommonlyusedwhileproducingmicrofluidicdevicesisalsopertinentandbeneficialtootherenzymaticreactionsinmicrofluidicdevices.

2.Materialsandmethods

2.1.Materialsinvestigated

ThefollowingmaterialsweretestedforPCRcompatibility:polymethyl-methacrylate(PMMA)(castacrylicsheetsClarexA

fromNittoJushiKogyoCo.Ltd.),polycarbonate(PC),polyvinylchloride(PVC),polypropylene(PP,froma200␮lPCRtube),poly-tetrafluoroethylene(PTFE),curedpolydimethylsiloxane(PDMS),threekindsofwaxwithmeltingtemperatures(Tm)of56◦C(whitewax:paraffinfromNacalaiTesque),60◦C(yellowwax:shiftwaxfromNikkaSeiko)and80◦C(blackwax:waxWfromApiezon),sil-icon(originsiliconwafer),SiO2of560nmthickness(carriersiliconwafer),quartz,pyrexglass,indiumtinoxide(ITO)glass,soda-limeglass,curedSU-8epoxy-basednegativephotoresist(SU-8),Nor-landOpticalAdhesives61(NOA61)and68(NOA68)exposedtoultravioletlight(UV)lightfor2min,driedepoxyandacrylicglues,metallicirontubes0.9mmindiameterand12.9mminlength,andmineraloilformolecularbiology(Sigma).TenmicrolitersofmineraloilwereusedinthepertinentPCRcompatibilitytest.Theothersolidmaterialsweremanuallybrokenintosmallfragmentsusingsurgicalscissors,andasampleofsize>5mm3wasaddedtoeachPCRreactiontube.Assuming(accordingtoourobserva-tions)thatthesamplewasfragmentedintomorethan10andupto100pieces,wecalculatedthetotalsurfaceareaofthematerialtobeintherangeof4.7×101–1.8×102mm2.Forthe10␮lmineraloilused,thetotalsurfaceareawas∼3.5×101mm2.Duetodiffer-entmaterialmechanicalproperties,fragmentationdidnotresultinthesamesizefragments;thereforeitisdifficulttoprovidepreciseSAVR.TheapproximatecalculatedvalueofSAVRforusedmateri-alsvariesfrom9.4×100to3.5×101mm2/␮l(oilSAVRvaluewas∼3.5×100mm2/␮l).ThatisanorderofmagnitudehigherthanfortheSAVRof1.5×100mm2/␮linaconventionalPCRreactiontube(e.g.,Perkin-ElmerMicroAmpReactionTube)[1].

2.2.PCRmethodology

PCRisusedtoamplifyselectedsectionsofDNA.Two-temperaturePCRisfast,efficient,andapplicabletovaryingconditions.Forcurrentprimerpair,theoptimizedconditionswereTa=71◦CandTd=91◦C.ThePCRwasperformedaspublishedin[22,27],exceptthanthefinalconcentrationsfortheprimerswere0.75␮MandfortheSpeedStarHSDNApolymerasetheywere0.025U/␮l.Distinguishingtheinhibitoryeffectofstronglyinhibit-ingmaterialswouldbedifficultifthePCRreactionvolumeislow.Basically,thereactionwouldbeimmediatelyinhibitedwithoutnoticingthedifferencebetweenvariousmaterials.Toclearlydis-tinguishtheinhibitoryeffectofstronginhibitorsthePCRreactionvolumewassetat30␮l.ThisvolumealsofacilitatedrecoveringthePCRmixafterincubationwiththematerial.

DetectingthePCRproductwasachievedbyrunningsamplesin4%agarosegelcontainingSYBRSafeDNAstain(LifeTechnolo-gies)andbysubsequentgelimaging.ThebandrelativeintensitywasquantifiedusingImageJversion1.43software(developedattheNationalInstitutesofHealth)bysubtractingthebackgroundnoiselevel,etc.andmeasuringtheareaofthepeak.Theobtainedvalueforthebandintensitywasdefinedastherelativebandinten-sity(RBI).TorelatetheRBItothesurfaceareaandthevolumeofthematerial,wealsodefinedtheweightedRBI(WRBI)astheratiobetweenRBIandSAVR.TheWRBIhelpstoclarifypossiblevaria-tionsintheresults,anditreducestheuncertaintyintroducedbyourfracturingmethodontheSAVR.

