Hybrid Integrated Circuit/Microfluidic Chips. for the Control of Living Cells and Ultra Small. Biomimetic Containers

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1 HybridIntegratedCircuit/MicrofluidicChips forthecontroloflivingcellsandultra Small BiomimeticContainers ADissertationPresented by DavidAaronIssadore to TheSchoolofEngineeringandAppliedSciences Inpartialfulfillmentoftherequirements forthedegreeof DoctorofPhilosophy inthesubjectof AppliedPhysics HarvardUniversity Cambridge,Massachusetts May2009

2 2009byDavidIssadore Allrightsreserved.

3 DavidAaronIssadoreAdviser:RobertWestervelt HybridIntegratedCircuit/MicrofluidicChipsfortheControlof LivingCellsandUltra SmallBiomimeticContainers Abstract Thisthesisdescribesthedevelopmentofaversatileplatformfor performingbiologyandchemistryexperimentsonachip,usingtheintegrated circuit(ic)technologyofthecommercialelectronicsindustry.thiswork representsanimportantsteptowardsminiaturizingthecomplexchemicaland biologicaltasksusedfordiagnostics,research,andmanufacturinginto automatedandinexpensivechips. HybridIC/microfluidicchipsaredevelopedinthisthesisto simultaneouslycontrolmanyindividuallivingcellsandsmallvolumesoffluid. Takinginspirationfromcellularbiology,phospholipidbilayervesiclesareused topackageplvolumesoffluidonthechips.thechipscanbeprogrammedto trapandposition,deform,setthetemperatureof,electroporate,andelectrofuse livingcellsandvesicles.thefastelectronicsandcomplexcircuitryoficsenable thousandsoflivingcellsandvesiclestobesimultaneouslycontrolledonthechip, allowingmanyparallel,well controlledbiologicalandchemicaloperationstobe performedinparallel. iii

4 Thehybridchipsconsistofamicrofluidicchamberbuiltdirectlyontopof acustomic,thatusesintegratedelectronicstocreatelocalelectricandmagnetic fieldsabovethechip ssurface.thechipsoperateinthreedistinctmodes, controlledbysettingthefrequencyoftheelectricfield.electricfieldsatkhz frequenciesareusedtoinduceelectroporationandelectrofusion,electricfields atmhzfrequenciesareusedfordielectrophoresis(dep)totrapandmove objects,andelectricfieldsatghzfrequenciesareusedfordielectricheating.in addition,magnetophoresis,usingmagneticfieldscreatedwithdccurrentonthe chip,isusedtodeformandtopositionobjectstaggedwithmagnetic nanoparticles. TodemonstratethesefunctionstwocustomhybridIC/microfluidicchips andadropletbasedpdmsmicrofluidicdevicewithexternalelectronicsare presented.thelaboratoryfunctionsdemonstratedonthesechipsprovide importantbuildingblocksforaversatilelab on a chipplatformthatcanbebuilt onthewell developedictechnologyofthecommercialelectronicsindustry. iv

5 TableofContents Abstract iii Acknowledgements...vii AbbreviationsandSymbols.ix ListofTableandFigures... xi Chapter1.Introduction Lab on a Chip Motivation HybridIC/MicrofluidicChips Concept OverviewofThesis..7 Chapter2.Theory:DielectricandMagneticControlof MicroscopicObjects TheDielectricPropertiesofWaterandSolutions Dielectrophoresis DielectricModelsforCellsandVesicles TransmembranePotentialDifferenceanditsApplications DielectricHeating Magnetophoresis..34 Chapter3.HybridIntegratedCircuit/MicrofluidicChips Overview DielectrophoresisChip(Fabutron1.0) OperatingPrincipals CharacteristicTimes IntegratedCircuitDesign Capabilities ExperimentalApparatus Fluidics Electronics Optics ThermalManagement ComputerControl.54 Chapter4.AHybridIntegratedCircuit/MicrofluidicPlatformto ControlLivingCellsandpLBiomimeticContainers Overview Methods TheHybridIntegratedCircuit/Microfluidic ChipPlatform UnilamellarVesicles 60 v

6 4.2.3DielectrophoresisofVesicles SuspendedinWater ElectroporationandElectrofusion Demonstrations TrappingandMovingCellsandVesicles TriggeredReleaseoftheContentsofVesicles ElectroporationofCells TriggeredFusionofVesicles DeformingVesicleswithDielectrophoresis Discussion. 76 Chapter5.HybridMagneticandDielectrophoretic IC/MicrofluidicChip Overview DescriptionoftheChip FieldSimulations ChipArchitecture Demonstrations Dielectrophoresis:Trapping andpositioningvesicles Magnetophoresis:Trapping andpositioningmagneticbeads DielectrophoresisandMagnetophoresis: DeformingVesicles Discussion. 93 Chapter6.MicrowaveDielectricHeatingof DropsinMicrofluidicDevices Overview ModelofDielectricHeatingofDrops TheMicrofluidicDevice Demonstration Discussion. 115 Chapter7.Conclusions Summary FutureDirections. 119 WorksCited AppendixA.DataSheetandUsersGuidefor thedepchip(fabutron1.0) AppendixB.DataSheetandUsersGuidefortheHV DEP MagneticChip(Fabutron2.0) AppendixC.FabutronControlSoftware vi

7 Acknowledgements FirstIwouldliketoexpressmyappreciationandgratitudeformyadviser,Bob Westervelt.Bob sdeepunderstandingofphysicsandhisuniqueandoftentimes unusualperspectiveonresearch,science,andtechnologyhavechallengedme intellectuallyandhelpedmedevelopasascientist.hehasbeenaconstant sourceofinsightfulideasandthoughtfulfeedbackandhasgivenmethesupport andfreedomthatineededtodrivemyownresearch.iamverygratefulthati endedupinhislab.itseemsthatiwasverylucky. IwouldliketothanktheWesterveltLabfolksthatIhavehadthepleasureto workwithandsharespacewithoverthelast5years.firstandforemost,i dlike tothanktomhunt.tomhelpedmefinddirectioninmyresearchandkeptme ontrackwhenmyresearchwaslackingfocus.healsotaughtmethepleasuresof roadbiking.i dliketothankkeithbrownwhojoinedthegrouptwoyearsafter me.keithhasbeengreattoworkwith,heprovidedmewiththekeyfeedback thatisooftenneededandhisearnestenthusiasmandscientificrigoroften improvedmywork.also,heisanadmirablewormsplayer.jonathan,ognjen, Lori,andAlmalchiandtherestoftheundergradsthathaveworkedtheirway throughthewesterveltlabhavekeptlifeinterestingandwereapleasureto workwith.erin,halvar,andjesseareawesomepeopletosharealabwithandi amgratefultothemforkeepingmesomewhatknowledgeableaboutcold temperaturephysics.alex,nan,andtinaarenewcomerstothegroupandseem tobekeepingthegoodtraditionsalive. vii

8 IwouldalsoliketothankthepeopleIhadthepleasuretocollaboratewith outsideofthewesterveltgroup.thomasfrankeatuniversityofaugsburgin Germany sexpertiseinpreparingvesicleswaskeytothesuccessofthisthesis. KatieHumphryintheWeitzGroup sabilityinsoftlithographywasessentialfor themicrowavedropheatingproject.rickrogersandrosalindasepulvedaatthe SchoolofPublicHealthprovidedcellsandgreatadviceonbiologyformanyof theprojectsinthisthesis. The5yearsthatI vespentincambridgehavebeenavariedlot,andiowealotto myfriendsandfamilyforgettingmethroughitinonepiece.firstandforemost, myparentshavealwayssupportedmeandhavebeensupremelyunderstanding, evenattimeswheni msureiwasintolerable.i dliketothankmybrotherfor alwaysbeingsupportive,mygrandmomchipforremindingmewhat s importantinlife,andtheoforgenerouslysharingwithmehisenlightened philosophyonlife. Alan,myroommateof5years,isresponsibleformyeducationonallofthetopics thatiwasn tlearninginschoolsuchastheworkingsofevilhedgefunds, bosanovamusic,anddjango.mikewasafantasticadditiontomagdaddyand significantlyimprovedourqualityoflife.benandclemensbothmightaswell havelivedwithusandaregreatfriends.andfinally,imani.it dbeinappropriate towritehereallthatiamthankfulforaboutyou.but,iamverygratefulandmy lifeissomuchfullerbecauseofyou. viii

9 AbbreviationsandSymbols a Particleradius B MagneticField C Capacitance Cmem CM χ CMOS Specificmembranecapacitance ClausiusMosottiFactor MagneticSusceptibility ComplimentaryMetalOxideSilicon D DiffusionConstant DC DEP ΔT E Directcurrent Dielectrophoresis ChangeinTemperature ElectricField ε RelativePermittivity ε o ε' ε'' VacuumPermittivity RealComponentofthePermittivity ImaginaryComponentofthePermittivity f Frequency F Force gm GUI HV SurfaceConductivity GraphicalUserInterface HighVoltage η Viscosity I/O IC j 1 kb Input/Output IntegratedCircuit Boltzmann sconstant L Thelengthofapixel ix

10 LD CharacteristicLengthintheHeatingModelforDrops MOSIS MetalOxideSemiconductorImplementationService MUX Multiplexer n Concentration NMR NuclearMagneticResonance PBS PhosphateBufferSaline R Resistance Re ReynoldsNumber RF RadioFrequency ROI RegionofInterest SPICE SimulationProgramwithIntegratedCircuitEmphasis SRAM StaticRandomAccessMemory σ Conductivity T Temperature ΔTSS τdiff τmem τmove τss τmw τw SteadyStateChangeinTemperature CharacteristicTimeforanObjecttoDiffuseaHalfPixellengthL/2 CharacteristicChargingTimeofaVesicleorCell smembrane CharacteristicTimetoMoveanObjectaPixelLengthL CharacteristicTimetoReachSteadyStateTemperature DielectricRelaxationTimeofavesicleorcell DielectricRelaxationTimeofWater µ o VacuumPermeability V Volume V Voltage VTM ω TransmembraneVoltage AngularFrequency x

11 ListofFigures Figure1.1aJackKilby sic...6 Figure1.1bAnIntelQuad CoreIC Figure1.1cASimpleMicrofluidicChip...6 Figure1.1dAComplexMicrofluidicChip..6 Figure1.1eAHybridIC/MicrofluidicChip.6 Figure1.2TheDEPChipSpelling LabonaChip withyeastcells...9 Figure1.3aTheDEPHybridIC/MicrofluidicChip Figure1.3bTheHighVoltageDEP/MagneticChip Figure1.3cTheMicrowaveDielectricDropHeatingChip Figure2.1FrequencyDomainPlotoftheEfficacy ofdep,electroporation,anddielectricheating Figure2.2aPermittivityofWatervs.Frequency...21 Figure2.2bPermittivityofAqueousSolutionswithVarying Conductivityvs.Frequency Figure2.3aLumpedCircuitModelforthePermittivityofaVesicle. 27 Figure2.3bClausius Mosottivs.InteriorConductivityforaVesicle Figure2.3cClausius Mosottivs.frequencyforaVesicle.. 27 Figure2.4aTransmembraneVoltagevs.FrequencyforaVesicle.. 31 Figure2.4bSchematicofElectrofusion..31 Figure2.5DielectricHeatingPowerDensityvs.Frequencyfora SolutionwithVaryingConduct Figure3.1aPlotofSimulatedElectricFieldStrength AboveTheDEPChip.40 Figure3.1bPlotoftheForceExperiencedbya VesicleonTheDEPChip...40 Figure3.2aSchematicoftheArchitectureoftheDEPChip xi

12 Figure3.2bSchematicofanindividualDEPPixel 45 Figure3.2cMicrographoftheDEPArray,ShiftRegister, androwdecoder...45 Figure3.3aDemonstrationofTrappingandPositioning YeastontheDEPChip.47 Figure3.3bDemonstrationofTrappingandPositioning DropsofWaterinOilontheDEPChip...47 Figure3.3cTheDEPChipSpelling Harvard withyeastcells...47 Figure3.4aFlowChartoftheExperimentalApparatusSurrounding thehybridic/microfluidicchips..53 Figure3.4bMicrographoftheDEPChip sic..53 Figure3.4cPhotographoftheHybridIC/MicrofluidicChip initschipcarrier Figure3.4dPhotographoftheExperimentalApparatus SurroundingtheHybridIC/MicrofluidicChips. 53 Figure3.5ScreenShotsoftheGUIthatControlsthe HybridIC/MicrofluidicChip.55 Figure4.1ASchematicofUnilamellarVesiclesandEmulsions 59 Figure4.2aPlotoftheConcentrationGradientofaSubstance ReleasedfromaVesicle...66 Figure4.2bPlotoftheConcentrationGradientofaSubstance ConsumedontheSurfaceofaCellorVesicle Figure4.3aDemonstrationofIndependentlyTrappingand MovingSeveralVesiclesontheDEPChip Figure4.3bTheDEPChipDrawing H withvesicles.. 69 Figure4.3cSimultaneouslyTrappingandMoving YeastCellsandVesiclesontheDEPChip...69 Figure4.4aTriggeringtheReleaseoftheContents ofavesicleonthedepchip Figure4.4bElectroporatingaYeastCellontheDEPChip...73 Figure4.4cElectrofusingTwoVesiclesontheDEPChip.. 73 xii

13 Figure4.5DeformingVesiclesontheDEPChip Figure5.1aMicrographoftheHV DEP/MagneticIC 83 Figure5.1bPlotoftheSimulatedElectricFieldStrengthAbove TheHV DEP/MagneticChip..83 Figure5.1cPlotoftheSimulatedMagneticFieldStrengthAbove TheHV DEP/MagneticChip.. 82 Figure5.2aMicrographoftheHV DEP/MagneticIC,showing thecircuitarchitecture..85 Figure5.2bSchematicofaDEPPixelontheHV DEP/MagneticChip...85 Figure5.2cSchematicofaMagneticWireonthe HV DEP/MagneticIC Figure5.3TrappingandMovingVesicles onthehv DEP/MagneticChip Figure5.4TrappingandMovingMagneticBeads onthehv DEP/MagneticChip Figure5.5DeformingVesiclesontheHV DEP/MagneticChip Figure6.1aSchematicofMicrofluidicDielectricHeater..102 Figure6.1bPlotofElectricFieldSimulationsforthe MicrofluidicDielectricHeater 102 Figure6.1cCalibrationCurvefortheCdSeTemperatureSensor Figure6.2aFlowDiagramoftheMicrowaveDielectricHeater Figure6.2bMicrographoftheDropMaker..107 Figure6.2cMicrographoftheDropSplitter 107 Figure6.2dMicrographoftheMicrowaveHeatingDevice Figure6.2ePhotographoftheExperimentalSetupofthe MicrowaveDielectricDropHeater Figure6.3aFluorescenceImageofDropsHeating..110 Figure6.3bLine averageoffluorescenceintensityvs.distance Figure6.3cPlotofChangeinTemperaturevs.DistanceandTime Figure6.4aPlotofChangeinTemperaturevs.MicrowavePower. 114 xiii

14 Figure6.4bPlotofLog LinearplotofChangein Temperaturevs.Time.114 ListofTables Table1.1ListofFunctionsthatarePerformedinthisThesis Table7.1Lab on a ChipFunctionsDemonstratedinthisThesis xiv

15 Chapter1.Introduction Lab on a chip:atechnologythatminiaturizesandintegratesthecomplexchemical andbiologicaltasksusedfordiagnostics,research,andmanufacturingonto automated,portable,andinexpensivechips. 1.1Lab on a Chip Motivation Amajorchallengeofthe21 st centuryistobetterdiagnoseandtreatinfectious diseaseforthelargeportionoftheworld spopulationthatiscurrentlyunderserved. Diseases,suchasHIV/AIDS,Tuberculosis,andMalaria,forwhichthereexists interventions,continuetokillmillionsandinfectmillionsmoreeachyeardue,in part,toourinabilitytodiagnosediseaseseffectivelyinpoorcountries.(urdeaetal., 2006)TheBillandMelindaGatesFoundationandtheNationalInstituteofHealth havedeclaredtheassessmentofdiseaseinpoorcountriesagrandchallengefor PublicHealth.Thesechallengesread:(Varmusetal.,2003) 1. Developtechnologiesthatpermitquantitativeassessmentofpopulationhealth statistics. 2. Developtechnologiesthatpermitquantitativeassessmentofindividualsfor multipleconditionsorpathogensatpointofcare. Withthesegoalsinmind,researchersaroundtheworldaredevelopingtechnology tomeasurebiologicalandchemicalmarkersforinfectiousdiseaseininfrastructurepoorpartsoftheworld.(ahnetal.,2004,chinetal.,2007,andmartinezetal.,2008) Thelaboratoriesthatperformthechemicalandbiologicalteststodiagnosedisease 1

16 intheworld swealthiernationsrequireresourcesthatarenotreadilyfound elsewhere,suchascentrallylocatedairconditionedbuildings,cleanwater, electricity,accesstoexpensivereagents,andwell trainedtechnicians.(chinetal., 2007)Assuch,technologytoassessdiseaseinpoorcountriesfaceschallengesnot ordinarilyfoundinamodernlaboratoryspace. Lab on a chiptechnologyoffersapowerfultooltobringexistingmedicaland environmentaltestsfromthelaboratoryintothefieldandtheclinic.(figeysetal., 2000,Petraetal.,2006,Chinetal.,2007)Attheforefrontofthistechnologyarethe micro fabricatedpipes,valves,pumps,andmixersofmicrofluidicsthatareleading tointegratedlab on a chipdevices.(whitesidesetal.,2001andstoneetal.,2004) Inadditiontothepotentialforlow costmedicine,theminiaturizationofthe handlingofliquidandbiologicalsampleshasenabledadvancesinfieldssuchas drugdiscovery,geneticsequencingandsynthesis,cellsorting,andsinglecellgene expressionstudies.(tabelingetal.,2005,yageretal.,2006,andmartinezetal., 2008)Theseintegratedmicrofluidicdevicesareleadingaparadigmshiftinfluid handlingthatisanalogoustowhatintegratedcircuitsdidforelectronicshalfofa centuryago.(leeetal.,2007)however,alab on a chipthatcanperformthe complex,multi stepexperimentsthatarecurrentlyperformedinlaboratories,akin toamicroprocessorforfluids,remainsachallenge.(leeetal.,2007) Thepotentialimpactofalab on a chipmicroprocessorcouldbeenormous.imagine adevicethesizeofanipodthatcostlessthan$100andcanperformnumerous complexbiologicalandchemicaltestsonsmallsamplesofbodilyfluids,drinking 2

17 water,orair.suchadevicewouldmakemedicalandenvironmentaltesting inexpensive,portableandautomated.testingcouldbeperformedinthefield,at home,orataclinicbyanon expert.theresultsoftestswouldbequantitativeand consistentsuchthatrelevantdatacouldbecollectedforbothpersonalandpublic healthstatistics.(chinetal.,2007)and,thedevicecouldeasilybeconnectedtothe Internetsuchthatrelevantdatacouldbeshared.Suchadevicecouldfundamentally changethewaythatpeopleinteractwiththechemicalandbiologicalinformationof theirsurroundingsandoftheirownbodies. Inadditiontoitsapplicationsinthedevelopingworld,lab on a chipdeviceshavea bigroletoplayinthefutureofhealthcareintheunitedstates.currently,15.2%of theunitedstate sgdpisspentonhealthcareandthatfractionisexpectedtogrow to19.5%by2020.(u.s.departmentofhealthandhumanservices,2007)this amountofspendingisbelievedtobeunsustainable,especiallyasthepopulation growsandages.(keehanetal.,2008)lab on a chipdevicescanbeusedtolower thepriceofmedicaldiagnosticsandmonitoring,enablingdiseasestobedetectedin theirearlierstageswheretreatmentiseasierandfarlessexpensive.(tudosetal., 2001) Inthisthesisalab on a chipplatformisdevelopedthatcancontrolsinglecellsand verysmallvolumesoffluidtoperformsimultaneous,programmableexperimentson achip.incontrasttotechnologythatattemptstomakelab on a chipdevicesultralowcostbybeinglowtech,(martinezetal.,2008)thechipsdescribedinthisthesis 3