2.3.Totalreactioninhibitionexperiment

AsindicatedinFig.1,twoPCRmastermixeswerepreparedanddistributedamong24wellsina96-wellplate.ThefirstPCRmixwaspreparedwithoutBSA;thesecondcontainedBSAatafinalconcentrationof2␮g/␮l.ThePCRmixwasaddedtothematerialfragmentstotestthePCRcompatibility.Thetubeswerebrieflyvor-texedtomixthematerialwiththePCRsolution,incubatedonicefor30min,andthenPCRwasperformedonabenchthermocycler.The

R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

351

Fig.1.ThePCRcompatibilityassay.BoththetotalreactioninhibitionexperimentandDNAorpolymeraseadsorptionexperimentsaredescribed.ThePCRisperformedusingbenchthermocycler,andsubsequentimagingisdoneonagarosegel.ThecontrolexperimentwasdonerunningPCRwithoutincludinganyofmaterial(“noadditives”part,seeFig.2).

methodweusedtodeterminewhichPCRcomponentwasinhibitedbyourtestedmaterialswastosimplyomitoneofthePCRcompo-nentsduringthePCRsetup.Thecomponentwasthenaddedandincubatedwiththetestedmaterialfor30min.UsualPCRprepa-rationtimesdonotexceedmorethan30min.Additionally,forupto2hwedonotexpectchangesinadsorption,becausepreviousresearchshowsthatBSAadsorptionisrestrictedtoamonolayerwithincubationtimesoflessthan2h[28].Thehighesttemperaturereachedintwo-temperaturePCRwas91◦C.WithanevenhigherTd,weexpectstrongermaterialinhibitiononPCR,asmultiplesourceshaveshownthatamountofsurfaceadsorbedproteinsincreasesatelevatedtemperatures[29–32].Withtwo-temperaturePCRwecouldusealowerTd[33],whichwouldenableawiderchoiceofmaterialsforthePCR.AfterPCR,thematerialswereremovedand,forvisualization,theamplificationproductswereloadeddirectlyontothegel.

fragmentedmaterial.ThetubeswerebrieflyvortexedinordertomixthePCRsolutionwiththematerial,afterwhichtheywereincu-batedonicefor30min.Then,10␮lofthePCRmix,separatedfromthematerial,wasextractedandtransferredtothenewtubes.A0.3␮l(1.5U)quantityoftheSpeedStarpolymerasewasaddedtothetubeslackingthepolymerase.Onemicroliter(correspondingto2,000,000dsDNAmolecules)oftemplatesolutionwasaddedtothetubeswiththePCRmixturelackingtemplateDNA.PCRwasper-formedwiththecyclingconditionsTa=71◦CandTd=91◦Ceachfor20s,for35cycles.Theproductswerethenloadedontothegelforvisualization.

3.Resultsanddiscussion

3.1.PCRinhibitionphenomena

2.4.DNAandpolymeraseadsorptionexperiments

AfterextractingthePCRmixture,themissingPCRcomponentwasaddedtocompletethePCRmixture.PCRwasperformedtodetermineifthesignalwasasintenseasthecontrolPCR.Ifthesignalisatthesameintensity,thenthecomponentundertest,whichwasincubatedwiththetestedmaterial,wasnotadsorbed.AdiminishedsignalindicatesthatthematerialadsorbssomeofPCRcomponentundertest,asthatparticularPCRcomponentwasincubatedwiththetestedmaterials.

AsshowninFig.1,twodifferentPCRmixeswereprepared,bothwithoutadditiveBSA.FortheDNAadsorptionexperiment,thePCRmixwaspreparedwithoutthepolymerase,whereas,forthepoly-meraseadsorptionexperiment,templateDNAwasomitted.ThirtymicrolitersofthePCRmixwasdistributedintotubescontaining

AlthoughsomeliteraturereportsidealBSAconcentrationsrang-ingbetween0.5and1.0␮g/␮l,BSAhasbeenutilizedonchipsatconcentrationsashighas2.5␮g/␮l[2,24,34,35].Infact,inourpresentexperiments,thepositivecontrolsrevealedthatBSAatthe2␮g/␮lconcentrationhadnonegativeinfluenceonthePCR.PreviousresearchshowedthatBSAadsorptionisrestrictedtoamonolayerwithincubationtimesoflessthan2handatconcentra-tionslowerthan10␮g/␮l[28].

Toassessamaterial’scompatibilitywithPCR,weincludedawiderangeofmaterialsusedinmicrofluidics.PCRwiththosemate-rials,butwithoutBSA,revealedwhichmaterialsarePCR-inhibitory.SuccessfulPCRproducesmoreDNA;thefluorescentbandonthegelismoreintense,and,therefore,theRBIvalueishigher.WhenmeasuringandquantifyingthevaluesofRBI,virtuallynosignal(RBI<1.9×102)wasdetectedinPCRwithPMMA,waxes(Tm56◦Cand60◦C),ITOglass,SU-8,NOA61,epoxy,andacrylicglues.Only

352R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

Fig.2.Amplificationof71bpCMVfragment.MoreintensebandindicatessuccessfulPCR(noorlittlematerialinhibition).

aweaksignal(RBI<8.4×102)wasobtainedinPCRwithPC,PVC,silicon,siliconwithalayerof560nmSiO2,andthemetaltubes.However,ifBSAwasincludedinthePCRmix,astrongsignal(RBI>1.4×103)wasobtainedforthePMMA,PC,PVC,wax(Tm56◦C),silicon,siliconwithalayerof560nmSiO2,SiO2quartz,ITOglass,NOA61,andthemetaltubes.Weconclude,therefore,thatwax(Tm60◦C)(RBI=1.9×102),SU-8(RBI=5.5×101),andtheepoxyglue(RBI=1.1×102)arePCR-inhibitorywithorwithoutadditiveBSA.MostPCR-friendlymaterialsexhibitsimilarsignalsregardlessoftheinclusionornotofBSAinthePCRmixture;thesematerialsarePP,PTFE,PDMS,wax(Tm80◦C),SiO2quartz,pyrexandsoda-limeglasses,NOA68,andmineraloil(Figs.2and3andTable1).