18 usecutting edgetechnology.thechipsremaininexpensive,however,byusingthe integratedcircuit(ic)technologyofthecommercialelectronicsindustry. 1.2HybridIC/MicrofluidicChips Concept Integratedcircuitsarecentraltomanyofthetechnologicalwondersofthe21 st century.builtonaslabofcrystallinesiliconnobiggerthanasquarecentimeterin area(roughlythesizeofaquarter)aniccontains100sofmillionsoftransistorsand amazeofwiresthatconnectthetransistorsintocomplexcircuitstoperformbillions ofoperationspersecond.thetransistorsandwiresoficsarefabricatedwithnanolithography(asopposedtooneatatime),whichallowicstobecomplex,small,fast, andalso,inexpensive.(lee,1998)integratedcircuitsareubiquitousintoday s technology.theyarethemicroprocessorsincomputers,theradiofrequency(rf) circuitsincellphones,thefilmindigitalcameras,andthemicrocontrollersinheart patients pacemakers.integratedcircuits,havingbeeninventedonly50yearsago, haveprofoundlychangedthewayhumansuse,store,andcommunicateinformation. InFig.1.1atheoriginalICisshown,builtbyJackKilbyin1958,itcontainsonlya singletransistor.(kilby,1976)infig.1.1bamodernpentiumquad coreic,thatcan befoundintoday slaptopsisshown,itcontains820milliontransistors.(intel) Inaspiritanalogoustotheminiaturizationofelectronicsthatleadtotoday sics, modernresearchersareminiaturizingthefluid handlingtoolsofbiologyand chemistrylaboratoriesintosmall,inexpensivechipstoperformautomated experiments.agrowinglibraryofmicrofluidicelementsforlab on a chipsystems havebeendevelopedinrecentyearsfortaskssuchasthemixingofreagents, 4

19 detectingandcountingcells,sortingcells,geneticanalysis,andproteindetection. (Whitesidesetal.,2001,Stoneetal.,2004,Tabeling,2005,andYageretal.,2006) InFig.1.1catypicaltwo channelglassmicrofluidicchipusedtocontrollablymix twosubstancestogetherfrommicronitcorp.isshown.figure1.1dshowsan exampleofoneofthemorecomplexmicrofluidicchipsbuilttoday,fromthequake GroupatStanford,thatconsistsofhundredsofpneumaticallycontrolledgatesand valvesandisusedtoperformgeneticanalysisonmicrobesinthehumanmouth. (Marcyetal.,2007) Thecomplexity,smallfeaturesize,andlowcostofICscanbeappliedtobiological andchemicalapplicationsbycombiningicswithmicrofluidicstoformhybridic/ microfluidicchips.complimentary metal oxide semiconductor(cmos)optical sensorshavebeencoupledwithmicrofluidicstomakea microscopeonachip that achievesenhancedresolutionandsensitivitybybringingmicroscopicobjects directlytotheopticalsensors.(eltoukhy,etal.,2006andcuietal.,2008)electrical sensorarrayshavebeenbuiltonic/microfluidicchipstostimulateandmeasure largearraysofindividualneuralandcardiaccells.(debusscherreetal.,2001and Eversmannetal.,2003)And,hybridIC/microfluidicchipshavebeenusedtotrap andmovedielectric(gascyoneetal.,2004andhuntetal.,2008)andmagnetic objects(leeetal.,2006)alongprogrammablepathsforchemistryandbiology experimentsonachip.figure1.1eshowsahybridic/microfluidicchipthattraps andmovesobjectsalongarbitrarypathswithdepusingalargearrayofpixels.the theoreticalframeworkforhowdepisusedtotransportcellsandvesiclesonachip isdetailedinchapter2ofthisthesis. 5

20 Figure1.1(a)TheoriginalICbuiltbyJackKilbyatNationalInstrumentsin 1958(Kilby,1976)(b)amicrographoftheIntelquad coreprocessor,whichhas 820milliontransistos(IntelCorp.),(c)atwochannelglassmicrofluidic chip(micronitcorp.),(d)anexampleofacomplexmicrofluidicdevicethatconsists ofhundredsofpneumaticallycontrolledgatesandvalvesandisusedtoperform geneticanalysisonmicrobesinthehumanmouth,(marcyetal.,2007)(e)a photographofanic/microfluidicchip,the DEPChip, andamicrographofthe arrayofpixelsthatareusedtotrapandmoveobjectswithdielectrophoresis(dep). (Chapter3) 6

21 1.3OverviewofThesis Thisthesisdescribesthedevelopmentofaversatilelab on a chipplatformusing hybridic/microfluidicchips.thechipsperformawiderangeoffunctionsonliving cellsandplbiomimeticcontainersforbiologyandchemistryexperimentsonachip. Previousworkhasbeendonedevelopingprogrammablelab on a chipplatformsto controlsmallvolumesoffluidandcells.pneumaticcontrolhasbeenusedtocreate reconfigurablemicrofluidiccomponentssuchasvalves,latches,pumps,and multiplexers.(ungeretal.,2000)recently,similarstructureshavebeendeveloped thatreplacethecumbersomepneumaticlineswithelectronicallyactivated componentsthataremadeofshapememoryalloys(smas)andwhichcanbebuilt ontopofcommercialprintedcircuitboards(pcbs).(vyawahareetal.,2008) Dropletshavebeentrapped,moved,mixed,andseparatedusingboth electrowetting(leeetal.,2002andpollacketal.,2002)anddielectrophoresis (DEP)(Vykoukaletal.,2001andPeteretal.,2004)withelectronicsthatareexternal tothefluidicsystem.hybridintegratedcircuit(ic)/microfluidicchipshavebeen developedthatharnessthematuretechnologyoficstomakegeneralpurpose, programmablefluid handlingsystems.(leeetal.,2007,gascyoneetal.,2004,hunt etal.,2008)thehighlylocalizedelectricandmagneticfieldsthatcanbecreatedby ICshavebeenusedtotrapandmovesmallvolumesofwatersuspendedinoil(Hunt etal.,2008),livingsinglecellssuspendedinwater (Gascyoneetal.,2004,Huntetal., 2008),andmagneticallytaggedbiologicalobjectssuspendinwater(Leeetal.,2007) alongprogrammablepaths.figure1.2showsahybridic/microfluidicchip 7

22 simultaneouslypositionthousandsofyeastcellswithdeptospell LabonaChip. ThechipshowninFig.1.2isdescribedinfulldetailinChapter3ofthisthesis. 8

23 Figure1.2ThecoverofLabonaChipfeaturingourhybridIC/microfluidicchip simultaneouslypositioningthousandsofyeastcellswithdeptospell Labona Chip. 9

24 Thereareseveralkeyfunctionsthatarenecessarytoperformexperimentsonachip thatareanalogoustotypicallaboratoryprocesses.anyworkingmedicalor researchlaboratoryhasbasicequipment:testtubestokeepssamplesseparate, pipettestomovesamplesaroundthelaboratory,mixerstobringsamplesand reagentstogether,andheaterstocontrolthetemperatureofexperiments.inthis thesisbasiclaboratoryfunctions,aswellasseveralfunctionsthatarenotpossiblein amacro scalelaboratory,areperformedonic/microfluidicchipsusingelectricand magneticfieldscreatedabovetheic ssurface.intable1.1thefunctionsperformed onhybridic/microfluidicchipsinthisthesisarelisted. Reagentsandsamplesarekeptseparated,suchthattheycanbetransportedaround the laboratory andmixedatthepropertimeinanexperiment,bypackagingthem inplcontainersasisdemonstratedinchapter4.inspirationistakenfromcellular biologyandphospholipidbilayervesiclesareusedtopackageplvolumesoffluidon thechip.vesiclesarecommonlyusedincellsforpackagingquantitiesofsubstances forintercellulartransport,tostoreenzymes,andasareactionchamber.(albertset al.,2007)unilamellarvesiclesmadewithelectroformationproviderobustpl containersthatareimpermeableandstableforawiderangeofsalinity,ph,and otherenvironmentalconditions.(chiuetal.,1999) Vesiclesandcellsaretransportedacrossthe laboratory usingdepwithelectric fieldsatmhzfrequenciesabovethechip ssurface,asisdemonstratedinchapters3, 4,and5.Thesmallfeature sizeoficsallowsmicrometer sizedvesiclesand individuallivingcellstobeindependentlytransported.thefastelectronicsand 10

25 complexcircuitryoficsallowthousandsoflivingcellsandvesiclestobe simultaneouslytrappedandmovedonthechip,allowingmanyparallel,wellcontrolledbiologicalandchemicaloperationstobeperformedinparallel.the theoreticalframeworkfordepisintroducedinchapter2. Vesiclesandcellsarecontrollablypermeabilized,fused,andreleasedusingkHz frequencyelectricfieldsabovethechip ssurface,asisdemonstratedinchapter4. Thevesiclescanbetriggeredtoreleasetheircontentslocallyintothesolutionand mixtheircontentswithothervesiclesusingelectricfieldscreatedbythechip. ElectricfieldsatkHzfrequenciesinducevoltagesacrossthevesicle smembrane inducingelectroporationorelectrofusion.electroporationcanalsobeperformed onlivingcells,inducingthecellstotake upsubstancesfromthesolution.the complexcircuitryofthechipallowsspecificvesiclesorcellstobetargetedfor electroporationorelectrofusionwithoutharmingsurroundingcellsorvesicles.the chipcantime multiplexthekhzfrequencyelectricfieldswiththemhzfrequency electricfieldsusedfordep,suchthatobjectsremaintrappedinplace,astheyare electroporatedorelectrofused.thetheoreticalframeworkforelectroporationand electrofusionisexplainedinchapter2. AhybridchipthatcansimultaneouslyperformDEPandmagnetophoresisis demonstratedinchapter5.magnetophoresis,usingmagneticfieldscreatedonthe chip,providesacomplimentarymethodtodepfortransportingsubstancesacross the laboratory. Theabilitytotrapandmovemagneticobjectsalongprogrammed 11

26 pathsisausefultooltocontrolthepositionofobjectsthatcannotbetrappedwith DEP,butwhichcanbetaggedwithmagneticparticles.(Leeetal.,2007) Themechanicalenvironmentofindividualcellsandvesiclescanbedefinedusing thechip,afunctionalitythatisnotpossiblewithmacro scalelaboratorytools,asis demonstratedinchapters4and5.in vitroexperimentsoftensufferfornot controllingthemechanicalenvironmentofcells,anaspectthatplaysanimportant rolein vivo.(seifritz,1924,curtisetal.,1964andwangetal.,1994)severaldep pixelscanbepatternedunderneathasinglevesicleorcelltocontrolitsmechanical environmentbychangingtheshapeofthedeptrap,asisdemonstratedinchapter4 withunilamellarvesicles.magneticfields,createdwithdccurrent,areusedtotrap andmovemagneticallypermeableobjectssuchasironoxidenanoparticles.these particlescanbeimplantedintovesiclesorlivingcellstoapplylocalforces selectivelytothelocationofthenanometersizedparticles.thistechniqueis demonstratedbycontrollablydeformingvesiclesimplantedwithironoxide nanoparticleswithmagneticfieldscreatedbythechipinchapter5. Thetemperatureofsmallcompartmentsoffluidcanbelocallyandrapidly controlledusingdielectricheatingwithelectricfieldsatghzfrequencies,asis demonstratedinchapter6.electricfieldswithafrequencyf=3ghzareusedto heatthermallyisolatedpldropswithdielectricheating.changesintemperature ΔT=0 30 Careachievedinacharacteristictimeτs=15ms. 12

27 Table1.1AlistofthefunctionalitiesdemonstratedonthehybridIC/microfluidic chipsinthisthesis. 13

28 ThreedevicesaredescribedinthisthesisandareshowninFig.1.3.InChapter3a customic/microfluidicchipthatconsistsof128x256(32,768)11x11µm 2 pixels, eachofwhichcanbeindividuallydrivenwith5vpeak to peakradiofrequency(rf) voltageswithfrequenciesfromdcto11mhz,(hunt,etal.,2007)isdevelopedintoa lab on a chipplatform.thischipiscalledthedepchip(fabutron1.0)anda micrographofitisshowninfig.1.3a. InChapter5asecondcustomIC/microfluidicchipispresentedthatcantrapand moveobjectswithbothdepandmagneticforces.thechipsconsistsof 60x61(3,660)30x38µm 2 pixels,eachofwhichcanbedrivenwitha50vpeak topeakrfvoltagewithfrequenciesfromdcto10mhz.underneaththedeppixel array,thereisamagneticgridthatconsistsof60horizontalwiresand60vertical wiresrunningacrossthechip,eachofwhichcanbesourcedwith120matocreate localmagneticfields.thischipiscalledthehv DEP/MagneticChip(Fabutron2.0) andamicrographofitisshowninfig.1.3b. InChapter6APDMSbasedmicrofluidicdevice,fabricatedwithsoft lithography,is usedtodemonstraterapidandlocaldielectricheatingofdrops.thedeviceconsists ofanintegratedflow focusingdropmaker,dropsplitters,andmetallineelectrodes tolocallydelivermicrowavepower.thischipiscalledthemicrowaveheateranda photographofitisshowninfig.1.3c. 14

29 Figure1.2(a)AmicrographoftheDEPChip(Fabutron1.0),(b)amicrographofthe HV DEP/MagneticChip(Fabutron2.0),(c)aphotographofthePDMSmicrofluidic device(microwaveheater)usedtodemonstratemicrowavedielectricheatingof drops. 15

30 Chapter2.Theory:DielectricandMagnetic ControlofMicroscopicObjects 2.1Overview Thischapterdescribeshowelectricandmagneticfieldsareusedtocontrol microscopicobjectsonhybridintegratedcircuit(ic)/microfluidicchips.electric fieldsatkhzfrequenciesandbelowareusedtoinduceelectroporationand electrofusion.electricfieldsatmhzfrequenciesareusedfor dielectrophoresis(dep).electricfieldsatghzfrequenciesareusedfordielectric heating.and,magneticfieldscreatedwithdcelectricalcurrentsareusedtotrap andmoveobjectswithmagnetophoresis. Thefrequencydependenceofthedielectricpropertiesofvesicles,cells,and solutionsenablethedielectricphenomenonusedinthisthesistobeindependently accessedwithelectricfieldsatdifferentfrequencies.thefrequencydependenceof DEP,electroporation,anddielectricheatingisshowninFig.2.1.Thestrengthof eachoftheseeffectsisplottedinunitlessvaluesthataredescribedindetaillaterin thischapter.atthemhzfrequencieswheredepworksbest,bothheatingand electroporationisminimal,enablingvesiclesandcellstobetrappedandmoved withoutdamage.attheghzfrequencieswhereheatingworksbest,both electroporationanddepareminimal,enablingcellsandvesiclestobeheated withoutinadvertenttrappingordamage.atthekhzfrequencieswhere electroporationandelectrofusionworkbest,heatingisminimalandthedepforceis 16

31 negative,enablingvesiclesandcellstobepermeabilizedandfusedwithout significantheatingorinadvertenttrapping. Figure2.1.Asemi logplotoftheefficacyofheatingp/pmax(red),dielectrophoresis CM(ω)(green),andelectroporationV TM /V max TM (blue)versusthefrequencyfofthe appliedelectricfield.thethreefrequencydomainsthatareusedfor electroporation,dep,anddielectricheatingarelabeled. 17

32 Inthischapterthevariousdielectricandmagneticphenomenonthatareusedto controllivingcellsandvesiclesonourchipsareexplained.insection2.1water s uniquedielectricproperties,whichplayanimportantroleinourabilitytocontrol objectswithelectricfields,areintroduced.insection2.2dielectrophoresis,the forcethatisusedtotrapandmoveobjectswithelectricfields,isintroduced.in Section2.3ageometriclumped circuitmodelisdevelopedtodescribethedielectric propertiesofcellsandvesicles.insection2.4thislumped circuitmodelisusedto explaintheinducedvoltagethatformsacrossthemembranesofcellsandvesicles, whichisutilizedinthisthesisforelectroporationandelectrofusion.insection2.5 dielectricheatingwithmicrowavefrequencyelectricfieldsisintroduced.and finally,insection2.6magnetophoresisisintroducedasamethodtocompliment DEPfortrappingandmovingobjects,utilizingmagneticdipolesinsteadofelectric permittivity. 2.1TheDielectricPropertiesofWaterandSolutions Tounderstandthedielectricpropertiesofcellsandvesiclesitisnecessarytofirst understandthedielectricpropertiesoftheirmostimportantingredient,water. Wateristheprimaryconstituentofcellsandisthemostcommonlyusedsolventin chemicalandbiochemicalexperiments.thedcdielectricconstantofwaterεs=78ε0 (att=25 C)isverylargecomparedtothatofmostotherorganicandinorganic solventsεs<10ε0,duetoitspermanentmoleculardipolemoment.waterwill typicallydominatethedielectricpropertiesofanyobjectthatismadeofit.(murrell etal.,1994) 18

33 Thedielectricresponseofwatervarieswiththefrequencyoftheappliedelectric field.(grant,etal.,1978)thepermittivityisdescribedbyboththemagnitudeand thephaseofthepolarizationrelativetotheappliedfieldandcanberepresentedasa complexfunctionε(ω) =ε (ω)+jε (ω),witharealcomponentε andanimaginary componentε.figure2.2ashowsaplotoftherealε andimaginaryε components ofthepermittivityofwaterplottedversusfrequency.therealcomponentofthe permittivityofwaterε hasaconstantvalueofεs=78ε0untilacornerfrequency definedbythedielectricrelaxationofwater1/(2π τw) 18GHz,atwhichpointthe permittivitymonotonicallydrops.theimaginarycomponentε approacheszero everywhere,exceptataresonantfrequencydefinedbythedielectricrelaxationtime ofwaterτw.thepermittivityofwatercanbedescribedbyanequationwithadebye form:(a.stogryn,1971) ε W = ε + ε ε s 1 jωτ 2.1 W whereεs=78.4ε0isthelowfrequencydielectricconstantofwater,ε =1.78ε0isthe opticaldielectricconstant,andτw=9.55psisthedielectricrelaxationtimeat T=25 C.Thedielectricrelaxationtimeτwcanbeunderstoodasthecharacteristic timethatittakesforwatermoleculestorealignthemselvestoaninstantaneous appliedelectricfield. Thedielectricresponseofwaterdependsontheconcentrationofionsinthe solution.dissolvedionschangetheconductivityofthesolutionσ byactingasfree 19