Next,wedeterminedwhichreactioncomponentsareinhibitedbytheincludedmaterials.Theoretically,everyPCRreactioncom-ponentcouldbetestedforpossibleinhibitionwithinmicrofluidicschannels;however,wedecidedtorestrictthechoicetothetest-ingofmaterialspossiblyinhibitoryofthekeycomponentsofthePCRmixture,whicharetheDNAandthepolymerase.InordertosimulatenaturalPCRconditions,BSAwasnotincludedinthePCRmixture.

ThePCRreactioncontainstemplateDNA,primersandfreedNTPs.SinceprimersanddNTPareinexcess,weconcentratedontemplateDNA.Also,templateDNAismuchlongerthaneitherprimersordNTP.Thus,iftemplateDNAwereadsorbed,theshorterDNAmoleculeswouldbeadsorbedaswell,duetothekineticnatureofadsorption(i.e.,smallermoleculesaremorepronetobeingadsorbed).

Ourresultsshowedthattherewasneartotaladsorptionoftem-plateDNAwhenthewax(Tm60◦C)wasused(RBI=9.2×101).Incontrast,whenNOA61,mineraloilandacrylicgluematerialswereemployed,significantadsorptionoccurred(RBI<1.5×103).AsshowninFigs.2and4andTable2,theotherexaminedmate-rialsdidnotexertanynoticeableeffectonthetemplateDNA(RBI>3.0×103).DNAisapolyanionicmolecule;therefore,itisnotexpectedtobindtohydrophobicsurfaces[36].OurobservationthatDNAisnotadsorbedinnoticeableamountsisinagreementwiththepreviousliterature[37].AdditionalinhibitorsthatreducethefinalPCRproductbyinteractingwithDNA,suchashumicacid,col-lagen,andmelanin,arealsodiscussedin[37].Someinhibitors,suchashematinandmelanin,mightaffecttheprocessivity(therateofextension)ofthepolymeraseduringprimerextension[38].

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353

Fig.3.PCRinhibitionthroughvariousmaterials.ComparisonofPCRmixwithoutBSA(redbars)orcontainingBSA(bluebars).ThecalculatedWRBIrangeisindicatedforeachofmaterial.LowerWRBIvaluesindicateinhibition,whereashigherWRBIvaluesmeanslessinhibition.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

WecharacterizedtheSpeedStarpolymeraseadsorptionontotheinvestigatedmaterials.Here,weobservedmoresignificanteffectsonthePCRefficiency.Thepolymerase-inhibitionexper-imentsindicatethatfollowingmaterialsdonothavestrongeffects(RBI>1.1×103)onpolymerase:PC,PP,PTFE,PDMS,silicon

withalayerof560nmSiO2,SiO2quartz,pyrex,andsoda-limeglass.Slightpolymeraseinhibition(RBI<9.2×102)wasobservedwithPMMA,PVC,waxes(Tm56◦Cand80◦C),silicon,andNOA68.Averystrongorneartotalinhibition(RBI<1.8×102)wasobservedwithwax(Tm60◦C),ITOglass,SU-8,NOA61,

Table1

PCRinhibitionthroughvariousmaterials.ComparisonofPCRmixwithoutBSAorcontainingBSA.ThemeasuredRBIandcalculatedrangeofWRBIareindicated.N.A.—notapplicable.

Material

RBI,standard

WRBI,standard

RBI,+BSA

WRBI,+BSA

PMMAPCPVCPPPTFEPDMS

Wax(Tm56◦C)Wax(Tm60◦C)Wax(Tm80◦C)Silicon

˚SiO25600A

SiO2quartzPyrexglassITOglass

Soda-limeglassSU8NOA61NOA68EpoxyglueAcrylicglueMetaltubesMineraloilNoadditives

6.0×1017.7×1026.9×1021.4×1031.4×1032.2×1031.1×1021.1×1021.0×1032.2×1024.4×1022.9×1033.3×1039.1×1012.8×1038.2×1011.9×1022.9×1039.1×1016.0×1018.4×1021.2×1031.8×103

1.7×100∼6.4×1002.2×101∼8.2×1012.0×101∼7.4×1014.1×101∼1.5×1024.1×101∼1.5×1026.4×101∼2.4×1023.2×100∼1.2×1013.3×100∼1.2×1013.0×101∼1.1×1026.4×100∼2.4×1011.3×101∼4.7×1018.3×101∼3.1×1029.5×101∼3.6×1022.6×100∼9.7×1008.1×101∼3.0×1022.4×100∼8.8×1005.4×100∼2.0×1018.4×101∼3.2×1022.6×100∼9.7×1001.7×100∼6.4×1002.4×101∼9.0×1013.4×102∼3.6×102N.A.

1.4×1031.6×1032.5×1032.7×1032.8×1032.9×1031.5×1031.9×1023.2×1033.2×1034.1×1033.5×1034.1×1033.3×1034.1×1035.5×1014.5×1034.5×1031.1×1025.9×1023.0×1033.4×1034.1×103

3.9×101∼1.5×1024.6×101∼1.7×1027.1×101∼2.6×1027.6×101∼2.8×1027.9×101∼2.9×1028.4×101∼3.1×1024.4×101∼1.6×1025.3×100∼2.0×1019.2×101∼3.5×1029.1×101∼3.4×1021.2×102∼4.3×1021.0×102∼3.8×1021.2×102∼4.4×1029.4×101∼3.5×1021.2×102∼4.4×1021.6×100∼5.9×1001.3×102∼4.8×1021.3×102∼4.8×1023.0×100∼1.1×1011.7×101∼6.4×1018.6×101∼3.2×1029.4×102∼1.0×103N.A.

3R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

Fig.4.PCRinhibitionthroughmaterialinteractionwithtemplateDNA—wherethepolymerasehasbeenaddedaftertherestofthePCRmixhasbeenincubatedwiththematerialundertest.Thisistoavoidinteractionbetweenthematerialundertestandthepolymerase(redbars).PCRinhibitionthroughmaterialinteractionwithpolymerase—wheretheDNAhasbeenaddedaftertherestofthePCRmixhasbeenincubatedwiththematerialundertest.ThisistoavoidinteractionbetweenthematerialundertestandtheDNA(bluebars).ThecalculatedWRBIrangeisindicatedforeachofmaterial.LowerWRBIvaluesindicateinhibition,whereashigherWRBIvaluesmeanslessinhibition.SeeSection2.4forfurtherdescriptionofthemethod.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

metaltubes,mineraloil,epoxy,andtheacrylicglues(Fig.2andTable2).

ThepossiblevariationoftheSAVRbetweendifferentmateri-alsduetothefracturingprocessleadstoalimitedestimateoftheSAVR.WithoutapreciseestimateoftheSAVR,thecompo-nentslistedinTables1and2shouldbecomparedwithcaution.ThedifferenceforPCR-compatibilitymaybealsoduetotheSAVR;i.e.,aslightlyincompatiblematerialwithahighSAVRmightappearworsethanaveryincompatiblematerialwithalowSAVR.Tothisend,theWRBIvalueshavebeenplottedtopro-videanestimateoftheuncertaintypresentinthemeasurements(Figs.3and4).

3.2.Materialinhibitoryeffect

SiliconhasbeenwidelyusedtofabricatePCRchips,asithasaveryhighthermalconductivityandiseasytofabricate.OurfindingthatbaresiliconhasastrongerinhibitoryeffectonthepolymerasethanSiO2(RBI=2.2×102versusRBI=4.4×102with-outBSA)isinlinewiththeliterature[1,2,4].Surprisingly,SiO2quartz(RBI=2.9×103),whichalreadyhasbeenusedinmicroflu-idics[39–41],doesnothaveasstrongofaneffectassiliconorsiliconwitha560nmlayerofSiO2.BecauseSiO2hasatleast12crystallineforms[42]whichmaybeproducedbybothoxidationorchemicalreactiondeposition,itsinhibitionpropertiesneedfurtherinvestigation.

Wealsocomparedthreekindsofoxides:pyrex,soda-lime,andITO.Accordingtoearlierfindings,pyrexandsoda-limeglasses(RBI=3.3×103and2.8×103,respectively)allowamplifi-cationwithoutadditiveBSA[7],whereasITOglassrequiresthatBSAbeincludedinthePCRmix(RBI=9.1×101withoutBSAand3.3×103withBSA)[43].SU-8wasfoundtobeinhibitorybothwithandwithoutBSA(RBI<8.2×101)inthePCRmix-ture.UntreatedSU-8hadalreadybeenshowntobeinhibitory[11,44].

NOA61andNOA68areliquidphotopolymersthatarecuredbyexposuretoUV.WhereasNOA61ismoresuitableasanadhesiveforglassandmetal,NOA68isanexcellentchoiceforplastics.NOA68hasahigherviscosity(22,000CPS)thanNOA61(300CPS).Despitesuchsmalldifferences,commercialproductNOA68issignificantlymorePCR-friendlythanNOA61(RBI=2.9×103vs.1.9×102).FortheNOA61material,thePCRamplificationwassuccessfulonlywiththeadditiveBSA.Acrylicglue,foritspart,ismorePCR-friendlythanepoxyglueandperformsbetterwhenBSAisincluded(RBI=5.9×102),whereasthePCRwiththeepoxymaterialwasafailure(RBI=1.1×102).Therefore,werecommendthatacrylicgluebeusedinsteadofepoxy.