34 chargecarriers.theeffectofionsinthesolutioncanbeincorporatedintothe permittivityofthesolution:(a.stogryn,1971) ε W = ε + ε ε s + j σ 1 jωτ W ε o ω 2.2 Figure2.2bshowsaplotoftheimaginarycomponentofthepermittivityε plotted versusfrequencyonalog logscaleforsolutionswithseveraldifferent conductivitiesσ.forsolutionswithafiniteconductivity,theimaginarycomponent ofthepermittivityε decreaseswithincreasingfrequencyuntilacharacteristic frequency1/τi.thecriticalfrequency1/τ 1 isdefinedbythedielectricrelaxation timeτi=εs/σthatarisesfromconductivityofthesolutionσandthelowfrequency dielectricconstantofthesystemεs.astheconductivityofthesolutionσis increased,thecharacteristicfrequency1/τiispushedtohighervalues,ascanbe seeninfig.2.1b.forlowfrequenciesω<1/τ1theimaginarycomponentofthe permittivityε =σ/ωε0isafunctionofonlytheconductivityσ andnoothermaterial propertiesofthesolution.inthelimitofwaterhavingzeroconductivityσ=0the imaginarycomponentofthepermittivityε increasesmonotonicallywithfrequency untilthecharacteristicfrequencyofwaterτw.therealpartofthedielectric constantisslightlyreducedwiththeadditionofelectrolytes,aswaterboundtothe ionshaveasmallerdielectricresponsethanfreewatermolecules.(a.stogryn, 1971) 20

35 Figure2.2.(a)Therealε andimaginarypartsε ofthepermittivityofwaterεw plottedversusfrequency,(b)theimaginarycomponentε ofthepermittivityof solutionsofwaterplottedversusfrequencyforvariousconductivitiesσ.the conductivityσ increasesgoingfromlefttoright(asisshownwiththeblackarrow) withvaluesof0s/m,0.03s/m,0.06s/m,0.14s/m,0.3s/m,0.62s/m. 21

36 2.2Dielectrophoresis Dielectrophoresisistheinducedmotionofadielectricobjectinanon uniform electricfield.anyobjectwithapermittivitythatisdifferentthanthesurrounding mediumcanbecontrolledwithdep.thedepforceonasphericalobjectis:(jones, 1995) F DEP = 2πε m a 3 CM(ω) 2 E RMS 2.3 whereaistheradiusoftheparticle,εmisthedielectricconstantofthemedium,and CM(ω)istheClausius Mosottifactor,arelationbetweenthefrequencydependent complexpermittivityoftheparticleandthemedium. % CM(") = Re # p $# m ( ' * &# p + 2# m ) 2.4 whereεpisthecomplexdielectricconstantoftheparticle.whencm(ω)<0,the mediumismorepolarizablethantheparticleandtheparticleispushedawayfrom thelocalmaximumoftheelectricfieldandthisiscallednegativedep.positivedep occurswhentheparticleismorepolarizablethanthefluidcm(ω)>0andthe particleispulledtowardthemaximumoftheelectricfield.theclausius Mosotti factorcm(ω)variesbetween 0.5and1. Themotionofmicroscopicobjectsisopposedbytheviscousdragofthesolutionon theparticle.atmicrometerlengthscalesinertiaisverysmallcomparedtoviscous drag(thereynoldsnumberre 10 3 ).Theviscousdragonaspherecanbe describedwithastokesdragforce F drag = 6πηa v whereηisthedynamicviscosity 22

37 ofthemedium,aistheradiusofthesphere,and v isthevelocityofthesphere relativetothemedium.thecombinationoftheexpressionforthedepforceona sphere,eq.2.3,withthestoke sdragforcegivesthevelocityofaparticle v DEP moved withdep, v DEP = ε m a2 CM(ω) E 2 RMS 3η. 2.5 ThelowReynoldsnumberofthesystemallowstheaccelerationoftheparticletobe ignoredbecausethesystemissufficientlyover dampedthattheparticlereachesits terminalvelocityataratethatiseffectivelyinstantaneous. 2.3DielectricModelsforCellsandVesicles Thedielectricpropertiesofcellsandvesiclescanbedescribedwithsimplemodels. Thepermittivityofcellsandvesiclesaresetbytheintrinsicpropertiesofthe object sconstituentmaterialsandalsotheobject sgeometry.bothvesiclesandcells canbemodeledasspheresofwaterwrappedinathin,impermeablemembrane,as isshowninthemodelinfig.2.3a.(jones,1995)thesolutioninsidethevesiclehasa permittivitythatisdefinedbytherealpartofthepermittivityεcanda conductivityσc.thethinshellsurroundingthevesiclehasacapacitanceperunit areacmemandasurfaceconductivitygm. Fromthemodeldescribedabove,alumped elementcircuitmodelisdevelopedto describevesiclesandcells.thecapacitanceacrossthemembraneisdefined C=2cmema,whereaistheradiusofthevesicle,andthesurfaceconductivityis 23

38 assumedtobenegligible.(jones,1995)theinteriorofthevesicleisdefinedby R=1/σcandC=εcinparallel.Bycombiningthelumpedcircuitelementsassuminga sphericalvesicleandanelectricfieldinradialdirections,asisshowninfig.2.3a,an effectivepermittivityfortheobjectisarrivedat % j#$ " eff = c m a c +1 ( ' * & j#($ m + $ c ) +1). 2.6 whereτm=cma/σcistherelaxationtimeofthechargesthatbuilduponthe membraneandτc=εc/ σcisthedielectricrelaxationtimeofthesolutioninsidethe object.asimilarmodelcanbedevelopedtounderstandtheyeastcell,whereathin dielectriclayerisaddedtothemodeltorepresentthecellwallthatisnotpresentin mammaliancellsandvesicles.(jones,1995andhunt,2007) TheDEPforceonavesicleorcellsuspendedinasolutioncanbecalculatedusingthe dielectricmodeldevelopedabove.thesolutionhasarealpermittivityεsand conductivityσs.theeffectivepermittivityforthevesicleorcellεefffoundineq.2.6 iscombinedwiththeexpressionforthecmfactorineq.2.4: % " 2 ' ' (# CM(") = Re s # m $ # c # m ) + j"(# ' m " 2 ' ' & (# s # m + 2# s # m ) $ j"(# m $ # s $ # m ) $1 ( * + 2# s + # m ) $ 2) 2.7 Therearenowtwomorecharacteristictimesinthesystem,thedielectricrelaxation timeofthesolutionoutsideofthevesicleτs=εs/σsandtherelaxationtimeofthe chargesontheoutsideofthevesiclebuildinguponthemembraneτm =cma/σs. 24

39 AvesiclecanbetrappedandmovedwithpositiveDEPatRFfrequenciesifthe internalconductivityσcislargerthantheexternalconductivityσs.infig.2.3bthe CMfactor,Eq.2.7,isplottedversustheinteriorconductivityσcofavesicleorcell suspendedinasolutionwithσs=10 3 S/m,εs =εc,a=5µm,andf=1mhz.ifthe conductivityinsidethevesicleσc>10 3 S/mthentheCMfactorispositiveandthe vesicleexperiencesapositivedepforce.thecmfactor,andasaresultthedep force,plateaustoitsmaximumvalueaboveaconductivityσc=1s/m. TheDEPforceonvesiclesandcellsdependsonthefrequencyoftheappliedelectric field.infig.2.3cthecmfactor,eq.2.7,isplottedversusfrequencyforavesiclewith aninteriorconductivitygreaterthanthatofthesolutionσc>σsthedepforceis negativeatlowfrequencies,positiveatintermediatefrequencies,andnegativeat highfrequencies. ThefrequencyresponseofCM(ω)canbeunderstoodheuristicallybyanalyzingthe lumpedcircuitmodelinfig.2.3a.forfrequenciesslowerthantheinterfacial chargingtimeofthemembraneτmem=acmem(1/σc+1/2σs),thedepforceis negative(cm<0).attheselowfrequenciesthemembranehasahighimpedance andcausesthevesicletoactlikeaninsulatingsphere,makingitlesspolarizable thanthemedium.atintermediatefrequencies,abovethechargingtimeofthe membraneτmemandbelowtheinterfacialdielectricrelaxationtimeofthe 25

40 vesicle τmw=(εp+2εm)/(σc+2σs),cm>0andthedepforceispositive.atthese intermediatefrequenciesthemembraneistransparenttotheelectricfieldandthe solutioninsidethevesicleactslikeaconductor,makingthevesiclemorepolarizable thanthemedium.atfrequenciesabovetheinterfacialdielectricrelaxationtimeτmw ofthevesicle,thedepforceisnegative(cm<0).atthesehighfrequenciesthe vesicleistransparenttotheelectricfield,makingthevesiclelesspolarizablethan themedium. 26

41 Figure2.3(a)Alumpedcircuitmodelforavesicleoralivingcellwitharadiusa, thatconsistsofaninternalsolutionwithaconductivityσcandadielectric constantεc,wrappedinathinshellwithaconductivitygmemandacapacitanceper unitareacmem,(b)theclausiusmosottifactorcmplottedversustheinternal conductivityofavesiclesuspendedinasolutionwithanexternalconductivity σs=10 2 S/minafieldwithafrequencyω=1MHz,(c)CMofavesiclewithan internalconductivityσc=0.1s/msuspendedinasolutionwithaconductivity σs=10 2 S/mfactorplottedversusthefrequencyωoftheappliedelectricfield. 27

42 2.4TransmembranePotentialDifferenceanditsApplications InadditiontotheabilitytotrapandmoveobjectswithDEP,electricfieldscanbe usedtoelectroporatevesiclestoreleasetheircontents,fusetwovesiclestogether, orpermeabilizecellmembranes,asisdemonstratedinchapter4.thesetasksare accomplishedbyusinglowerfrequencyelectricfields(khzfrequenciescomparedto themhzfrequenciesusedfordep)toinducevoltagesacrossthemembranesof vesiclesandcells.alargetransmembranevoltagecancauseporestoformin membranes(electroporation)orcausetwovesiclestofusetogether(electrofusion). Theelectroporationofavesicleorcelloccurswhenthereisalargepotential differenceacrossthemembraneofacellorvesiclevtm.ingeneral,electroporation andelectrofusionoccurwhenthemaximumtransmembranevoltagev TM max isgreater than1v.(sugaretal.,1987)themaximumvoltagev TM max inducedacrossthe membraneofasphericalvesicleinanexternalelectricfieldis:(grosseetal.,1992) 3 E a V max TM = 2 1+ (ωτ mem ) whereτmem=acmem(1/σc+1/2σs)istheinterfacialchargingtimeofthemembrane, asisdescribedintheprevioussection.atypicalvesicleorcellhasacapacitanceper unitareacmem=10 2 F/m 2.(Jones,1995)Foravesiclewitharadiusa=5μmwith aninternalconductivityσc=0.1s/msuspendedinasolutionwithaconductivity σs=10 2 S/m,thereisaninterfacialchargingtimeτmem=20μs.Thetransmembrane voltagevtmisplottedversusfrequencyinfig.2.4foravesicleorcell,asisdescribed insection2.3,inanelectricfield E = 3 V /µm.atthefrequenciesf 1MHzusedfor 28

43 DEPthevesicleorcellwillhaveaninducedtransmembranevoltageofVTM 1mV. Thissmalltransmembranevoltagedoesnothaveasignificanteffectonthe membraneofavesicleorcell.(olofssonetal.,2003)atfrequenciesslowerthanthe interfacialmembranechargingtimeτmem,atransmembranevoltageontheorderof VTM 1Vforms.ThismuchlargertransmembranevoltageVTMcantrigger electroporationorelectrofusion. Ithasbeenshownintheliteraturethatelectricfieldpulsescantriggerthefusion (electrofusion)betweenadheringvesiclesorcells.(sugaretal.,1987andtressetet al.,2007)themodelforelectrofusionisamulti stepprocessthatisillustratedin Fig.2.4b.Figure2.4b(i)showstwovesiclesthataretrappedandmovedtogether withdep.figure2.4b(ii)showsthevesiclesbroughtintotightcontact,partially squeezingoutthewaterlayerbetweenthetwovesicleswithdep.electricfield pulseswithadurationlessthanτmemarethenappliedtocreatepores (electroporation)inthetransmembranecontactarea,asisshowninfig.2.4b(iii).if theporedensityislargeenoughthenporescanthennucleateintoastableholein thecontactareaasisshowninfig.2.4b(iv).(tressetetal.,2007)onourchip electricfieldsatmhzfrequenciesareusedtoholdvesiclesincontactwithdepwhile time multiplexedelectricfieldpulseswithfrequencieslessthanthemembrane chargingtimeω<1/τmemtriggerthefusion,asisdemonstratedinchapter4. Inducedvoltagesacrossacell smembranevtmcanaffectthecell sviabilityand physiologicalstate.inalivingcell,ionpumpsmaintainatransmembranevoltageof VTM=70mVacrossthecellmembrane.Itisageneralruleofthumbthataslongas 29

44 theinducedtransmembranevoltageismuchlessthanthenormalphysiological transmembranevoltagevtm<<70mv,thenitwillhaveonlyasmalleffectoncell physiology.(olofssonetal.,2003)acelltrappedinadeptrapwithanelectricfield strength E = 3V /µm andafrequencyf=1mhzwillhaveaninduced transmembranevoltagevtm 1mV.Therefore,acellinaDEPtrapshouldremain healthyuntilthefrequencyoftheappliedelectricfieldispurposelyloweredto induceelectroporationorelectrofusion. 30

45 Figure2.4(a)Thetransmembranepotentialofamodelofavesicleorcellwithan internalconductivityσc=0.1s/m,suspendedinasolutionwithaconductivity σs=10 2 S/m,witharadiusa=5µmandacapacitanceofCmem=10 2 F/m 2 isplotted versusthefrequencyfofanappliedelectricfield E = 3 V /µm,(b)astep by step illustrationofthefusionoftwovesicles. 31

46 2.5DielectricHeating Dielectricobjectscanbeheatedwithtime varyingelectricfields.inducedand intrinsicdipolemomentswithinanobjectwillattempttoalignthemselveswitha time varyingelectricfield.theenergyassociatedwiththisalignmentisviscously dissipatedasheatintothesurroundingsolution.thepowerdensitypabsorbedby adielectricmaterialisgivenbythefrequencyoftheappliedelectricfieldf,the imaginarycomponentofthepermittivity(thelossfactor)ε ofthematerial,the vacuumpermittivityε0,andtheelectricfieldstrength E withthe expression(bengtssonetal.,1974): P = ωε o ε'' E Thelossfactorofthematerialdependsonthefrequencyoftheelectricfieldandthe characteristictimeτwofthedielectricrelaxationofthematerial,withthe expression: ε''= (ε s ε ) 1+ (ωτ) 2 + σ ωε o 2.10 whereεs=78.4ε0isthelowfrequencydielectricconstantofwater,ε =1.78ε0isthe opticaldielectricconstant,τw=9.55psisthecharacteristicrelaxationtimeofwater att=25 C(asisshowninFig.2.1),andσistheconductivityofthesolution. (Murrelletal.,1994) Thepowerthatamaterialabsorbsfromatime varyingelectricfielddependsonthe frequencyofthefield.infig.2.5thepowerdensityp[w/m 3 ]absorbedbywaterin 32

47 anelectricfieldwithamagnitude E =10 5 V /misplottedversusfrequencyfor severalsolutionswithdifferentconductivitiesσ.fordeionizedwaterthepower densityincreasesmonotonicallyuntilitplateausatf=18ghz,thefrequency associatedwiththecharacteristicrelaxationtimeofwaterτw.forsolutionswitha finiteconductivityσ,thepowerdensityhasaconstantvalue P = ε o E 2 σ at frequenciesbelowacriticalfrequencysetbythedielectricrelaxationtimeofthe solutionτs= εs/σs.atfrequenciesabovethedielectricrelaxationtimeofthe solutionτsthepowerincreasesmonotonicallyuntilitplateausatf=18ghz.for biologicalsolutionsσ 0.5S/m,thedielectricrelaxationtimeofthesolutionis τs 1.4ns. Duetowater slargedielectriclossatghzfrequencies,microwavepoweris absorbedmuchmorestronglybywaterthanpolymers,glass,silicon,andmost objectsthatmicrofluidicdevicescouldbeconstructedwith.thisallowsmicrowave powertobedeliveredtosolutionswithoutsignificantlyheatingthesurrounding microfluidicdevice.effectiveheaterscanbebuiltthatworkatf=3.0ghz,a frequencyveryclosetothatofcommercialmicrowaveovens(2.45ghz),thatis belowthefrequencyassociatedwiththerelaxationtimeofwaterbutwherewater stillreadilyabsorbspower(asisdemonstratedinchapter6). 33

48 Figure2.5.ThepowerdensityP(W/m 3 )plottedversusthefrequencyfofthe appliedelectricfield.severalsolutionswithdifferentconductivityσ areplotted, increasinggoingfromthebottomtothetopwithvaluesof0s/m,0.03s/m, 0.06S/m,0.14S/m,0.3S/m,and0.62S/m. 2.6Magnetophoresis Magnetophoresisprovideatechniquetotrapandmoveparticlesthatcompliments DEP.Whereasalmostallmaterialshavesomedielectricresponse,mostobjectsare completelytransparenttomagneticfields.assuch,magnetophoresishasthe benefitthatforcescanbeappliedspecificallytomagneticparticleswhilenot affectingthesurroundingdielectricobjects.thistechnique(asisdemonstratedin Chapter5ofthisthesis)isusefulforapplyinglocalforcestomicroscopicobjects. Aparticlewithamagneticmoment m inamagneticfield B experiencesaforce, F MAG = m B

49 Foraparamagneticparticlewithamagneticsusceptibilityχ,themoment m = VχB /µ o isproportionaltotheexternalmagneticfieldstrength B,thevolumeof theparticlev,andthevacuumpermeabilityµ0.theforceonaparamagneticparticle is:(leeetal.,2004) F MAG = Vχ µ o B Intheworkpresentedinthisthesis,superparamagneticparticlesareused. Superparamagnetismisaformofmagnetismwhereverysmallferromagnetic particlesbehavelikeparamagnets.theparticlesaresmallenoughthatthermal fluctuationscanceltheirnetmomentwhenthereisnofieldapplied,butanapplied fieldwillcausetheparticlestoalign.assuch,superparamagneticparticlesmaybe describedwithahighmagneticsusceptibilityχatfieldslowerthantheirsaturation value.(beanetal.,1959andleeetal.,2005) 35

50 Chapter3.HybridIntegratedCircuit/ MicrofluidicChips InthischapterhybridIC/microfluidicchipsandtheexperimentalapparatusthat surroundthemarepresented.twohybridchipsaredescribedinthisthesis.inthis chapterthedielectrophoresis(dep)chip(thefabutron1.0)isintroduced.(huntet al.,2008)thehighvoltagedielectrophoresis/magneticchip,(hv DEP/Magnetic Chip,TheFabutron2.0)whichcombinesdielectricandmagnetictrappingontoa singlechip,isdescribedinchapter5.thedetailsoftheexperimentalapparatus surroundingbothchipsarepresentedinthischapter,includingthefluidics, electronics,optics,thermalmanagement,andcomputercontrol. 3.1Overview HybridIC/microfluidicchipscombinetheinexpensivecomplexityandsmall featuresizeoficswiththebiocompatibleenvironmentofmicrofluidicstoperform programmablebiologicalandchemicalexperimentsonthemicrometerscale.(leeet al.,2007)inthischapterachipisdescribedthattrapsandmovesindividual microscopicobjectsalongprogrammablepathswithdielectrophoresis(dep).the DEPchipconsistofalargearrayofmicrometer scalepixelsthatcanbeindividually addressedwithradiofrequency(rf)voltages,creatingelectricfieldsabovethe chip ssurface.thechipcansimultaneouslyandindependentlycontrolthelocations ofthousandsofdielectricobjectssuchaslivingcellsorpldropsoffluid,allowing thousandsofbiologicalandchemicalexperimentstobecontrolledinparallel. 36

51 3.2DielectrophoresisChip(Fabutron1.0) TheDEPChipconsistsofacustomICandafluidcellthatholdsliquiddirectlyonthe chip ssurface,asisshowninfig.1.1e.electroniccircuitsintegratedintotheicare usedtocreatelocalelectricfieldsabovethechip ssurface.theseelectricfieldscan beusedtopositionobjectswithdeporrelease,permeabilize,andfuseobjectswith electroporationandelectrofusion.thecomplexcircuitryandfastelectronicsofthe DEPchipallowmanylivingcellsandvesiclestobecontrolled,allowingmany parallel,well controlledbiologicalandchemicaloperationstobeperformed. TheDEPchipconsistsofanarrayofelectrodes,eachofwhichcanbeindividually drivenwithavoltage,tocreateanelectricfieldabovethechip ssurfaceasisshown intheinsetinfig.1.1e.specifically,thedepchipconsistsof128x256(32,768) 11x11μm 2 pixels,eachofwhichcanbeindividuallydrivenwitha5vpeak to peak radiofrequency(rf)voltagewithfrequenciesfromdcto11mhz.underneatheach pixelisastaticrandomaccessmemory(sram)element.thestateofthesram elementdetermineswhetherthepixelisdrivenbytheexternalrfvoltagesource (thepixelturnedoff)orbythelogicalinverseoftherfvoltage(thepixelturnedon). TheRFvoltagebetweenthepixelscreatesanelectricfieldabovethechip ssurface thatisusedtotrapandmoveobjectswithdep.theentirearrayofpixelscanbe updatedhundredsoftimesinasecond.theicisdesignedusingcadencedesign softwareandisfabricatedwithacommercial0.35μmprocesswith4metallayers throughmosis(process:tsmc35_p2).thedetailedspecificationsofthedepchip areoutlinedintheappendixintheformatofadatasheet.(appendixa) 37

52 3.2.1OperatingPrincipals TheDEPChipusespositiveDEPtotrapandmovedielectricobjects.Byshiftingthe locationofthepixelsthatareturnedon,thelocationoflocalelectricfieldmaxima aremovedaroundthearray.infig.3.1aquasi staticfiniteelementsimulations (Ansoft:Maxwell11)areshownoftheelectricfieldmagnitude E 5μmabovethe chip ssurface.twopixelsinthecenterofthearrayaresetto5vandtherestofthe pixelsaresetto0v.adielectricobjectwitharadiusa=5µmisschematically shownbeingpulledtowardsthemaximumofthefield.thesimulationsshowthat theobjectwillexperienceamaximumfieldof E 5 V /µm.(hunt,2007) TheexpectedDEPforceonanobjectabovethechip ssurfacecanbecalculatedby combiningtheelectricfieldsimulationsshowninfig.3.1awiththedielectricmodels presentedinchapter2.theforceonadielectricobjectwitharadiusa=5µm, describedbythemodelinsection2.3,suspendedinasolutionwithaconductivity σs=10 2 S/m,inanelectricfieldwithafrequencyf=1MHzandanRMSmagnitude showninfig.3.1a,isplottedinfig.3.1b.thedielectricobjectistrappedatthe interfaceoftwopixelsontheleftthatareturnedonandtwopixelsontherightthat areturnedoff.themagnitudeofthetrappingforceisf~1nnandtheforceis localizedwithintwopixellengthsofthetrap.alineisfittotheforcecurveatthe locationofthetrap,andtheeffectivespringconstantisfoundtobek=520pn/µm. 38

53 3.2.2CharacteristicTimes Theratethatthedevicecanperformoperationsislimitedbythetimethatittakes formicroscopicobjectssuspendedinsolutionabovethechip ssurfacetoreactto theelectricormagneticfields,notthespeedoftheelectronics.therearetwo importantcharacteristictimesthatdescribethemicroscopicobjectssuspendedin solutiononourchips:thetimethatittakesforaparticletodiffuseawayifatrapis turnedoffτdiffandthetimethatittakesforaparticletomovebetweentrapswhen onepixelisturnedoffandthenextisturnedonτmove.aparticlesuspendedina solutiontakesatimeτdiff=l 2 /16Dtodiffusethedistanceofhalfofapixel lengthl/2.theself diffusionconstantdisdefinedbythestokes Einstein EquationD=kBT/6πηa,wherekBTisthethermalenergy,ηistheviscosityofthe solution,andaistheradiusoftheparticle.thetimethatittakesforana=1µm particlesuspendedinwatertodiffusel/2=5µmisτdiff 1sec.Thediffusion timeτdiffislongerforbiggerparticles. Thetimethatittakesforaparticletomovefromonepixeltothenextτmoveis calculatedusingtherelationshipbetweenthedepforceandvelocityineq.2.5and theplotofthedepforceversusdistanceinfig.3.1b.thetimethatittakesforan a=1µmparticleonthedepchiptomovefromonepixeltothenextisτmove 10ms. Forlargerparticlesthetimethatittakestomoveparticlesbetweenpixelsτmove increases.theicoperatesatsub mstimescales(asisdescribedbelow)suchthat theiccanchangeitsstatemanytimesinthetimethatittakesforaparticletoreact tothechangeinfield. 39

54 Figure3.1.(a)Afiniteelementsimulationoftheelectricfieldstrength5μmabove thechip ssurface,(b)aplotoftheforceonadielectricobject(asmodeledin chapter2.3)attheinterfaceoftwopixelsontheleftthatareturnedon(inyellow) andtwopixelsontherightthatareturnedoff(inwhite). 40

55 3.2.3IntegratedCircuitDesign ThissectionoutlinesthedetaileddescriptionoftheDEPchipgiveninTomHunt s Thesis.(Hunt,2007andHuntetal.,2008)Thechip s128x256(32,768)pixelsare addressedwithlogicandmemorybuiltintotheic,suchthatonly8datalines(not includingthetwoclocksandfourcontrolsignals)arerequiredtoupdatetheentire array.aschematicofthesramarrayandthelogicandmemorythatsurrounditare showninfig.3.2a.thesrammemoryisorganizedas128wordsx256bits.the bitsofeachwordarereadinandoutofthechipwithasequentiallyloadedtwophaseclockedshiftregister.eachwordisaddressedtoaspecificrowinthearray usinga7bitrowdecoder.therowdecodertakes7binaryinputsandusesthemto choose1of2 7 (128)rowsoftheSRAMarray.Amicrographofthechip,showinga sectionofthearrayofdeppixelssurroundedbythecontrolelectronics,isshownin Fig.3.2c. AschematicofthecircuitunderneatheachDEPpixelisshowninFig.3.2b.The SRAMmemoryelementsunderneatheachpixelareaddressedwithaword lineand itsvaluesetwithabit line.thesramelementcontrolsamultiplexer(mux)that routesoneoftwosignals,producedoffofthechip,tothe11x11µm 2 pixelabove. GenerallythetwosignalsareRFvoltagesthatare180 outofphase,butcanbeset arbitrarily.theoutputofthemuxgoesthroughavoltage amplifierbeforedriving thepixel.thevoltage amplifierisatwotransistorinverter,appropriatelysizedto drivethecapacitiveloadofthepixel.thevoltage amplifiershaveanon resistance ofron 10kΩ,drivingapixelcapacitanceoflessthanCload 50fF,whichyieldsa 41

56 sub nsrctime.thevoltageonthepixelcanhaveapeak to peakvoltageof5vand abandwidthofdc 11MHz.Thebandwidthcouldhavebeenlarger(DC GHz)if greatercarewastakeninthedesignofthechiptobuffertherflinesthroughoutthe circuit. Thechip saddressingarchitectureisaresultoftrade offsbetweenmaximizingthe refreshrateofthesramarray,minimizingthesizeoftheelectronicsneeded underneatheachpixel,andminimizingthenumberofinputandoutputpinsonthe chip.therefreshrateofthechipisimprovedbyallowingregions of interest(roi) tobeupdatedwithoutupdatingtheentirearray.onthechip,wordsthatare256 bitsinlengthareupdatedwithrandom access.foreachpixeltobefullyrandomaccesswouldrequiretwoextratransistorsunderneatheachpixel,increasingthe pixelsizeandcomplexityofthecircuit.therefreshrateismaximizedbybreaking thearrayintowordswiththesmallestnumberofbits.inthelimitingcase,thechip wouldbeasfastasitcouldbeifeachpixelhadawiretotheoutside world. Conversely,thenumberofpinsisminimizedbyincreasingthesizeofeachword.In thelimitingcase,thechipwouldhavetheminimumnumberofpinsiftheentire arraywasupdatedinserialwithashiftregister. Inourdesign,witha128wordx256bitarray,theshiftregisterisupdatedata maximumrateof1bit/0.1µsmakingittake~26µstoupdateasinglewordonour chipand~3.3mstoupdatetheentirearray.thus,thechipcanrefreshitsentire statefasterthanthetimeτmove>10msthatittakesfortheparticlesabovethe chip ssurfacetoreacttothefields.theupdaterateofthechipiscurrentlylimited 42

57 bytheelectronicssurroundingthechip.withthecurrentelectronicstherefresh rateofthechipis400mstoupdatetheentirearray.thechipisinterfacedtoa computerandtheinterfaceisslowerthanthechipitself,asisexplainedinmore detailbelow.anewgenerationofgraduatestudentsiscurrentlydesigninga microcontrollerinterfacetothechipthatcanrefreshthechipatitsmaximumrate. Thedesignofthechip,usingtheCadencesoftwarepackage,ishierarchicaland incorporatesdigitalandanalogtestingsuchthat(ifdonecorrectly)thelayoutis guaranteedtomeetdesignspecificationsandthedesignrulesofthefabrication process.thechipdesignbeginswithbreakingthechipintoseparablemodules:the pixel,theshiftregister,therowdecoder,asinglesramelement,etc Theindividual modulesaredesignedintermsofothermodules,continuingdownwardsinthe hierarchyuntilreachingthetransistor level.eachtransistorcorrespondstothe layoutofmasks(dopingandmetallayers,vias,etc ).Themodulesarecombinedat themask layoutlevelbymanuallyplacingthemodulesandmanuallyconnecting themwithmetallayers.thedesignsoftwarechecksthatthecircuitsatthemasklevelmatchesthecircuitsatthemodule level.theelectricaltestingbeginsatthe bottomofthehierarchyatthemodule level,usingspicemodelsforeachelement, totestthedigitalandanalog(bothtimedomainandfrequencydomain)responseof thecircuits.oncethelayoutforamoduleisdrawn,additionalunintended capacitancesinthesystemareextractedfromthelayoutandincorporatedintothe testing.thenewlayoutcanbemodifiedbasedontheresultsofthetesting,andthis cyclecanberepeatediniterationsuntilthedesignspecificationsofthemoduleare met.themodulesarecombinedandthesystemistestedworkingfromthebottom 43

58 ofthehierarchytothetop,untiltheentirechipislaidoutandfullytested.forthe chipsdesignedinthisthesis,thedesignprocesstakesabouttwomonthsfromstart tofinishandresultsinachipwithn 300,000transistors. 44

59 Figure3.2.(a)AschematicofthearchitectureoftheSRAMarrayandthelogicand memorythatareusedtoreadandwritetoitonthedepchip,(b)aschematicofthe circuitunderneatheachdeppixel,showingthesrammemoryelement,a multiplexer(mux)thatdirectseithertherfsignaloritslogicalinversetoavoltage driverthatconnectstotheelectrode,(c)amicrographofa250x200µm 2 section showingacornerofthechipwithaportionofthedeparrayintheupperleftcorner, therowcontrolcircuitsonthebottom,andthebitcontrolcircuits,includingthe shiftregister,ontheright. 45

60 3.2.4Capabilities TheDEPchiphasbeenusedtotrapandmovedielectricobjectssuchasliving cells,(fig.3.1aandfig.3.1c)dropsoffluidimmersedinoil,(fig.3.1b)andvesicles usedasbiomimeticcontainers(chapter6).inadditiontotheabilitytotrapand moveobjectswithdep,electricfieldscanbeusedtoelectroporatevesiclesto releasetheircontents,fusetwovesiclestogether,orallowcellstotakeup substancesfromthesolution.(chapter6)thesetasksareaccomplishedbyusing lowerfrequencyelectricfields(khzfrequenciescomparedtothemhzfrequencies usedfordep)toinducevoltagesacrossthemembranesofvesiclesandcells,asis describedinchapter2.inadditiontowhathasbeendemonstratedbyourgroup, anyapplicationthatcouldbenefitfromaprogrammableelectricfieldcontrolledon thelengthscaleofl=10µmwithanrfbandwidthcouldbeperformedonthedep chip,suchaselectro optics(lopez,1970),electro wetting(pollacketal,.2000),or electro chemistry(nyholm,2005). 46

61 Figure3.3(a) Atimesequenceofyeastandratalveolarmacrophagesbeingtrapped andmovedwithdep.theblackarrowsindicatethedirectionthatthecellsare movedbetweenframes.thecellsmoveatarateof~50µm/sec,(b)atimeseriesof dropsofcoloredwaterbetweenalayeroffluorocarbonoilandhydrocarbonoil trapped,moved,split,andmergedwiththechip,(c)amicrographoftheentirechip beingusedtopositionthousandsofyeastcellstospell Harvard. (Huntetal.,2008) 47

62 3.3ExperimentalApparatus TheapparatusthatsurroundstheIC/microfluidicchipsisdescribedinthissection. Theapparatusconsistsofaninterfacetocontrolthechipwithacomputerandan opticalmicroscopewithavideocameratoobservewhathappensonthechip.a blockdiagramoutliningtheorganizationoftheapparatussurroundingthechipsis showninfig.3.4a.fluidicsbringsamplesandreagentstothechip.thechip communicatestotheoutsideworldthroughelectricalconnectionsonacustom printedcircuitboard.theprintedcircuitboardconnectstoacomputerthrougha digitalinput/output(i/o)card.thecomputercanbothwriteandread outthe stateofthechipthroughcustomcontrolsoftware.afluorescencemicroscope imagesthecontentsofthechip.theimagesaresenttothecomputerthrougha digitalcamera.agraphicaluserinterface(gui)providesanintuitiveplatformfor theusertointeractwiththechip.theoverallsystemisaimedatbeingrobust,easy, andflexibleenoughtousethatnewexperimentscanbequicklyprototyped,with minimumsetupandmaintenancetime Fluidics AfluidcellisbuiltdirectlyontopoftheICwithasiliconegasket(Invitrogen:p 24744)witha1.2mmholethatiscutwithaholepunch,asisshowninFig.3.4c.A 3x3mm 2 glasscoverslipisplacedontopofthefluidcelltosealit.thefluidcell canbefilledwithapipette.directlypipettingfluidontothechip ssurface,rather thansettingupamicrofluidicnetwork,keepstheexperimentalapparatusflexible, allowingmanyexperimentstobeattemptedinashorttime. 48

63 TheIC/microfluidicchipcanbeintegratedasacomponentinalargerPDMSor glassbasedmicrofluidicsystem.passivemicrofluidicscancoupletheoutsideworld, anditsmacroscopicfluidsamples,tothechipinarolethatisanalogoustotherole printedcircuitboardsplayforicsincomputers.theic/microfluidicchipcould behaveasaprogrammablecomponenttoperformtaskssuchassortingor combiningsamplesandreagents.insuchasetup,thefluidoutputofthechipcould befedtooutputchannelsofthepassivemicrofluidicdeviceforfurtherprocessingor collection Electronics InthissectiontheelectronicssurroundingtheDEPchiparedescribed. Electronicallythechipiseffectivelya32kbitSRAM;Assuch,theelectronics surroundingthechipareidenticaltotheread/writeelectronicssurroundingany SRAMarray.TheIC/microfluidicchipismountedonastandard84pinchipcarrier asisshowninfig.3.4c.theicismountedwithsilverpainttobothelectrically groundthesubstrateoftheicandtocreateathermallyconductiveconnectiontoa thermalreservoir.wirebondsconnecttheictothepinsofthechipcarrier.inall thechiprequires24wirebonds,includingredundantpowerlines.allofthewire bondsareonthesamesideoftheictomakeiteasiertoprotectthemfromthefluid. Thewirebondscanbecoveredwithepoxytoprotectthemfurtherfromthenearby fluid.howevermostoftenthebondsareleftunprotected,asthefluidcellisa sufficientbarrierbetweenthefluidandthewirebonds. 49

64 ThechipcarriersitsonacustomPCBwhichconnectstheICtothecomputer, providespower,andconnectstheictoanrfvoltagesource,asisshownin Fig.3.4d.ThePCBhasRCfiltersoneachoftheinputsandoutputsofthechipwitha cornerfrequencyf3db=100mhztoprotectthechipfromvoltagespikes.thedigital linesconnectfromthepcbtothecomputerthroughaproprietarynational Instrumentscable(NI:SHC68 68 EPM)tothedigitalI/Ocard(NI:PCI 6254).The RFvoltageisproducedoffofthechipandisbroughtontotheboardwithaBNC connector.thedepchiphasapowerconsumptionof0.5 2W,dependingonhow manypixelsareturnedonandthefrequencyoftherfvoltage Optics TheIC/microfluidicchipsitsunderafluorescencemicroscope,asisshownin Fig.3.4d.Fluorescencemicroscopyisapowerfulandwidelyusedtooltoimage biologicalandchemicalsystems.(pawley,2008)couplingfluorescencemicroscopy withthechipenablesaccesstoawelldevelopedtechniquetoimagethesamplesand reagentsthatthechipcontrols.thesiliconsubstrateoftheicisnotoptically transparentandsothemicroscopeoperatesbymeasuringreflectedlight.theicis notfluorescentatopticalfrequenciesandsobehavesasablackbackground.the microscopeviewsthesamplesthroughtheglasscoverslipofthefluidcell. ThemicroscopeapparatusconsistsofapillarmountedOlympusBX 52,hovering abovetheic/microfluidicchip,asisshowninfig.3.4d.longworkingdistance objectivesareusedtogiveenoughspaceforthefluidcell(olympus:lmplflseries). Thelightsourceisa100Wmercurylamp.Thedeviceismonitoredwithan 50

65 HamamatsuORCA ERcooledCCDcamera.ImagesaretakenwithMicroSuiteBasic Edition(Olympus)andStreamPixdigitalvideorecordingsoftware(Norpix). FuturedevicesthatutilizeIC/microfluidicchipsandhopetobeportablesurelycan notincludealaboratory sizedfluorescencemicroscope.recentworkhasshown thatfluorescencemicroscopycanbeperformedinmicrofluidicdevicesusingsmall LEDlightsourcesandphotodiodes.(Psaltisetal.,2006)Moreforwardlooking,a CCDarraycouldbeflippedandbondedontopoftheICformingafluidchannel betweenthetwochips.(cuietal.,2008andhengaetal.,2006) 3.3.5ThermalManagement Temperaturecontrolisessentialforexperimentsonbiologicalsystems.The temperatureofthechipiscontrolledusingheat sinks.typicalicshaveoperational temperaturesbetween65 Cand85 C.(Leeetal.,1998)Formanybiologicaland chemicalexperimentsthatmightbeperformedonthechip65 Cisfartoohot.As such,stepsaretakentocontrolthetemperatureofthechip. Inthefirstiterationofthermalmanagement,theceramicchipcarriersitsontopofa machinedcopperblockwithathinlayerofthermalgrease(arcticsilverinc.:arctic Silver).Airisblownwithafanoverthelargesurfaceareaofthecopperblock s bottomside.thetemperatureofthedepchip stopsurfacewiththechipturnedon is40 C 50 Ccomparedto90 Cwhennoheatsinkisattached.Thechip temperatureismeasuredwithaninfrarednon contacttemperaturesensor(control Company4477). 51