MetaltubingreducesthePCRyieldthroughinhibitionofthepolymeraseratherthanbybindingtotemplateDNA(RBI=1.5×102vs.3.0×103).Panaroetal.observedthatPCRisinhibitedwithothermetals,suchasstainlesssteel,titanium[45],1.0×101mm2sur-faceareaplatinum[46],and1.2×101mm2goldnanoparticles[47].

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Table2

PCRinhibitionthroughmaterialinteractionwithtemplateDNAorthepolymerasecorrespondingly.ThemeasuredRBIandcalculatedrangeofWRBIareindicated.N.A.—notapplicable.

MaterialPMMAPCPVCPPPTFEPDMS

Wax(Tm56◦C)Wax(Tm60◦C)Wax(Tm80◦C)Silicon

˚SiO25600A

SiO2quartzPyrexglassITOglass

Soda-limeglassSU8NOA61NOA68EpoxyglueAcrylicglueMetaltubesMineraloilNoadditives

RBI,interactionwithDNA

WRBI,interactionwithDNA

RBI,interactionwithpolymerase

WRBI,interactionwithpolymerase

4.6×1034.0×1035.4×1034.1×1033.5×1034.5×1033.7×1039.2×1024.3×1033.4×1033.8×1033.7×1033.3×1034.2×1034.3×1034.1×1031.5×1033.9×1034.2×1039.4×1023.0×1037.6×1029.8×102

1.3×102∼5.0×1021.2×102∼4.3×1021.5×102∼5.8×1021.2×102∼4.4×1021.0×102∼3.8×1021.3×102∼4.8×1021.1×102∼4.0×1022.6×100∼9.8×1001.2×102∼4.5×1029.7×101∼3.6×1021.1×102∼4.1×1021.0×102∼3.9×1029.6×101∼3.6×1021.2×102∼4.5×1021.2×102∼4.5×1021.2×102∼4.3×1024.3×101∼1.6×1021.1×102∼4.2×1021.2×102∼4.5×1022.7×101∼1.0×1028.7×101∼3.3×1022.1×102∼2.2×102N.A.

3.4×1021.5×1039.1×1021.7×1031.6×1031.7×1033.2×1021.0×1029.2×1024.3×1021.5×1031.1×1032.0×1031.8×1021.7×1031.0×1028.1×1013.9×1038.0×1018.4×1011.5×1023.4×1012.0×102

9.7×100∼3.6×1014.3×101∼1.6×1022.6×101∼9.7×1014.8×101∼1.8×1024.6×101∼1.7×1024.9×101∼1.8×1029.1×100∼3.4×1012.9×100∼1.1×1012.6×101∼9.8×1011.2×101∼4.6×1014.3×101∼1.6×1023.2×101∼1.2×1025.7×101∼2.1×1025.3×100∼2.0×1014.9×101∼1.8×1022.9×100∼1.1×1012.5×100∼9.5×1001.1×101∼4.2×1012.3×100∼8.6×1002.4×100∼9.0×1004.4×100∼1.6×1019.6×100∼1.0×101N.A.

Toovercomethisinhibition,theysuggestaddingexcessiveEDTAfollowedbythesameamountofMg2+[48].

MineraloilhaslittleinfluenceonPCRperformance(RBI=1.2×103withoutBSAand3.4×103withBSA),whichisunderstandablebecausemineraloilisinertand,assuch,doesnotinteractwithmagnesiumions.MineraloilhashadalongassociationwithPCR;thefirstthermocyclersuseditinplaceoftopheaterstocoverPCRmixturesandavoidevaporation.Somepolymerstreatedbynativeormolecularbiologymineraloilmayhavenegative(Buna-N,silicone)orpositive(Santoprene)effectonPCR[45].Becausemineraloilhasmanykinds,weadvisethatfurthertestingbeconductedtodeterminethebestkindforPCR.

Amongthesixdifferentplasticstested,theleastinhibitoryarePP,PTFE,andPDMS(RBI>1.4×103withoutBSA).Conventionaltubes,multiwellplates,andmicrochipsforPCRarefabricatedfromPP[49].PTFEisasyntheticfluoropolymer,aninertmaterialthathasalreadybeenusedinmicrofluidicsPCR[18].PDMS,too,iswellknown,andithasextensiveuseinmicrofluidics,includ-ingPCR[46,50,51].WhenBSAwasincludedinthereactionmix,PMMA,PC,andPVCwerefoundtobesuitableformicrofluidics(RBI>1.4×103).