66 TheseconditerationoftheheatsinkwasdesignedfortheHV DEP/Magnetic Chip(Chapter5)andutilizesawatercooler.Withwatercoolingthetemperatureof thechipcanbecontrolledbysettingtheflowrateandtemperatureofthewater bath.thewatercoolerisconstructedwithamachinedcopperpiecethatis connectedwithathermallyconductiveepoxy(loctite383and7387)toa commercialcpuwatercooler(dangerden,mc).thetopofthecopperblockis polishedandimmediatelycoveredwithathermallyevaporatedlayerof 8nmTi/100nmgoldtokeepcopperoxidefromgrowing.Thechipcarriersitson topofthemachinedcopperblockwithathinlayerofthermalgrease(arcticsilver). Thewatercoolerisdrivenwithasubmersiblepump(BecketCorp,Versa)witha flowrateof92gallons/hour.thehv DEP/MagneticChip(Chapter5)has temperaturesensorsintegratedintotheic.theanalogoutputofthesensorscanbe placedintoafeedbackloopwiththewater coolertocreateacontrolsystemto maintainthechiptemperature. 52

67 Figure3.4.(a)Aschematicoftheexperimentalapparatussurroundingthe IC/microfluidicchip.Thegreenboxesrepresentcomponentsthatareelectrical, bluerepresentfluidic,orangerepresentoptic,andyellowrepresentswherethe componentscometogether,(b)anopticalmicrographoftheic(b)84pinchip carrierholdingtheic,ontopoftheicisaredfluidcell,(c)aphotographofthe experimentalsetupofthehybridic/microfluidicsystem. 53

68 3.3.6ComputerControl ComputersoftwareisusedtoprogramandtointerfacetheIC/microfluidicchip withauser.thecomputercommunicateswiththechipthroughanational InstrumentsPCIboard(NI:PCI6254).ThePCIboardiscontrolledwithcustomLab View(NI)software.MATLAB(TheMathWorks)codeisusedonthefront endto interfacewiththeuserandtotranslateauser sinputintothecommandsthatwillbe senttothechip.allmatlabandlabviewcodeisincludedinappendixc. TheGUIthatwedesignedisshowninFig.3.5.IntheGUIausercandrawobjects ontoabit mapthatcorrespondstothedeppixelarrayofthechip.objectscanbe created,recognizedbythecomputer,andmovedaroundthescreenwithacursor. Basicdefaultshapescanautomaticallybedrawnsuchashorizontal,vertical,and diagonallinesandchecker boardpatterns.inadditiontothestaticdrawingsthat canbedrawninthegui,multi framemovies,whereeachframeisastateofthechip, canbeimportedintoacompilerandplayedonthechip.(appendixc)tomaximize therefreshrate,thecodecansendcommandstowriteonlyword linesinthesram arraythathavechangedfromframetoframe. Thedigitalcameraonthemicroscopeconnectstothecomputerandisdisplayed withstreampix(norpix).currently,thecoordinatesystemonthevideo feedfrom thedigitalcameraandtheguiismanuallyalignedbytrappingandmovinganobject andthenfindingitonthemicroscope.thecombinationofimage recognition softwareandautomatedcontrolofthemicroscopestagecouldbeusedtoremove 54

69 thehumanfromthecontrolloop,andallowcomplexautomatedtaskssuchas sortingtobedoneinaclosed loop. Figure3.5.ScreenshotsoftheGUI,(a)ThemainscreenfortheGUIwhereobjects canbedrawnonabitmapthatrepresentsthestateofthechip.theblackandwhite stripesarethearbitrarypatternthatisbeingwrittentothechipinthescreen shot, (b)the advancedmode oftheguiwhereobjectsthatarecreatedinthemain screencanbemovedwithacursor. 55

70 Chapter4.AHybridIntegratedCircuit/ MicrofluidicPlatformtoControlLivingCells andplbiomimeticcontainers 4.1Overview ThischapterdescribeshowthehybridDielectrophoresis(DEP)Chip,introducedin Chapter3,canbeusedtoperformbiologyandchemistryexperiments.TheDEP chipsimultaneouslycontrolsmanyindividuallivingcellsandsmallvolumesoffluid. ThesmallvolumesoffluidarecontainedinpLsizedlipidvesicleswhichprovidea stablecontainerforaqueoussolutionsthatcanbesuspendedinwater.thevesicles canbemovedaroundthechip,fusedtogether,andhavetheircontentsreleasedinto thesolution.livingcellscanalsobemovedaroundthechipandcanbe electroporatedtoallowsubstancesfromthesolutiontoenterthecells.inaddition, thechipcancontrollablymechanicallydeformthecellsorvesicles.thesebasic functions,whicharedemonstratedinthischapter,canbestrungtogetherto performcomplexbiologicalandchemicallaboratorytasks. Previousworkhasbeendonetousehybridintegratedcircuit(IC)/microfluidic chipsforbiologyandchemistryexperiments.tomhunt sthesisdescribeshybrid chipsbeingusedtotrapandmovedropsofwatersuspendedinoil.(hunt,2007) TheDEPChipcanpositionthedrops,splitsthedropsintwo,andmergestwodrops together,demonstratingapotentialplatformforperformingprogrammable chemistryexperiments.(hunt,2007andhuntetal.,2008)intheseemulsion based 56

71 systems,dropsofwateraresuspendedinoilandarestabilizedusingasurfactant,as isshowninfig.4.1.(holtzeetal.,2008)dropbasedmicrofluidicchipshavebeen showntobeimportanttechnologyforperforminghigh throughputbiologicaland chemicalexperiments.(kosteretal.,2008)achallengeforthesesystems,however, isthatcellsmustalsobeencapsulatedindropsbecausetheoil basedcontinuous phaseisnotbiocompatible.inaddition,moleculeswithhydrophobictailscanleak frominsidethevesiclesintotheoil,thuslimitingthechemistrythatcanbedonein thesesystems.(hunt,2007andholtzeetal.,2008) Inthischapteraversatileplatformisdevelopedforbiologyandchemistry experimentsonthedepchip.thisplatformcansimultaneouslycontrolbothliving cellsandsmallisolatedvolumesoffluidsuspendedinwater.insection4.2.1the functionalityandcapabilitiesofthedepchiparereviewed.topackagesmall volumesoffluid,suchthattheycanbesuspendedinwater,inspirationistakenfrom cellularbiology.thepreparationofthevesiclesusedinthischapterisdescribedin Section4.2.2.Lipidvesicles,dropsofwatersurroundedbyathinphospholipid bilayermembrane,arecommonlyusedincellstopackagesubstancesfor intercellulartransport,tostoreenzymes,andtocreateisolatedchemicalreaction chambers. (Albertsetal.,2007)Artificiallyproducedunilamellarvesiclesmimic naturalvesiclesandproviderobustplcontainersthatareimpermeableandstable forawiderangeofsalinity,ph,andotherenvironmentalconditions. (Tressetetal., 2007)AschematicofaphospholipidvesicleisshowninFig.4.1.Thischapter demonstratestheuseofunilamellarvesiclesasarobustplcontainerforfluidson thedepchip. 57

72 Thefrequencydependenceofthedielectricpropertiesofvesiclesandcellsenable thedielectricphenomenonusedinthischaptertobeindependentlyaccessedwith electricfieldsatdifferentfrequencies.voltagesatmhzfrequenciesareusedfor DEP,totrap,position,anddeformcellsandvesiclesasisdescribedinSection Voltagepulseswithmspulse widthsareusedforelectroporationandelectrofusion, totriggerthereleaseofthecontentsofvesicles,fusetwovesiclestogether,and permeablizethemembranesofcellsasisdescribedinsection TheDEPChipisusedinthischapterdemonstratebasicfunctionsonlivingcellsand unilamellarvesicles,whichcanbestrungtogethertoperformcomplexbiological andchemicallaboratorytasks.insection4.3.1thedepchipisusedto simultaneouslyandindependentlytrapandmovemanyindividualvesiclesand livingcellswithdielectrophoresis(dep).insection4.3.2voltagespulsescreatedby thedepchipareusedtoselectivelyelectroporatevesiclestoreleasetheircontents, fusetwovesiclestogether,andpermeabilizelivingcells.insection4.3.3vesiclesare deformedbychangingtheshapeofthedeptraps.theplatformdemonstratedin thischaptercanbeusedforawiderangeofchemicalandbiologicalapplicationsand isastepforwardforthefieldofhybridic/microfluidics. 58

73 Figure4.1.Aschematicrepresentationofawaterdropinoilemulsionstabilized withsurfactantsisshownontheleft.aschematicrepresentationofaunilamellar vesicleisshownontheright.thephospholipidmoleculesthatformthesurfactant andthevesiclemembraneareshownwithredcirclesforthehydrophilicheadsand blacklinesforthehydrophobictails. 59

74 4.2Methods 4.2.1TheHybridIntegratedCircuit/MicrofluidicPlatform InthissectiontheDEPChip,whichisdescribedfullyinChapter3,isusedtocontrol livingcellsandsmallvolumesoffluidusingspatiallypatternedelectricfields.here, thedepchipanditscapabilitiesaresummarized.thehybridchipconsistsofa microfluidicchamberbuiltdirectlyontopofanic.theicconsistsofanarrayof electrodes,eachofwhichcanbedrivenwithanrfvoltagetospatiallypatternthe electricfieldmagnitudeabovethechip ssurface.underneatheachpixelisastatic randomaccessmemory(sram)element.thestateofthesramelement determineswhetherthepixelisdrivenbytheexternalrfvoltagesource(thepixel turned off )orbythelogicalinverseoftherfvoltage(thepixelturned on ).The arrayconsistsof128x256(32,568)11x11µm 2 pixels,eachofwhichcanbedriven withanrfvoltagewithabandwidthfromdc 11MHz.Theentirearrayofpixels canbeupdatedhundredsoftimesinasecond.amicrographoftheic/microfluidic chipisshowninfig.1.1b UnilamellarVesicles Lipidvesiclesaredropsofwaterencapsulatedbyathinphospholipidbilayer membrane.aschematicofthecross sectionofaphosphlipidunilamellarvesicleis showninfig.4.1.thephospholipidmembraneconsistsofamphipathic phospholipidsthatself assembleintobilayerssuchthattheirhydrophilicheadsface theinsideandoutsideofthevesicle,providingarobustcontainerforaqueous 60

75 solutions.(albertsetal.,2007)aunilamellarvesicleconsistsofasinglebilayer membrane,asopposedtoseveralassomevesiclesdo. ThevesiclesaremadethroughcollaborationwithThomasFrankeatUniversityof AugsburginGermany. Thevesiclesarepreparedwithliposomeelectroformation usingamodificationoftheangelovamethod.(angelovaetal.,1986)therecipe usedtocreatethevesiclesisasfollows.briefly,asmallamountoflipidin chlorophorm(ca.20microliterofa5mg/mlsolution)isdepositedontotwoindiumtin oxidecoatedglassslides.afterevaporationoftheorganicsolventforatleast 6hourstheslidesareassembledinparallelwithaseparating2mmteflonspacerto formthepreparationcellandincubatedforonehourinasaturatedwatervaporat roomtemperatureforprehydration.subsequently,theelectroformationcellisfilled with200mosmaqueoussolution(50mmsodiumchlorideandsucrose)andalow frequencyelectricfieldof500hzandamplitude1v/mmisappliedtotheitoslides.afterapproximately8hourslipidvesiclesofdiameterslargerthe20µmare harvested.theexternalsaltysolventisremovedbyrepeatedlywashingwithanisoosmoticaqueousglucosesolutionandgentlecentrifugationatlowrotation frequencies.thisprocedureprovidesthedielectriccontrastnecessaryfordep actuationandstillensurestheintegrityofthevesiclecontainers Dielectrophoresis(DEP)ofVesiclesSuspendedinWater ThehybridchipusespositiveDEPtotrapandmovecellsandvesiclessuspendedin water.anyobjectthathasacomplexpermittivitythatisdifferentthanthe surroundingmediumcanbecontrolledusingdep,(jones,1995)asisdescribedin 61

76 detailinchapter2. ByshiftingthelocationoftheDEPChip spixelsthatareturned on,thelocationoflocalelectricfieldmaximaaremovedaroundthearray.in Fig.3.1aquasi staticfiniteelementsimulations(ansoft:maxwell3d)areshownof theelectricfieldmagnitude E 5µmabovethechip ssurface.twopixelsaresetto 5Vandtherestaresetto0V.Avesiclewitharadiusa=5µmisshownbeing pulledtowardsthemaximumofthefield.thesimulationsshowthatavesicle experiencesamaximumelectricfieldstrength E 0.5 V /µm. Thedielectrophoreticresponseofvesiclescanbecontrolledbysettingthesalinityof thesolutioninsidethevesicleσintrelativetotheconductivityofthesolutiononthe outsideofthevesicleσsol,asisdescribedinfulldetailinchapter2.infig.2.3ba plotoftheclausius Mosotti(CM)factor,ameasureofthedifferenceinpermittivity oftheobjectandthemedium,isshownversustheinteriorconductivityofan a=5µmvesiclesuspendedinadeionizedsolutionwithaconductivityof σsol=10 3 S/m.Iftheconductivityinsidethevesicleisgreaterthanσint>10 2 S/m thenthecmfactorispositive,andthevesiclecanbetrappedandpositionedwith positivedep. Thedielectrophoreticresponseofcellsandvesiclesisafunctionofthefrequencyof theappliedelectricfield.infig.2.3caplotofcmversusfrequencyisshownfora vesiclewithinteriorconductivityσint=50ms/m.belowacut offfrequencyof ωd=50mhzandaboveacut offfrequencyofωmem=2khz,thecmfactorispositive andinvariabletochangesinfrequency.itisinthisfrequencybandthatpositive DEPisusedinthischapter.Thehighfrequencycut offissetbythedielectric 62

77 relaxationtimeτd=εp/σintofthesolutioninsidethevesicle.thelowfrequencycutoffissetbythechargingtimeofthemembraneofthevesicle τmem=acmem(1/σint+1/σsol) ElectroporationandElectrofusion Voltagepulsescreatedbythechipcanbeusedtoelectroporateandelectrofuse vesiclesandcells,asisexplainedinfulldetailinchapter2.electroporation generallyoccurswhenthereisatransmembranevoltagevtm>1v.(sugaretal., 1987)ThemaximumvoltageinducedacrossamembraneVTMofasphericalvesicle orcellinanexternalelectricfieldisgivenbyeq.2.8.thecharacteristiccharging timeofthemembraneisgivenbytheexpression:(jones,1995) τ mem = ac mem ( ) σ int 2σ sol 4.1 wherecmem=10 2 F/m 2 isthespecificmembranecapacitanceofaunilamellar vesicle.(jones,1995)foravesiclewitharadiusa=5µmandinternalconductivity σint=0.1s/minasolutionwithconductivityσsol=10 2 S/m,thereisamembrane chargingtimeτmem=20ms.electricfieldswithfrequencies1/ω<τmemareusedfor DEP.Atthef=1MHzfrequenciesusedforDEPthetransmembranevoltage VTM 10mVandnodamageiscausedtothemembrane.(Albertsetal.,2007and Grosseetal.,1992)Electricfieldscreatedwithvoltagepulseswithapulsewidth τ>τmemcreatetransmembranevoltagesvtm~1vandareusedtoelectroporateand electrofusevesiclesandcells. 63

78 Theabilitytoselectivelyelectroporatevesiclesenablesthechiptolocallyrelease substancesintothesolution.thislocalreleaseofsubstancescreatesagradientin theconcentrationasafunctionofthedistancerfromthecenterofthevesicle. Consideravesiclewitharadiusafilledwithaconcentrationn=nsofasubstance suspendedinasolutionthathasnoneofthesubstancen=0.thegradientinthe concentrationiscalculatedbysolvingthediffusionequationinspherical coordinatesaroundthevesiclewiththeboundaryconditionsthattheconcentration n=nsadistancer=afromthecenterofthevesicleandn=0atadistancer=. (DillandBromberg,2003)Thesolutiontothediffusionequationisplottedfora vesiclewithradiusa=5µminfig.4.2aandisgivenbytheexpression: n(r) = n a S r. 4.2 Whenacellorvesicleiselectroporated,suchthatareactionoccursatthe membrane ssurface,aconcentrationgradientisalsocreated.considera reactionthatoccursatthemembranesurfacewithareagentinthesolutionthat hasabulkconcentrationn.inthiscase,theconcentrationatthesurface disappearsbecauseitisconsumedbythereaction,andtheconcentrationatan infinitedistanceawayisthatofthebulkconcentration.theboundaryconditions arethenthatn=n atadistancer= formthecenterofthevesiclesandn=0at adistancer=a.thesolutiontothediffusionequationisplottedforavesicle withradiusa=5µminfig.4.2bandisgivenbytheexpression: n(r) = n (1 a r )

79 Theconcentrationgradientsdependonlyontheconcentrationofthesubstances, theradiusofthevesicle,andthedistancefromthevesicle.theabilitytocreate well controlledconcentrationgradientsinthesolutionisatoolthatcanbeusedfor deliveringreagentsandsamplestocellsortootherreagentsforchemistryand biologyexperimentsonthechip. Thevoltagepulsesusedforelectroporationcanbetime multiplexedwiththe voltagesusedfordep.thisispossiblebecauseduringthedurationofthems voltagepulses,duringwhichthedepforceisturnedoff,cellsorvesiclesdonot diffuseasubstantialamount.aparticlesuspendedinasolutiontakesτdiff=l 2 /16D todiffusethedistanceofhalfofapixellengthl/2,asisexplainedinchapter3.the timethatittakesforaparticlewitharadiusa=1μmsuspendedinwatertodiffuse L/2=5μmisτdiff 1sec,whichismuchlongerthanthetimethattheDEPtrapis turnedoff.thediffusiontimeτdiffisevenlargerforbiggerparticles. Electricfieldpulsescanalsobeusedtriggerthefusionofvesicles,asisexplained infulldetailinchapter2.themodelforelectrofusionisamultistepprocess. TwovesiclesarebroughtintotightcontactwithDEP.Electricfieldpulsesare thenusedtoinduceatransmembranevoltageacrossthecontactareaofthetwo vesicles.thetransmembranevoltagecauseselectroporationonthecontact area, andiftheporedensityislargeenoughthentheporesnucleateandthevesicles fuse. (Sugaretal.,1987)Onthechip,voltagesatMHzfrequencyareusedtohold thevesiclesincontactwithdepwhiletime multiplexedelectricfieldpulsesare usedtotriggerthefusion. 65