Thevariouswaxesdonotformachemicallyhomogeneousgroup.Allwaxesarewater-resistantmaterialscomposedofvar-ioussubstances,includinglong-chain(from12to38carbonatoms),hydrocarbons,ketones,alcohols,aldehydes,sterolesters,alkanoicacids,terpenes,andmonoesters,whicharesolidoverawidetemperaturerange.Wax,notonlychemically,butalsosomeofitsby-products,caninfluencePCRperformance.Becausewaxhasbeenreceivingmoreattentioninthemicrofluidicsfield,weincludeditinourassay[22,52–55].Wetestedthreekindsofwaxobtainedfromthreecompanies.WeobservedthatwaxwithTm=80◦CisthemostPCR-compatible(RBI=1.0×103with-outBSAand3.2×103withBSA).Also,amplificationcouldbeperformedbyaddingBSA(RBI=1.5×103)whenusingwaxwithTm=56◦C.WaxwithTm=60◦C,however,wasfoundnottobecompatiblewiththePCRreaction,asittotallyinhibitedthereactiondespitetheinclusionofBSA(RBI=1.9×102).Inourpreviouswork,wedemonstratedthatwaxwithTm=60◦CstillcanbeusedforPCRbydoublingthepolymeraseconcentration[22].

Thisstudyoftheinhibitoryeffectofvariouscommonmicroflu-idicsmaterialshasprovidedanewrapidtestingmethodusingonlyaPCRcycler,andithasconfirmedandexpandedthelistoftestedmaterials.

3.3.AchievingsuccessfulPCR

Inthispublication,weprovideasimplemethodforanalyzingtheinhibitionmechanism(DNAtemplatelossversuspolymeraseloss)thatenablestheextrapolationofamaterial’sPCRcompatibil-ityfromthisworktootherstudies.

Indeed,thesimplePCRinhibitiontestdescribedhereincanbeusedtochoosethemostfriendlymicrofluidicsmaterialsortoeval-uatethewiderangeofadditivestobeincludedinPCR.Inourexperiments,wedemonstratedthatBSAenhancesthePCRprod-uctyieldformostofthetestedmaterials;themostvisibleeffects(RBIchangedifferenceismorethan9times)werewithPMMA,wax(Tm=56◦C),silicon,siliconwithalayerof560nmSiO2,ITOglass,NOA61,andacrylicglue.Somepolymeraseswereadsorbedonthesurfacemorethanothers[7],but,byincreasingtheDNAorthepolymeraseconcentration,PCRamplificationproductswereobtainedevenwithoutanyadditives(resultsnotshown).Insuchcases,theentireDNAorthepolymerasewasprobablynotinhib-itedoradsorbed,sotheremainingfreeDNAorpolymerasewassufficientforsuccessfulamplification.Regardless,carefulselectionoftherightPCRcomponentshelpstoimprovetheproductyield.Further,oil,intheformofwater-in-oildroplets,helpstominimizethecontactofPCRreactioncomponentswithwallmaterials,and,becausemineraloilisnon-inhibitory,weadviseusingit.Addition-ally,duetotheeffectivelyreducedinteractionbetweenmaterialsandthePCRcomponents,two-temperaturePCRwithalowdena-turingtemperature(Td)allowedforwidermaterialchoiceinchipproduction.Employingreal-timePCR,moreover,couldhelpunder-standthegeneralmechanismofthatinteraction.Real-timePCRexperimentshavebeenconductedforSiandSiO2inhibitoryeffects[5],aswellastodeterminetheeffectsonthevariousPCRinhibitors[38].

QuantifyingthePCRcompatibilityofthetestedmaterialsispossiblebymeasuringtheSAVRandrelatingittothechangesofthePCRoutcome.Althoughobtainingpowderofvariousmaterialsbyphysicallysmashingispossible,thismethodmakesestimating

356R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

theSARVdifficult.Thesurfaceareaofnanoparticlesassmallas5nmcanbeeasilycalculatedandtheareacanbecorrelatedtothePCRinhibitoryeffect.Wanetal.showedthatthePCRproductyieldismodulatedbythetotalsurfaceareaofgoldnanoparticlesregardlessofthesize[47].Gonzalesetal.testedtubingofvariousfluoropolymers.Heconcludedthattubingupto40cm(internaldiameter5.0×102␮m,innersurfacearea6.3×102mm2)canbeusedsafely,whilethelongertubingof3meters(innersurfacearea4.7×103mm2)adsorbstheDNAandSybrGreenI,aDNAstainsubstantiallytothetubingwallsresultingtoseverelysubsequentPCRinhibition[56].Knowingthetubingdiameter,thesurfaceareacanbecalculated.Forexample,thesurfaceareaofconventional200␮lPCRtubeis3.0×101mm2.However,itsnoteasytoobtaintubingofsomematerialsandperformtestPCRforinhibition.Panaroetal.machineda1mmdiameterand25mmlongchannelintoplasticmaterials,fortheincubationofPCRmixture.Healsouseda16-gaugepunchtocutsmallpiecesofgasketmaterialindiameterof1mmwithatotalsurfaceareaof4.0×101mm2[45].OurinhibitioninvestigationmethodprovidedmeansforafastPCRinhibitiontestofawiderrangeofmaterialsthanpreviouslytested.Theresultsaresuitableforsemiquantitativemeasuringofinhibi-tion,aschoosingsimilaramountsofvariousmaterialswillprovideonlyanestimatevalueofSAVR(thetotalmaterialsurfaceareawas4.7×101–1.8×102mm2).However,ourmethodiswellsuitableforindicatingmaterialsthatareseverelyproblematicformicroflu-idics.InahigherSAVRenvironment,moreDNAorpolymerasemaybeneededtoavoidtheinhibitoryeffect,asevenseeminglyfriendlymaterialsmaybecomeproblematicduetotheadsorptionofPCRcomponents.Inourcurrentstudy,onlyPCRwiththeadditionof60◦Cyellowwaxdidnotyieldanyamplification.Wealreadydemonstratedthetotalreactioninhibitionphenomenonforvariouspapermaterialsimpregnatedwithwaxoracrylicglue.Althoughwechosealowerendpolymeraseconcentrationof0.025U/␮l,thepolymeraseconcentrationcanbedoubledto0.05U/␮l[22,27].Atpresent,awidevarietyofplasticsisusedformicrofluidics,andweexpectadditionalmaterials,suchaspaper-basedchips,tobeintroducedinthenearfuture[22,27,53,,57–61].