80 Figure4.2(a)Aplotoftheconcentrationgradientcreatedaroundavesicle, shownastheorangecircle,thatisreleasingasubstanceatitssurface,(b)aplot oftheconcentrationgradientcreatedaroundavesicleorcell,shownasthe purplecircle,thatisreactingwithasubstanceinthesolutionatitssurface. 66

81 4.3Demonstrations 4.3.1TrappingandMoving InthissectionitisshownhowthehybridIC/microfluidicchipscantrapandmove individualvesiclesandcells,bothsuspendedinwater,alongarbitrarypaths. Figure4.3ashowshowindividualvesiclescanbeindependentlytrappedandmoved alongindependentpaths.figure4.3a(i)showsthreevesiclesthatare independentlytrappedonthechipwithdep.infig.4.3a(i iii)thevesicleontheleft isbroughtbetweenthetwoothervesiclesat70µm/sec.infig4.3a(iv vi)the vesicleonthebottomismovedupwards,positioningthevesiclesintoatriangle. ThisdemonstrationshowsthattheDEPChipcanindependentlycontrolseveral objectsatonce,enablingcomplexexperimentstobeperformedinparallelonthe hybridchip. Figure4.3bshowshowthehybridIC/microfluidicchipcansimultaneouslytrap andmovemanyvesicles.infig.4.3bhundredsofvesiclesaresimultaneously positionedintoan H,demonstratingthatthechipcancontrolthepositionofmany vesiclesatonce.toaccomplishthis,manydeppixelsareturned on tospell H and thevesiclessedimentontotheinterfaceofthepixelsthatareturned on andthose thatareturned off.thisdemonstrationshowsthatthedepchipiscapableof simultaneouslycontrollingmanyindividualobjects. Figure4.3cshowshowthehybridIC/microfluidicchipcansimultaneouslytrapand movebothvesiclesandlivingcellsthataresuspendedinwater.yeastcellsare 67

82 culturedovernightinypdbroth(bdinc.)at37 C,andthenresuspendedina 250mMglucosesolution.Figure4.3cshowsavesicleandabuddingyeastcell trappedonthechip.thefluorescenceimageoftherhodaminefilledvesicleis superimposedontothebrightfieldimageoftheyeastcellsandiscoloredred.in Fig.4.3c(ii)thevesicleismovedupwardswhilethecellistrappedinplace.In Fig.4.3c(iii)theyeastcellismovedtotheleftwhilethevesicleisheldinplace.In Fig.4.3c(iv)theyeastcellismoveddownwardsasthevesicleissimultaneously movedtotheleft.thisdemonstrationshowsthatthedepchipcansimultaneously controlcellsandsmallvolumesoffluidheldinvesicles,bothsuspendedinwater. Thisenablesbiologicalandchemicalexperimentstobeperformedonthehybrid chipthatusebothcellsandsmallcompartmentalizedvolumesoffluid. 68

83 Figure4.3(a)TimesequenceofvesiclespositionedwithDEP.Pixelsareturnedon insequencetotrapandmovethevesicles.thegreenlineshowsthedirectionthat thechipmovesthevesicle.themaximumspeedofavesicleis70μm/sec.each frameisseparatedby~1sec,(b)amicrographofhundredsofvesicles simultaneouslypositionedtospellan H, (c)timesequenceofvesiclesandcells simultaneouslytrappedandmovedonthechip.thefluorescenceimageofthe vesiclesissuperimposedontothebrightfieldimageofthecellsandiscoloredred. Thegreenlineshowsthedirectionthatthechipismovingthevesiclesorcells.Each frameisseparatedby~1sec. 69

84 4.3.2TriggeredReleaseoftheContentsofVesicles InthissectionitisdemonstratedthatthehybridIC/microfluidicchipscan controllablyreleasethecontentsofvesicles.figure4.4ashowshowthehybrid IC/microfluidicchipcancontrollablyreleasethecontentsofavesicleintothe surroundingsolutionwhileholdingitinplacewithadeptrap.figure4.4ashowsa vesiclefilledwith4mmnaclsolutionsuspendedinaconcentratedfluorescently self quenchedfluorosceinsolution.a1mselectricfieldpulse,thatistime multiplexedwiththedepfiel,andrepeatsevery5ms,isturnedonattimet=0sec. Thevesicleiselectroporatedandtheconcentratedfluorosceinmixeswiththe solutioninthevesiclecausingthevesicletofluoresce.afterδt=8secthepulse sequenceisturnedoffandthevesicleisheldinadeptrapforδt=2minutes.the vesiclemaintainsitsfluorescence,demonstratingthatwhenthepulsesareturned offthevesiclehealsitselfandstopsmixingwiththesolution.afterδt=2minutes thepulsesequenceisturnedonuntiltheconcentrationoffluorosceininsidethe vesiclematchesthatofthesolutionandthevesicleceasestofluoresce.this demonstrationshowsthatthechipcanuseelectroporationtocontrollablymixthe contentsofvesicleswiththesolution.electroporationofvesiclescanbeusedto formchemicalgradientsinthesolutionsurroundingthevesicleortoallow substancesinthesolutiontomixwiththecontentsofthevesicle ElectroporationofCells InthissectionitisdemonstratedhowthehybridIC/microfluidicchipscan permeabilizeacell smembrane,allowingsubstancesinthesolutiontoenterthecell. 70

85 Figure4.4bshowsanindividualyeastcellheldinplacewithDEPandselectively electroporated,allowingsubstancesinthesolutiontoenterthecell.yeastcellsare culturedovernightinypdbroth(bdinc.)at37 Candresuspendedin250mM glucosesolution.a0.4%solutionoftheviabilitystain,trypanblue,isaddedtothe suspendedcells.whentrypanblueentersacellitstainsitblue.infig.4.4bayeast cellisheldinplacewithdep.becausethecellishealthy,trypanbluedoesnot enterthecellandthecelldoesnotturnblue.a1mspulsethatrepeatsevery5ms, thatistimemultiplexedwiththedepfield,isturnedonatt=0.afterδt=40sec thecellisobservedtoturndarkblue,asisshowninagrayscaleimageinfig.4.4b. Theabilitytoelectroporatecellsonthechipenablessubstancesinthesolutiontobe controllablyintroducedintoselectedcells.thisfunctionalitycouldbeusedto introducesubstancessuchasmolecularprobes,dna,ordrugsintoselectcells TriggeredFusionofVesicles InthissectionitisshownhowthehybridIC/microfluidicchipcanfusetwovesicles togetherandmixtheircontents.figure4.4cshowstwovesiclesthatarebrought intocontactwithdepandthenfusedtogetherwithelectrofusion.infig.4.4c(i) twovesiclesarebroughtintocontactwithdep.infig.4.4c(ii)thevesiclesare broughtintotightcontactwithdep,creatingalargecontactareabetweenthetwo vesicles.infig.4.4c(iii)a1mspulsethatrepeatsitselfevery5ms,multiplexed withthedepfield,isturnedonfor0.5seccausingthetwovesiclestofuseintoone. ThevesicleisstretchedintotheshapeoftheDEPtrap.InFig.4.4c(iv)thetrapis turnedoffandthefusedvesiclerelaxesintoasphericalshape. 71

86 ThefusionoftwovesiclesallowspLvolumesofsamplestobecontrollablymixed. Thevoltagepulsesusedtofusethevesiclesdonotleadtoanappreciablemixingof thecontentsofthevesiclewiththesolution.thevesiclesareobservedtofuseafter 0.5secofthepulsesequence.IntheexperimentshowninFig.4.4a,ittakes~4sec forasimilarvesicletohaveappreciablemixingwiththeoutsidesolutionusingthe samepulsesequence.thereforeinthetimethatittakestofusethevesiclesthere shouldnotbeappreciablemixingofthevesicleswiththesolution.theabilityto selectivelyfusetwovesiclestogetherenablessmallisolatedvolumesofsamplesto bemixedonthechip,animportantfunctionforperformingchemistryandbiology experiments. 72

87 Figure.4.4(a)TimesequenceofavesicleheldinplacewithDEPfieldswhileits contentsarereleasedintothesolutionwithelectroporation.thevesicleis suspendedinaconcentratedfluorosceinsolution.theelectroporationcausethe vesicletomixitscontentswiththesolution,makingthevesicletofluoresce.inthe firstthreeframes,eachseparatedby4seconds,thevesicleiselectroporated.the vesicleisheldinadeptrapfor2minutes.inthenextthreeframes,separatedby4 seconds,thevesicleiselectroporateduntilthecontentsofthevesiclefullymixes withthesolutionandthevesiclestopsfluorescing,(b)acellisheldinplacewith DEPandelectroplatedinasolutioncontainingTrypanBluestain.Theleftandright frameshowthecellbeforeelectroporationandafter40sec.ofelectroporation, respectively,(c)twovesiclesarebroughtintocontactwithdepandthenfused togetherwithelectrofusion.theschematiconthebottomshowwhichpixelsare turnedon.agreenpixelindicatesapixelthatisturnedonfordepandaredpixel indicatesapixelthatisturnedonwithvoltagepulsesmultiplexedwiththemhz frequencyvoltage. 73

88 4.3.5DeformingVesicleswithDielectrophoresis InthissectionitisshownhowthehybridIC/microfluidicchipcancontrollably deformobjects.figure4.5showshowchangingtheshapeofthedeptrapscan deformvesicles.severaldeppixelsareturnedonunderneathavesicle,causingthe vesicletobedeformedintotheshapeofthedeptrap.theleftcolumnoffig.4.5 showsamapofwhichpixelsonthechipareturnedon.themiddlecolumnshows simulations(ansoft:maxwell)oftheelectricfieldgenerated5µmabovethechip s surface.therightcolumnshowsmicrographsofvesiclesdeformedinthedeptrap. Thesamevesicleisusedforeachdemonstration.InFig.4.5athevesicleisheldina simpletrapof4pixelsandthevesicle scross sectioniscircular.infig.4.5bthetrap isspreadintotwodisplacedbars,andthevesicleispulledintoanoblongshape. MorecomplicatedpixelpatternsleadtoshapessuchasdiamondsinFig.4.5c, hexagonsinfig.4.5d,andsquaresinfig.4.5e.theabilitytodeformvesiclesusing DEPtrapsisanimportantproof of conceptforperformingrheologyandfor controllingthemechanicalenvironmentofobjectsonthechip.(riskeetal.,2006) Thistechniquecouldbeespeciallyusefulinfuturechips,thathavesmallerpixel sizes,forcontrollingthemechanicalenvironmentoflivingcells.(wangetal.,1994) 74

89 Figure4.5Vesiclesaredeformedintoavarietyofshapesbychangingtheshapeof thedeptrap.theschematicsontheleftshowwhichpixelsareturnedon.redand whitepixelsindicatepixelsthatareturnedonoroff,respectively.thecenter graphicsareplotsofthesimulatedelectricfieldstrength E 5µmabovethechip s surface.ontherightaremicrographsofthedeformedvesicles. 75

90 4.4Discussion Theplatformthatispresentedinthischaptertakesadvantageofthecapabilitiesof ICsandthepropertiesofunilamellarvesiclestomakeaversatileplatformfor biologicalandchemicalexperimentsonachip.thischapterhasdemonstrateda platformthatcantrap,move,deform,fuse,andlocallyreleasethecontentsofpl volumevesiclessuspendedinwater.onthesamechip,atthesametimeandinthe samesolution,theplatformcantrap,move,andpermeabilizelivingcellssuspended inwater.thisplatformprovidesanimportantstepforwardforperforming biologicalandchemicalexperimentsonahybridic/microfluidicchipby demonstratingfunctionsthatareimportantbuildingblocksformorecomplextasks. Tomovethislaboratory on a chipplatformfromthelaboratoryintoaportable, inexpensivetoolforbiologicalandchemicaltesting,furtherfunctionalitymustbe included.forinstance,whiletheplatformcanperformcomplexoperationsonmany vesiclesandcells,thesamplesthemselvesarepreparedonlaboratorybench equipmentandthenpipettedontothechip.integratingthehybridchipintoa complexmicrofluidicnetwork,wheresamplepreparationcouldbeperformed,could solvethisproblem.anotherhurdleforthehybridchipisthatitisconnectedto large,laboratorysizedequipment,suchasafluorescencemicroscopeandadesktop computer.furtherintegrationoftasksintothehybridchipssuchaslocal temperaturecontrol, (Issadoreetal.,2009)NMRsensors, (Leeetal.,2008) measurementofeletrogeniccells, (DeBusschereetal.,2001)andintegrated 76

91 optics(cuietal.,2008)wouldopenupmoreapplicationsandleadtoamore versatileandself sufficientplatform.(leeetal.,2007) 77

92 Chapter5.HybridMagneticand DielectrophoreticIC/MicrofluidicChip 5.1Overview Thischapterdescribesthedevelopmentofahybridintegratedcircuit(IC)/ microfluidicchipthatusesbothdielectrophoresis(dep)andmagneticforcesto controllivingcellsandsmallvolumesoffluid.thechipthatispresentediscalled thehighvoltagedielectrophoresis/magneticchip(hv DEPChip,Fabutron2.0). ThehybridchipisfabricatedusingaspecialhighvoltageICprocessthatenables DEPforcesthatareroughly100xasstrongasthosedemonstratedontheDEPChip inchapter3.amicrographofthehv DEP/MagneticChipisshowninFig.5.1a. ThecombinationofbothDEPandmagneticforcesonthesamechipexpandsthe capabilitiesofhybridic/microfluidictechnology,asisdemonstratedinthis chapter. TheHV DEP/MagneticChipissimilartotheDEPChipdescribedinChapter3,but includesadditionalfunctionality.theicincludesanarrayofpixelsthatcaneachbe addressedwitharadiofrequency(rf)voltagetolocallyapplydepforces,amatrix ofwiresthatrunsunderneaththedeppixelarraytoapplylocalmagneticforces, andintegratedsensorstolocallyreportthetemperatureofthechip.thelargearray ofdeppixels,magneticwires,andtemperaturesensorsarecontrolledusingstatic randomaccessmemory(sram)andlogicthatarebuiltintotheic.figure5.1a showsamicrographoftheic. 78

93 Previoushybridchipshavebeendevelopedtocontrollivingcellsanddropsoffluid usingelectricormagneticfields.chipshavebeendevelopedtosimultaneously controlthepositionofindividualpldropsoffluid(huntetal.,2008)andlivingcells (Manaresietal.,2003andHuntetal.,2008)usingdielectrophoresis(DEP)for biologicalandchemicalexperimentsonachipasisshowninchapter4.chipshave alsobeendevelopedtocreatelocal,programmablemagneticfieldsusingmatrixesof wires(leeetal.,2004)andarraysofcoils(leeetal.,2006)tocontrolcellsand biologicalobjectsimplantedwithmagneticnanoparticles.magneticactuationof nanoparticleshasbeenusedtoapplyprecisemechanicalstressesoncell membranes,(berryetal.,2003)manipulateandactivateindividualmechanically sensitiveionchannelsandsurfacereceptorsonspecificcells,(dobsonetal.,2008) andtotrapandsortcellsandothermagneticallytaggedobjects.(berryetal.,2003) ThischapterdemonstratestheHV DEP/MagneticChipuseitsDEPpixelarrayto positionplvolumesoffluidencapsulatedinlipidvesiclesinsection5.3.1,useits magneticmatrixtopositionmagneticbeadsinsection5.3.2,anduseitsdeppixel arrayandmagneticmatrixtogethertodeformmicroscopicobjectsimplantedwith magneticnanoparticlesinsection5.3.3.unilamellarvesiclesareusedasbiologically inspiredplcontainersforfluidsthatareimpermeableandstableforawiderangeof salinity,ph,andotherenvironmentalconditions,asisdescribedinchapter4.(chiu etal.,1999andtressetetal.,2007) 79

94 5.2DescriptionoftheChip 5.2.1FieldSimulations Thechipcreateselectricfieldsaboveitssurfacewitha60x61pixelarrayof 30x38µm 2 electrodesthatcanbeindividuallyaddressedwith50vpeak to peak voltagesatfrequenciesfromdcto10mhz.thepixelsareseparatedfromone anotherby0.8µm.infig.5.1btheresultsofquasi staticfiniteelementsimulations (Maxwell3D,Ansoft)oftheelectricfieldareshown.Twopixelsareheldat50V relativetothesurroundingpixels.theelectricfieldisplotted5µmabovethechip s surface.themaximummagnitudeoftheelectricfieldis E = 3 V /µm. Thechipuseselectricfieldstotrapandmovedielectricobjects,suchascellsorpL volumesoffluid,withdep.(manaresietal.,2003andhuntetal.,2008)aspherical objectinanelectricfield E willexperienceaforcegivenbyeq.2.3(jonesetal., 1995)Foranobjectwitharadiusa=5µmwiththedielectricpropertiesofaliving cellorvesiclesuspendedindeionizedglucosesolution,a F 1 nn forcepullsthe objecttowardstheactivatedpixelsfromalocationonepixel lengthaway.avesicle filledwithsalinesolutionhasdielectricpropertiessimilartothatofalivingcellin electricfieldsatmhzfrequencies,andthusbehavessimilarlyinthedepfield.(chiu etal.,1999) BeneaththeDEPpixelsisamagneticgridthatconsistsof60wiresthatrun horizontallyand60wiresthatrunverticallyacrossthechip,whichareusedtoform amagneticfieldabovetheic ssurface.thewirescanbeindividuallysourcedwith ±120mAor0mA.Fig.5.1cshowstheresultsofquasi staticsimulations5µmabove 80

95 thechip ssurface.twowiresthatruninperpendiculardirectionsacrossthechip aredrivenwith120maandallotherwiresarenotdrivenwithcurrent.the maximummagnitudeofthemagneticfieldis B =6mT. Thechipusesmagneticfieldstotrapandmoveobjectswithmagneticsusceptibility differentthanthesurroundingmedium.asphericalobjectinamagneticfield B willexperienceaforcegivenbyeq.2.12.onthechipanironoxidebeadwitha radiusa=1µm(bioclone:ff102)willexperiencea10pntowardsthemaximumof themagneticfield,whichexistswherethetwoactivatedwiresintersect.objectscan betrappedandmovedalongarbitrarypathsbychangingthelocationwherethe activatedwirescross.currentcanbedriventhroughseveralwiresinthearray simultaneouslytocreatemorethanonefieldmaximum,allowingthechiptotrap andmovemanymagneticobjectsindependently.(leeetal.,2004) 81

96 Figure5.1.(a)Amicrographoftheintegratedcircuit,(b)themagnitudeofthe electricfield E,fromaquasi staticelectricfieldsimulation,plotted5µmabove thechipsurface.thepixelsareshownasbluetilesthatcoverthesurfaceofthechip. Twopixelsareheldat50Vrelativetothesurroundingpixels,(c)themagnitudeof themagneticfield B,fromsimulation,plotted5µmabovethechip ssurface.the wiresareshownasbluestripesrunningacrossthesurfaceofthechip.twowires aresourcedwith120maandallsurroundingwiressetto0ma. 82