OurresultsshowthatmaterialselectionformicrofluidicPCRchips,whicharecharacterizedbylargeSAVR,isavitalpartofopti-mizingPCRoutcome.Forexample,anSU-8basedmicrofluidicPCRsystemwouldbeexpectedtoprovidelessthanonetenthofthesignal(WRBI1.6×100–5.9×100)asthesamesystemfabricatedinPDMS(WRBI4.4×101–1.6×102).Thusmaterialselectionisasimportantanoptimizationparameter,asprimerandpolymeraseselectionorreactioncomponentandconditions(temperature,time)optimization[33].ThetypeofPCRcompatibilitytesttreatedinthispaperenablesthemosteffectualchoiceofmaterialsforuseinbiology-relatedexperiments.

4.Conclusions

Aspartofthecurrentminiaturizationtrend,biologicalreactionsandprocessesarebeingadaptedtomicrofluidicsdevices.BecausePCRistheprimarymethodemployedinDNAamplification,itsminiaturizationiscentraltoeffortstodevelopportabledevicesfordiagnosticsandtestingpurposes.Aproblem,however,isthePCR-inhibitoryeffectduetotheinteractionbetweenPCRreagentsandthesurroundingenvironment,theeffectsofwhichareincreasedinhigh-SAVRmicrofluidics.Somematerialsarepoorlystandard-izedwithbatch-to-batchvariations.Inthisstudy,weintroducedasimpletestforassessingthecompatibilityofmaterialsinPCR.Becauseofitsspeedandsimplicity,wehavebeenabletocomparethePCRcompatibilityofawiderrangeofmaterialsthananyprevi-ousstudy.Thistestdoesnotrequirebulkyorexpensiveequipmentusedforprotein–surfaceinteractionmeasurements,suchasAFM,

SEMorTEM,spectrophotometricproteinconcentrationmeasure-ment,FTIRorXPS.OurtestcanbeeasilyconductedincommonPCRtubesusingastandardbenchthermocycleravailableineverymolecularbiologylaboratoryinordertoclearlyidentifymateri-alsthatinhibitthePCRreactioncomponents.ThePCRcomponentadsorptionisassessedinnaturalPCRconditions,duringthecyclinginhightemperatures.Thetestcanbeperformedonalargenumberofmaterialsamplesinparallel,andadatabaseoftestedmaterialscanbecreatedandshared.Furthermore,thebiocompatibilityofmaterialscanbemeasuredonaDNA,RNA,enzymatic(protein)orcellularlevel.

Inkeepingwithotherworkintheliterature,weconfirmedthatincludingBSAinthePCRreactioncansignificantlyimprovemostofthetestedmaterials’surfacecompatibility,whichimprovesreactionperformanceandyieldoutcomes.Thisfindingisespe-ciallyimportantbecausemostbiologicalreactionsoccuronthesurface.Ourproposedstrategyformaterial-surfacePCRcompat-ibilitytestingcanbeusedforotherbiologicalprocessesaswell.Suchcompatibilityiscentraltoeverysuchprocess.Asimilartestcanbeperformedforcellstudiesmeasuringtheinhibitionofmate-rialsoncellgrowth[10,63].Insuchatest,boththecellsandthecellmediumcomponentscanbetestedfortheadsorptiononthemate-rialsurface.Thusotherapplications,forexample,high-throughputscreening[,65]ortranscriptomeanalysis[66,67]couldbenefitfromourdescribedapproach.

Acknowledgements

TheauthorswouldliketoacknowledgethefinancialsupportprovidedbytheResearchGrantsCouncil(GrantNo.HKUST603208and660207).ThispaperisbasedonworkpartiallysupportedbyAwardNo.SA-C0040/UK-C0016madebyKingAbdul-lahUniversityofScienceandTechnology(KAUST).