97 5.2.2ChipArchitecture Thechip slargearrayof60x61(3,660)deppixels,60horizontaland60 vertical(120)magneticwires,and16temperaturesensorsareaddressedwithlogic andmemorybuiltintotheic,suchthatonly6datalines(notincludingtwoclocks andthe4controllines)arerequiredtoupdatetheentirechip.figure5.2ashowsa micrographofasectionofthechipthatshowsthedeppixelarray,thecurrent driversforthemagneticmatrix,andthedigitallogicthatisusedtoupdateandread fromthearray.thestateofthechipisstoredinanintegratedsrammemorythat controlsthedeppixels,magneticwires,andthetemperaturesensors. TheSRAMmemoryisorganizedinto32wordsthathave128bits.ThereisanSRAM memoryelementunderneatheachdeppixel,temperaturesensor,andcurrent driverforthemagneticwires.thememoryarrayisupdatedbyselectingaword line andthenloadingitsstateusinga128bitshiftregister.theshiftregisterusesatwophaseclockingscheme.wordselectionisdonewithastandard5bitrowdecoder. TheentireSRAMarraycanbeupdatedatarateof~50Hzandindividualwordsin thesramcanbeupdatedatarateof~1.5khz.thelogic,memory,andsurrounding electronicsthatareusedtoupdatethechiparesimilartothatusedforthedepchip describedinchapter3. AschematicforthecircuitunderneatheachDEPpixelisshowninFig.5.2b.The stateofthesrammemoryelementcontrolsthe2:1multiplexer(mux)thatdirects eithertherfsignaloritslogicalinversetothevoltagedrivingcircuitthatconnects tothepixel.aschematicforeachmagneticlineisshowninfig.5.2c.themagnetic 83

98 lineshavedrivingcircuitryonbothends.bysettingthememoryelementsoneither sideofthewireacurrentof±120maor0maflowsthroughthewire.interspersed throughoutthearrayare16distributedtemperaturesensorsthatcanbe individuallyaddressedtoberoutedtoananalogvoltageoutput. ThehybridchipisconstructedbybuildingasimplefluidcelldirectlyontopoftheIC withasiliconegasket.thefluidcellispreparedbycuttinga1.2mmholeoutof Press to Seal TM 0.5mmthicksiliconesheetsfromInvitrogen(Invitrogen:p 24744) withahole punch.thesiliconegasketisplacedontotheicunderastereoscope withtweezers.a3x3mm 2 glasscoverslipsealsthefluidcell.theintegrated circuitwasdesignedatharvardusingcadencedesignsoftware(cadence)and fabricatedinacommercialfoundryonahighvoltage0.6µmprocess(x Fab XC06 MIDOX). TheexperimentalsetupsurroundingthehybridIC/microfluidicchipissimilarto thatofthedepchipdescribedinchapter3.thedevicesitsonachipcarrierona customprintedcircuitboard(pcb)connectedtoacomputerthroughapci card(nationalinstruments:pci 6254).Thepatternssenttothechiparecreated usingaguiwritteninmatlab(themathworks)andsenttothepcicardwitha customlabview(nationalinstruments)program.thedevicesitsunderan OlympusfluorescencemicroscopewithanOrca ER(Hammamatsu)digitalcamera thatconnectstothecomputer. 84

99 Figure5.2.(a)AmicrographofapartoftheintegratedcircuitshowingtheDEP array,themagneticcurrentdrivers,andthelogicandmemoryusedtoupdatethe SRAMarray,(b)aschematicofthecircuitryunderneatheachDEPpixel,showingthe SRAMmemoryelement,amultiplexer(MUX)thatdirectseithertheRFsignalorits logicalinversetoavoltagedriverthatconnectstotheelectrode,(c)aschematicof thecircuitryforeachmagneticwireinthematrix,showingthesrammemory elements,andthemuxsthatconnecttothecurrentdriverstodrivethewireswith either±120maor0ma. 85

100 5.3Demonstrations ThissectiondemonstratestheHV DEP/MagneticchipusingDEPtotrapandmove dielectricobjects,magneticforcestotrapandmovemagneticallytaggedobjects,and magneticanddepforcesusedtogethertodeformobjectsimplantedwithmagnetic nanoparticles.section5.3.1showshowthehybridchipcanbeusedtotrapand moveobjectsalongprogrammablepathswithdep.section5.3.2showshowobjects thataretaggedwithmagneticbeadscanbetrappedandmovedalong programmablepathsusingthemagneticmatrix.section5.3.3demonstratesthe utilityofhavingbothelectricandmagneticforcesonthesamechipbyholdinga unilamellarvesicleembeddedwithironoxidenanoparticleinplacewithdepand controllablypullingathintetherfromthevesicle smembraneusingthemagnetic matrix. Thevesiclesarepreparedwithliposomeelectroformationusingamodificationof theangelovamethod,asisdescribedindetailinchapter4.(angelovaetal.,1986) Theunilamellarvesiclesareloadedwith4mMNaClandsuspendedinaglucose solutionwithmatchedosmolarity,whichprovidescontrastinthecomplexdielectric responsetofacilitatedepforces.(huntetal.,2008)thevesicles membranesare stainedwithrhodaminesuchthatthevesiclesareeasilyobservableundera fluorescencemicroscope Dielectrophoresis: Trapping and Positioning Vesicles ThissectiondemonstrateshowtheHV DEP/MagneticChipsimultaneouslyand independentlycontrolsthepositionofobjectswithdep.figure5.3shows 86

101 unilamellarvesiclesthataretrappedandmovedalongprogrammedpathsusingthe DEPpixelarray.Figure5.3ashowstwovesiclesthatareindependentlytrappedon thechipwithtwosetsofdeppixels.infig.5.3(a d)thevesiclesaremovedat speedsupto80μm/secbysequentiallychangingthepixelsthatareturnedon.in Fig.5.3athevesicleontherightisindependentlymoveddownwardswhilethe vesicleontheleftisheldinplace.infig.5.3bboththeleftandrightvesicleare moveddownwardssimultaneously.infig.5.3cthevesicleontheleftis simultaneouslymovedtotherightwhilethevesicletotherightismovedtotheleft. Thechiphas3,660pixelsandtheentirearraycanberefreshedat~50Hz,enabling manyindividualvesiclestobecontrolledsimultaneously.theabilitytotrapand moveobjectswithdepisausefultooltocontrolthepositionofplvolumesoffluid andlivingcellsthatarenottaggedormodified. 87

102 Figure5.3AtimesequenceoftheDEPpositioningofvesicles.Theframes(a d)are fluorescenceimagesoftherhodaminestainedmembranesofthevesicles.thedep pixelsareturnedoninsequencetopositionthevesicles.thedashedgreenline showsthedirectionthatthechipismovingthevesicles,theyellowsquareshows thedeppixelthatisactivated.themaximumspeedofavesicleis80μm/sec.each frameisseparatedbyroughly1sec. 88

103 5.3.2Magnetophoresis: Trapping and Positioning Magnetic Beads ThissectiondemonstrateshowtheHV DEP/MagneticChipcancontroltheposition ofobjectswithmagnetophoresis.theabilitytotrapandmovemagneticobjects alongprogrammedpathsisausefultooltocontrolthepositionofsamplesthat cannotbetrappedwithdepforces,butwhichcanbetaggedwithmagnetic particles.(leeetal.,2007)figure5.4showsthemagneticmatrixtrappingand movinganironoxidebeadwitharadiusa=1µm(bioclone:ff102)alongan arbitrarypath.infig.5.4atwoperpindicularwiresaredrivenwith120ma,such thatthebeadistrappedatthefieldmaximumthatoccursatthewire sintersection. InFig.5.4(b d)differentintersectingwiresareturnedontomovethebeadalonga programmedpathatspeedsof2µm/s.themagneticforceisnotaslocalizedasthe DEPforce.Alongthelengthofeachintersectingwire,particlesareattractedtothe activatedwire. 89

104 Figure5.4Timesequenceofmagnetictrappingandpositioningofamagneticbead. Magneticwiresinthematrixareturnedoninsequencetopositionthebead.The positionoftheparticleismarkedwithagreencircle.thedirectionthatthebeadis beingpulledismarkedwithagreenarrow.theredlinesshowswhichmagnetic wiresinthematrixhavebeenturnedon,theactualwiresareburiedunderthedep pixelsandarenotvisible.eachframe(a d)isseparatedbyroughly2sec. 90

105 5.3.3DielectrophoresisandMagnetophoresis:DeformingVesicles ThissectiondemonstrateshowtheHV DEP/MagneticChipcanusemagneticand DEPforcestogether.Figure5.5showthechipholdingavesicle,thatisimplanted withironoxidenanoparticles,inplacewithdepbodyforceswhilepullingathin tetherfromthevesicle smembranebyapplyingpointforcesonthemagnetic nanoparticleswithmagnetophoresis.thisdemonsrationshowsthatdepand Magnetophoresiscanbeusedtogethertoapplypointforceswithnmresolutionto micrometersizedobjects.(waughetal.,1992)infig.5.5aavesicleispositioned withdeppixels.twomagneticwiresthatcrossnearthedeppixelaresourcedwith 120mAandtheironoxidenanoparticlesinsidethevesiclearepulledtowardsthe magneticfieldmaximum.thevesicle smembraneislocallydeformedintoathin tether,asvesicleshavebeenshowntodointhepresenceofapointforce.(waughet al.,1992)intheproceedingframesfig.5.5(b f)themagneticfieldisturnedoffand tensioninthemembranepullsthetetherbackintothevesicle.(waughetal.,1992) TheabilitytolocallydeformmicroscopicobjectsusingDEPasabodyforceand magnetophoresisasapointforceisausefultooltoapplyprecisemechanical stressesonvesiclesandlivingcells.(berryetal.,2003,dobsonetal.,2008,and Tressetetal.,2007) 91

106 Figure5.5AvesicleisheldinplacewithaDEPpixelwhileatetherispulledusing themagneticmatrix.theyellowsquareshowsthedeppixelthatisactivated,and theredlinesshowthemagneticwiresthatareturnedon.thevesicleisimplanted withironoxidenanoparticles.afterthefirstframe(a)themagneticfieldisturned offandthetetherispulledbackintothevesicle.eachframe(a f)isseparatedby0.2 seconds. 92

107 5.4Discussion HybridIC/microfluidicchipsprovideaversatileplatformtocontrolsmallvolumes offluidandlivingcells.thecombinationofbothmagnetic (Leeetal.,2004andLee etal.,2007)anddepforces(manaresietal.,2003andhuntetal.,2008)ontoa singlechipfurtherexpandsthecapabilitiesandgeneralityoftheplatform. Dielectrophoresiscanbeusedtotrapandpositionuntaggedlivingcellsanddropsof fluidandmagneticforcescanbeusedtotrapandpositionobjectstaggedwith magneticnanoparticles.dielectrophoresisandmagnetophoresiscanbeused togethertoapplyprecisenm resolutionpointforcestomicrometer sizedobjects. Magneticactuationofnanoparticlesembeddedincellsorvesicleshasmanyexciting scientificapplications.thetechniquedemonstratedinthischapter,pullingathin tetherfromaunilamellarvesicleandobservingthevesiclepullthetetherbackinto itself,canbeusedasatooltostudythecomplexmechanicalpropertiesoflipid bilayers.(waughetal.,1992)anumberofexcitingapplicationsexistfor magneticallyactuatingcellstaggedwithmagneticnanoparticles,suchasapplying precisemechanicalstressesoncellmembranes(berryetal.,2003)ormanipulating andactivatingindividualionchannelsandsurfacereceptorsonspecificcells. (Dobsonetal.,2008) Thetechniquesdemonstratedinthischaptercanbecombinedwiththe laboratoryon a chip functionsdemonstratedinchapter4,tocreateageneral purpose platformforbiologyandchemistryexperimentsonindividuallivingcells.the techniquesdemonstratedinchapter4canbeusedtomixsmallvolumesofreagents 93

108 andsamplestogetherandtocontrolthechemicalenvironmentofindividualcells. Themagneticactuationofnanoparticles,demonstratedinthischapter,canbeused tocontrolcellsembeddedwithmagneticnanoparticlestoactivateordeactivate specificsurfacereceptorsorionchannels.thehybridchipscansimultaneously performmanyoftheseexperimentsinparallel,allowingastatisticalnumberof independentlycontrolledsingle cellexperimentstobecarriedout. 94

109 Chapter6.MicrowaveDielectricHeatingof DropsinMicrofluidicDevices 6.1Overview Inthischapteratechniqueispresentedtolocallyandrapidlyheatdropsofwater withmicrowavedielectricheating.thistechniqueaddsakeyfunctionalitytothe listoffunctionsthataredemonstratedinchapters3,4,and5,theabilitytocontrol temperature.waterabsorbsmicrowavepowermoreefficientlythanpolymers, glass,silicon,oroils,duetoitspermanentmoleculardipolemomentthathasalarge dielectriclossatghzfrequencies.thisselectiveabsorptionofmicrowavepowerby waterallowsthermalenergytobedirectlyinsertedintosmallvolumesoffluidina microfluidicdevicewithoutsignificantlyheatingthesurroundings.therelevant heatcapacityofsuchasystemisthatofasinglethermallyisolatedpicoliter scale dropofwater,enablingveryfastthermalcycling. Inthischaptermicrowavedielectricheatingisdemonstratedinamicrofluidic devicethatintegratesaflow focusingdropmaker,dropsplitters,andmetal electrodestolocallydelivermicrowavepowerfromaninexpensive,commercially available3.0ghzsourceandamplifier.thetemperaturechangeofthedropsis measuredbyobservingthetemperaturedependentfluorescenceintensityof cadmiumselenidenanocrystalssuspendedinthewaterdrops.characteristic heatingtimesaredemonstratedthatareasshortas15mstosteady state temperaturechangesaslargeas30 Cabovethebasetemperatureofthe 95

110 microfluidicdevice.manycommonbiologicalandchemicalapplicationsrequire rapidandlocalcontroloftemperatureandcanbenefitfromthisnewtechnique. Microwavedielectricheatingiswellsuitedtobeintegratedwiththehybrid integratedcircuit(ic)/microfluidicchipsdescribedinchapters3,4,and5.using modernictechnologyanddesigningthecircuitswithappropriateradiofrequency designprincipals,pixelscanbedrivenwithghzfrequencyvoltages.ahybrid IC/microfluidicchipwithelectrodesthatcanbedrivenwithvoltagesatGHz frequencies,couldindividuallycontrolthetemperatureofthermallyisolated,small volumesoffluidusingthetechniqueoutlinedinthischapter. Agrowinglibraryofelementsformicrofluidicchipshavebeendevelopedinrecent yearsfortaskssuchasthemixingofreagents,detectingandcountingcells,sorting cells,geneticanalysis,andproteindetection.(whitesidesetal.,2001,stoneetal., 2004,Tabelingetal.,2005,Yageretal.,2006,Martinezetal.,2008,Leeetal.,2007, Huntetal.,2008,Maltezosetal.,2005)Thereisonefunction,however,thatis crucialtomanyapplicationsandwhichhasremainedachallenge:thelocalcontrol oftemperature.thelargesurfaceareatovolumeratiosfoundinmicrometer scale channelsandthecloseproximityofmicrofluidicelementsmaketemperature controlinsuchsystemsauniquechallenge.(maltezosetal.,2005,leeetal.,2005) Muchworkhasbeendoneinthelastdecadetoimprovelocaltemperaturecontrolin microfluidicsystems.themostcommontechniqueusesjouleheatedmetalwires andthinfilmstoconductheatintofluidchannels.(nakanoetal.,1994,lagallyetal., 2000,Liuetal.,2002,Khandurinaetal.,2000)Thethermalconductivityof 96

111 microfluidicdevicescontrolthelocalizationofthetemperaturechangeandtendsto beontheorderofcentimeters.(nakanoetal.,1994andkhandurinaetal.,2000) Temporalcontrolislimitedbytheheatcapacityandthermalcouplingofthe microfluidicdevicetotheenvironmentandthermalrelaxationtimestendtobeon theorderofseconds.(nakanoetal.,1994andkhandurinaetal.,2000)alternative techniquestoimprove thelocalizationandresponsetimehavebeendeveloped, suchasthosethatusenon contactinfraredheatingofwateringlassmicrofluidic systems(odaetal.,1998)andintegratedmicrometersizepeltierjunctionsto transferheatbetweentwochannelscontainingfluidatdifferenttemperatures. (Maltezosetal.,2005)Fluidshavealsobeencooledonmillisecondtimescaleswith evaporativecoolingbypumpinggassesintothefluidchannels.(maltezosetal., 2006) Thefocusoftheresearchdescribedinthisthesisistointegrateelectronicswith microfluidicstobringnewcapabilitiestolab on a chipsystems.thischapter presentsatechniquetolocallyandrapidlyheatwaterindropbasedmicrofluidic systemswithmicrowavedielectricheating.thedevicesarefabricatedusingsoft lithographyandareconnectedtoinexpensivecommerciallyavailablemicrowave electronics.thisworkbuildsonpreviousworkinwhichmicrowaveshavebeen usedtoheatliquidinmicrofluidicdevices(shahetal.,2007,sundaresanetal.,2005, Sklavounosetal.,2006,andGeistetal.,2007)byachievingsignificantlyfaster thermalresponsetimesandagreatertemperaturerange.inourdevice,dropsof waterarethermallyisolatedfromthebulkofthedeviceandthisallows exceptionallyfastheatingandcoolingtimesτs=15mstobeattainedandthedrops 97

112 temperaturetobeincreasedby30 C.Thecouplingofmicrowaveelectronicswith microfluidicstechnologyoffersaninexpensiveandeasilyintegratedtechniqueto locallyandrapidlycontroltemperature. 6.2ModelofDielectricHeatingofDrops Duetowater slargedielectriclossatghzfrequencies,microwavepoweris absorbedmuchmorestronglybywaterratherthanpdmsorglass.thedevice describedinthischapteroperatesat3.0ghz,afrequencyveryclosetothatof commercialmicrowaveovens(2.45ghz),thatisbelowthefrequencyassociated withtherelaxationtimeofwaterbutwherewaterstillreadilyabsorbspower.itis inexpensivetoengineerelectronicstoproduceanddeliver3.0ghzfrequencies becauseitisnearthewell developedfrequenciesofthetelecommunications industry. Twoindependentfiguresofmeritdescribetheheater,thesteady statechangein temperatureδtssthatthedropsattainandthecharacteristictimeτsthatittakesto changethetemperature.thesteady statetemperatureoccurswhenthemicrowave powerenteringthedropequalstheratethatheatleavesthedropintothethermal bath.thethermalrelaxationtimedependsonlyonthegeometryandthethermal propertiesofthedropsandthechannelandisindependentofthemicrowave power. Todescribetheheaterasimplifiedmodelisusedinwhichthetemperatureofthe channelwallsdonotchange.thethermalconductivityoftheglassandpdms channelwallsismuchlargerthanthatofthefluorocarbon(fc)oilinwhichthe 98

113 dropsaresuspended,whichallowtheglassandpdmsmoldtoactasalargethermal reservoirthatkeepthechannelwallspinnedtothebasetemperature. Thedropismodeledashavingaheatcapacitythatconnectstothethermalreservoir throughathermalresistance.thedrophasaheatcapacityc=vcwthatconnectsto thethermalreservoirwithathermalresistancer=ld/akoil,wherevisthevolume andaisthesurfaceareaofthedrop,cwistheheatcapacitypervolumeofwater,ld isthecharacteristiclengthbetweenthedropandthechannelwall,andkoilisthe thermalconductivityoftheoilsurroundingthedrop.asteady statetemperature ΔTssisreachedwhenthemicrowavepowerPVenteringthedropisequaltothe powerleavingthedropδtsskoila/ld.thesteady statetemperatureδtss=pvris: ΔT SS = V A L D k oil P. 6.1 Thesystemhasacharacteristictimescaleτs=RCthatdescribesthethermal responsetimeofthesystem, τ S = V A L D k oil C W. 6.2 Thecharacteristictimescaleτsdescribesthethermalrelaxationtime,thetimeit takesforthedroptoreachanequilibriumtemperaturewhenthemicrowavepower isturnedonandthetimethatittakesforthedroptoreturntothebasetemperature ofthedevicewhenthemicrowavesareturnedoff. 99