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Biographies

RimantasKodziusgraduatedwithaPhDdegreefromtheUniversityofSalzburg(Austria,EU)withProf.RetoCrameri(SwissInstituteofAllergyandAsthmaResearch,Davos,Switzerland)andDr.GeraldWalter(MaxPlanckInstituteofMolec-ularGenetics,Berlin,Germany,EU)in2002.From2002to2006hedevelopedandestablishedCAGE(5󰀅-SAGE)technologyforlargescalehighthroughputgenepro-motermappingatRIKENinstituteinJapan.From2006to2009heworkedinthelaboratoryofNobelPrizeWinnerProf.SydneyBrennerandProf.ByrappaVenkateshattheInstituteofMolecularandCellBiologyinSingaporeoncreatinglibrariesforthecomparativegenomicsprojectonElephantshark.From2009hejoinedKingAbdul-lahUniversityofScienceandTechnology(KAUST)andspentoneyearinJointUniversityofScienceandTechnology(HKUST)/KAUSTMicro/Nano-FluidicsLaboratoryleadbyProf.WeijiaWen.Since2010heisworkingatKAUSTinSaudi

358R.Kodziusetal./SensorsandActuatorsB161 (2012) 349–358

Arabia.Hisresearchinterestsareintheareaofmolecularbiology,genomics,tran-scriptomics,surfacechemistry,microfluidicsystemsandbioMEMS.

KangXiaogothisBSdegreefromSchoolofLifeSciences,UniversityofScienceandTechnologyofChina(USTC)in2003.ThenhewenttoHKUSTforpursuingPhDinDepartmentofBiologyandgraduatedin2009.Afterwards,hejoinedDepart-mentofPhysicstodopostdoctoralresearch(February–October2009)undertheco-supervisionofProf.DonaldChoyChang(DepartmentofBiology)andProf.Wei-jiaWen(DepartmentofPhysics).ThenhejoinedmarinebiologyresearchgroupunderthesupervisionofProf.PeiyuanQianinDepartmentofBiologyofHKUST.Dr.Xiao’sresearchinterestincludestheapplicationofmicrofluidicsintobiologicalresearch,investigationsonmechanismsofapoptosisinmammaliancells,andlarvalsettlementanddevelopment.

JinboWureceivedhisBSdegreeinappliedchemistryfromShandongUniversity,Jinan,China,in2005.HecompletedhisMSdegreeinmaterialsscienceandengineer-ing(2007)inHKUST,.HecurrentlyworksasaPhDstudentatHKUST.Hisresearchinterestsincludemicro/nanofabrication,micro/nanofluidics,biochips.

XinYireceivedhisBSdegreeinphysicsfromtheBeijingNormalUniversity,China,2007.In2008,hejoinedtheNanoScienceandNanoTechnologyProgram,HKUST,andreceivedM.Phil.degreein2011.Hismainresearchareasaremicrofluidics,LOC(Lab-on-a-Chip),andbioMEMS.

XiuqingGongreceivedhisMSinanalyticalchemistryin2006fromtheUSTC.Since2006,hehasbeenaPhDstudentatHKUSTmajoringinmicrofluidicsscienceunder

thesupervisionofProf.WeijiaWen,wherehehasbeendoingresearchon3Dchipfabrication,organicorinorganicmaterialsynthesis,drugencapsulationandrelease,cellcultureandevolution,etc.HereceivedhisPhDdegreeinApril,2010andnowcontinueshispostdoctoralresearchinmicrofluidicareaatAndrewdeMello’sgroup(ImperialCollege,London,UK,EU).

IanG.Fouldsgraduatedin2007withhisPhDinelectricalengineeringfromSimonFraserUniversity,inBurnaby,BC,Canada.HewentontoserveinthemechanicalengineeringdepartmentatUniversityofVictoria,asapostdoctoralfellow,untilhejoinedKAUSTin2009asanassistantprofessorofelectricalengineering,inthedivisionofphysicalsciencesandengineering.Hisresearchinterestslayintheareaofmicrosystemsdesignandfabrication,withspecialemphasisonpolymerfabricationtechniques.

WeijiaWenearnedhisBS(1982)andMS(1988)degreesatChongqingUniversity.HecompletedhisPhD(1995)degreeinInstituteofPhysics,ChineseAcademyofScience,Beijing.HewasapostdoctoralfellowatHKUST(1995–1997)andUCLA(CaliforniaUniversityatLosAngeles)(1997–1999).HejoinedHKUSTin1999andcurrentlyisaprofessorintheDepartmentofPhysics,HKUST.ProfessorWen’smainresearchinterestsincludesoftcondensedmatterphysics,electrorheological(ER)andmagnetorheological(MR)fluids,field-inducedpatternandstructuretransitions,micro-andnano-fluidiccontrolling,microsphereandnanoparticlefabrications,thinfilmphysics,bandgapmaterials,metamaterialsandnonlinearopticalmaterials.