114 Thissimplifiedmodeldescribesseveralkeyfeaturesofthemicrowaveheater.The steady statetemperatureislinearlyproportionaltothemicrowavepower,whereas thecharacteristicthermalrelaxationtimeisindependentofthemicrowavepower. Thecharacteristictimeandthesteady statetemperaturearebothproportionalto thevolumetosurfaceratioofthedrops.atrade offrelationexistsbetweentherate ofheating1/τsandthesteady statetemperature,wherebyalargervolumeto surfaceratioreducesthethermalrelaxationtimeoftheheaterbutdecreasesits steady statetemperatureforagivenmicrowavepower,andviceversa.similarlyan increaseintheratioofthecharacteristiclengthbetweenthedropandthechannel wallandthethermalconductivityoftheoilld/koilincreasesthethermalrelaxation timeandincreasesthesteady statetemperature. 6.3TheMicrofluidicDevice Thedevicesarefabricatedusingpoly(dimethylsiloxane)(PDMS) on glassdropbasedmicrofluidics.microwavepowerislocallydeliveredviametalelectrodesthat aredirectlyintegratedintothemicrofluidicdeviceandthatrunparalleltothefluid channel,asisshowninfig.6.1a.thedropsarethermallyinsulatedfromthebulkby beingsuspendedinlowthermalconductivityoil.aschematicofthedeviceisshown infig.6.2a.syringepumpsprovidetheoilandwateratconstantflowratestothe microfluidicdevice.adropmakerandtwodropsplittersinseriescreatedropsthat areproperlysizedforthemicrowaveheater.themicrowavepoweriscreatedoff chipusinginexpensivecommercialcomponents. 100

115 Dropsarecreatedusingaflow focusinggeometry(annaetal.,2003)asisshownin Fig.6.2b.Afluorocarbonoil(FluorinertFC 40,3M)isusedasthecontinuousphase andtheresultingdropscontain0.1μmofcarboxylcoatedcdsenanocrystal (Invitrogen)suspendedinaphosphatebufferedsaline(PBS)solution.Asurfactant comprisedofapolyethyleneglycol(peg)headgroupandafluorocarbontail (RainDanceTechnologies)isusedtostabilizethedrops.(Holtzeetal.,2008)The wallsofthemicrofluidicchannelsarecoatedwithaquapel (PPGIndustries)to ensurethattheyarepreferentiallywetbythefluorocarbonoil.fluidflowis controlledviasyringepumps. 101

116 Figure6.1(a)Aschematicofthemicrowaveheater.Theblacklinesrepresentthe metallineswhichareconnectedtothemicrowavesource,thecenterfluidchannel carriesdropsofwaterimmersedinfluorocarbon(fc)oil,(b)acrosssectionofthe microwaveheaterwithaquasi staticelectricfieldsimulationsuperimposedis shown,theelectricfieldisplottedinlogscale,(c)thecalibrationcurveofthecdse nanoparticleswhichareusedastemperaturesensors,wherethecirclesaredata pointsandtheredlineisthefit. 102

117 Tomakedropssmallerthanthechannelheight,andthusseparatedfromthewalls ofthechanneltoensureadequatethermalisolation,dropsplittersareused(linket al.,2004)asisshowninfig.6.2c.thedropsplittersaredesignedtobreakeach dropintotwodropsofequalvolume.passingasphericaldropthroughtwodrop splittersinseriesdecreasestheradiusofadropbyafactorof(½) ⅔ =0.63.Drop splittersallowthedevicetobemadeinasinglefabricationstep,becausethey removethenecessityofmakingthedropmakerwithachannelheightsmallerthan therestofthedevice.(annaetal.,2003) ThemetalelectrodesaredirectlyintegratedintothePDMSdeviceusingalow melt solderfilltechnique.(siegeletal.,2006)themasksforthesoftlithographyprocess aredesignedtoincludechannelsforfluidflowandasettobefilledwithmetalto formelectrodes.afterinletholeshavebeenpunchedintothepdmsandthepdmsis bondedtoaglassslide,themicrofluidicdeviceisplacedonahotplatesetto80 C.A 0.02inchdiameterindiumalloywire(Indalloy19;52%Indium,32.5%Bismuth, 16.5%TinfromIndiumCorporation)isinsertedintotheelectrodechannelinlet holesand,asthewiremelts,theelectrodechannelsfillwithmetalviacapillary action.theresultingelectrodechannelsrunalongeithersideofthefluidasis showninfig.6.2d.tokeepthedropsfromheatingfromthefringeelectricfields beforethedropenterstheheaterthefluidchannelisconstrictedtopressthedrops againstthepdmswall,whichkeepsthedropsatthesametemperatureasthebase temperatureofthemicrofluidicdevice. 103

118 Theelectronicsthatcreatethemicrowavepowerareassembledusinginexpensive modules.themicrowavesaregeneratedwithavoltagecontrolledoscillator(zx S+,Mini Circuits)andamplifiedtoamaximumof11.7Vpeak to peakwitha maximumpowerof26dbmwithapoweramplifier(zrl 3500+,Mini Circuits).The microwaveamplifierconnectswithacabletoasubminiatureassembly(sma) connectormountednexttothemicrowavedeviceasisshowninfig.6.2e.copper wiresapproximately2mminlengthconnectthesmaconnectortothemetal electrodesinthepdmsdevice.ourelectronicsoperateat3.0ghzwherewater s microwavepowerabsorptionisroughly1/3asefficientasatthefrequency associatedwithwater srelaxationtime(~18ghz)butwhereelectronicsare inexpensiveandcommerciallyavailable.theelectronicsusedinoursystemcosts lessthan$us200andareeasytosetup. Finiteelementsimulationsareperformedtodeterminetheelectricfieldstrengthin themicrowaveheaterwhichisusedtocalculatethemicrowavepowerabsorbedby thedrops.figure6.1bshowsaschematiccross sectionofthemicrofluidicdevice wherethedropspassbetweenthemetalelectrodes.thechannelcross sectionhas dimensions50x50μm 2.Paralleltoand20μmawayfromeachsideofthefluid channelaremetallinesthatare100μmwideand50μmhigh.superimposedonthe schematicinfig.6.1bisaquasi staticelectricfieldsimulationoftheelectricfield (Maxwell,Ansoft).Fora12Vpeak to peaksignalacrossthemetallines,therms electricfieldwithinadropwitha15μmradiussuspendedinfluorocarbonoilis E ~ V/m.Theelectricfieldlinearlyscaleswiththevoltageacrossthemetal lineswhichallowsustocalculatethefieldwithinthedropforanyvoltage.the 104

119 simulatedelectricfieldiscombinedwitheq.2.1andeq.2.2tocalculatethe microwavepowerthatentersthedropswhichmaybecombinedwitheq.6.1to predictsteady statetemperaturechanges. Thetemperaturechangeofthedropsismeasuredremotelybyobservingthe temperature dependentfluorescenceofcdsenanocrystalssuspendedinthe drops.(maoetal.,2002)tocalibratethisthermometer,themicrowavepoweris turnedoffandahotplateisusedtosetthetemperatureofthemicrofluidicdevice. ThefluidchannelisfilledwithCdSenanocrystalssuspendedinwaterandthe temperatureofthehotplateisslowlyincreasedfrom25 Cto58 Cwhilethe fluorescenceintensityofthecdsequantumdotsismeasured.themeasured fluorescenceintensityisplottedasafunctionoftemperatureinfig.6.1c.alineisfit withaslope0.69%/ C±0.03%/ Ctothedataandthisslopeisusedtoconvert fluorescencemeasurementsintomeasurementsofthechangeintemperature.a lineisfittothedatausingaleast squarestechniqueandtheerroristheuncertainty inthecoefficientofthefit.acalibrationcurveistakenimmediatelybeforean experiment.thereisnoevidencethatthecdsenanoparticlesleakfromthedrops intotheoilorprecipitateontothemicrofluidicchannel.themicrofluidicchannel andtheoilinthewastelinearecheckedaftertheexperimentsandthereisno measuredfluorescencesignal.thedeviceismonitoredwithanhamamatsuorca ERcooledCCDcameraattachedtoaBX 52Olympusmicroscope.Imagesaretaken withmicrosuitebasiceditionbyolympusandanalyzedinmatlab(the MathWorks,Inc.).ThemicrofluidicdeviceisconnectedwithanSMAtothe 105

120 microwaveamplifierandsitsontopofahotplateunderneaththemicroscopeasis showninfig.6.2d. Thedevicesaretestedbymeasuringthetemperaturechangeofwaterdropsasthey travelthroughthemicrowaveheater.along exposurefluorescenceimageofmany dropstravelingthroughthemicrowaveheatershowstheensembleaverageofthe temperaturechangeofdropsateachpointinthechannel.aplotofthedropheating intimemaybeextractedfromthisimageusingthemeasuredflowrateofthedrops throughthemicrofluidicsystem.anexperimentisperformedwiththeconstant volumetricflowrateofthewaterat15μl/hrandtheoilat165μl/hr.abright field,shortshutterspeedimageistakenofthedropstravelingthroughthe microwaveheaterandthedrops averagediameterismeasuredtobe35μm.the microwaveheateristurnedonwithafrequencyof3.0ghzandapeaktopeak voltageof11v.alongexposure(2seconds)fluorescenceimageistakenofthe microwaveheaterthatisnormalizedagainstimagestakenwiththemicrowaves turnedofftoremoveartifactsthatarisefromirregularitiesinthegeometryofthe channel,thelightsource,andthecamera. 106

121 Figure6.2(a)Aschematicofthemicrowaveheatingdevice,showingtheoilin orange,thewaterinblue,andtheelectronicconnectionsinred,(b)amicrographof theflow focusingdropmaker,(c)thetwosetsofdropsplittersinseries,(d)a micrographofthemicrowaveheater,thedarkregionsthatrunparalleltothefluid channelarethemetallines,(e)aphotographofthemicrofluidicdevice,connectedto themicrowaveamplifierwithacablewithansmaconnector,ontopofahotplate, underneaththefluorescencemicroscope. 107

122 6.4Demonstration Thedropsareheatedtoasteady statetemperaturechangeδtssastheypass throughthemicrowaveheater.figure6.3ashowsthenormalizedfluorescence intensityofthedropsastheyenterthemicrowaveheatersuperimposedontoa bright fieldimageofthedevice.itcanbeseenthatasthedropsenterthechannel theiraveragefluorescenceintensitydropswhichshowsthattheyarebeingheated. Alineaverageofthenormalizedimageistakeninthedirectionperpendiculartothe fluidflowandisplottedagainstthelengthofthechannel,asinfig.6.3b.asthe dropsareheatedtheaveragefluorescenceintensityofthedropsfallsexponentially withdistanceto85%ofitsinitialintensityafterapathlengthof300μm.the fluorescenceintensitymeasuresthetemperaturechangeofthedrops,andsothe dropsareheatedinacharacteristiclengthof300μm. Thedropsareheatedtosteady statetemperaturechangesδtssaslargeas30 C abovethebasetemperatureofthemicrofluidicdeviceinonlyτs=15ms.the averagetemperaturechangeofthedropsasafunctionoftimeanddistancetraveled isplottedinfig.6.3c.forthisheatingpower,thetemperaturerisestoasteadystatevalueof26 Cabovethebasetemperatureof21 Cin15ms.Thecurvein Fig.6.3cisarrivedatbyusingthecalibrationcurveoftheCdSenanocrystals, Fig.6.1c,toconvertthefluorescenceintensityinFig.6.3bintoachangein temperatureδt.thesumoftheflowratesoftheoilandwaterareusedtocalculate thespeedofthedropsthroughthechannel,whichmaybeusedtotransform the 108

123 lengthinfig.6.3aintothetimethatthedropshavespentintheheater.eachdata pointconsistsoftheaverageof20independentlytaken2secexposures. 109

124 Figure6.3(a)A2secondexposureofthefluorescencesignalnormalizedtoan imagetakenwiththemicrowavesourceturnedoffsuperimposedontoabrightfield imageofthedevice,(b)thelineaverageofthenormalizedintensityplottedversus distancedownthechannel,(c)thetemperaturechangeδtplottedversustimeand versusthedistancethedropshavetraveleddownthechannel. 110

125 Thecombinedstatisticalerrorofthemeasurementoftheheatingisalsoplottedin Fig.6.3c.Theaverageerroris~1.4 C.Thestatisticalerrorinthesteady state temperatureisfoundtocomeprimarilyfromvariationsinthesizeofthedrops.the steady statetemperaturechangeofeachdropislinearlyrelatedtothevolume toarearatioofthedropandtothedistanceofthedroptothechannelwalls,asis describedineq.6.1.byobservingthedropsexitthemicrowaveheatingregionof thechip,thedropsarefoundtocoolwithacharacteristictimesimilartotheheating time.inaddition,bynarrowingthechannelattheexitofthemicrowaveheater,the dropsarebroughtclosertothechannelwallandthedropsreturntothebase temperatureoftheoilinlessthan1ms. Thesteady statetemperaturechangeδtssofthedropsmaybesetfrom0 Cto30 C byvaryingtheappliedmicrowavepowerasisdescribedineq.6.1.themicrowave poweriscontrolledbyexperimentallyvaryingthepeak to peakvoltageofthe appliedmicrowavevoltagewhichvariesthestrengthoftheelectricfieldinsidethe dropsasisdescribedbyoursimulations.aseriesofplotsofthechangein temperatureversustimefordifferentappliedpowersisshownintheinsetof Fig.6.4a,andshowssteady statetemperaturechangesrangingfrom2.8 Cto30.1 C, withanaverageerrorof1.5 C.Itisnoteworthythatalloftheheatingcurveshave anexponentialformandhavethesamecharacteristicrisetimeτs=15ms. Goodagreementisfoundbetweenthesteady statetemperaturechangesobserved infig.6.4afordifferentappliedmicrowavepowersandthemodelofmicrowave heatingoutlinedinthebeginningofthischapter.thesteady statetemperature 111

126 changeisplottedversusthemicrowavepowerdensityinfig.6.4aandisfitwitha line.asisexpectedfromeq.6.1thesteady statetemperaturechangeriseslinearly withappliedmicrowavepower.themicrowavepowerdensityiscalculatedusing theelectricfieldvaluesdeterminedfromsimulations(fig.6.1b).theonlyvariable ineq.6.1thatisnotmeasuredorthatisnotamaterialpropertyisthecharacteristic lengthscalebetweenthedropandthechannelwallld.thischaracteristiclength LD=28μmisestimatedusingthemeasuredsteady statetemperaturechange (Fig.6.4a)andtheknownmaterialproperties,usingEq.6.1. Goodagreementisfoundbetweentheobservationthatthetemperaturechange approachessteady stateexponentiallyintimeinfig.6.4bandthemodelfor microwaveheatingoutlinedinthebeginningofthischapter.tocomparethe model spredictionthatthedropsapproachequilibriumexponentiallyintimewitha singlerelaxationtimeconstant(eq.6.2)withourobservations,thechangein temperatureδtsubtractedfromthesteady statechangeintemperatureδtssis plottedversustimeonasemi logplotandfitwithaline.asispredictedbythe modelthedropsapproachequilibriumexponentiallywithasingletimeconstant. Thethermalrelaxationtimeconstantismeasuredtobeτs=14.7±0.6ms.Theonly variableineq.6.2thatisnotmeasuredisthecharacteristiclengthldbetweenthe dropandthechannelwall.thischaracteristiclengthld=35μmisestimatedusing themeasuredcharacteristictimeconstantandtheknownmaterialproperties,using Eq.6.2.Theindependentmeasurementsofthethermalrelaxationtimeandthe steady statetemperaturechangeversuspowergivetwoindependentmeasuresof thelengthldthatarewithin20%ofeachother.thedifferenceinthetwo 112

127 predictionsofldcanbeatleastpartiallyexplainedbynotingthattheheatcapacity ineq.6.2islargerthaniscalculated,duetothefactthatthechannelwallsarenota perfectheatsinkasthemodelassumes.theagreementbetweenthetwo independentmeasurementssupportsthesimplemodelformicrowaveheatingof dropsoutlinedinthebeginningofthischapter. 113

128 Figure6.4(a)Thesteady statetemperaturechangeofthedropsiscontrolledby varyingtheamplitudeofthemicrowavevoltage.intheinsetthegreencurveshows thedropheatingversustimeformicrowaveswithanamplitudeof11.7v,thered 11.0V,theyellow10.3V,thelightblue9.3V,thepurple8.5V,thegrey7.5V,andthe blue4.5v.thesteady statetemperaturechangeisplottedversusthepower density,ascalculatedbyeq.2.1,inthemainplot,(b)themagnitudeofthechangein temperatureδtsubtractedfromthemaximumchangeintemperatureδtssversus timeisplottedonalog linearscale.thetemperaturerisesasasingleexponential withacharacteristictime,τs=14.7±0.56ms.theinsetshowsthetemperature versustimeplotfromwhichthelog linearplotistaken. 114

129 6.5Discussion Inthischapteranintegratedmicrofluidicmicrowavedielectricheateris demonstratedthatlocallyandrapidlyincreasesthetemperatureofdropsofwater suspendedinoil.thelargeabsorptionofmicrowavepowerbywaterrelativetooil, glass,andpdmsallowslocalandrapidheatinginmicrofluidicdeviceswithout difficultfabrication.bothimprovingtheinsulationofdropsfromthechannelwalls andincreasingthevolumetosurfacearearatioofthedropswouldallowforlarger temperaturechanges.thestatisticalerrorinthesteady statetemperatureofthe dropscanbeimprovedbyreducingvariationsinthedropsizethatarisefrom fluctuationsintheflowfromthesyringepumps. Microwavedielectricheatingofdropsiswellsuitedforintegrationwiththehybrid integratedcircuit(ic)/microfluidicchipsdescribedinchapters3,4,and5.(leeet al.,2007)ifelectrodesonthechiparedrivenwithvoltagesatghzfrequencies,then onecanusethechiptolocallyheatsmallvolumesoffluidusingdielectricheating. Theadditionoflocallyaddressabletemperaturecontroltothelab on a chip functionsdemonstratedinchapters3,4,and5wouldfurtherexpandthe capabilitiesofthehybridchipplatformandwouldbeavaluabletoolforanumberof applications,includingdnaanalysis.(leeetal.,2007) Microwavedielectricheatinghasmanyexcitingscientificandtechnological applications.onenoteworthypotentialapplicationforrapid,localizedheatingin microfluidicdevicesispcr.(lagallyetal.,2000,liuetal.,2002,khandurinaetal., 2000,Odaetal.,1998,Maltezosetal.,2005,Maltezosetal.,2006)Themicrowave 115

130 microfluidicheatercanraisethetemperatureofdropsupto30 Cabovethebase temperatureoftheoilinwhichthedropsaresuspended.bysettingthebase temperatureoftheoilinourdeviceto65 Candappropriatelysettingthe microwavepower,a30 Cchangeintemperaturecouldcyclethetemperaturefrom 65 Cto95 CasrequiredforPCR.Drop basedpcr,whichwouldbeespeciallywell suitedforthistechnique,allowsfortherapidanalysisoflargepopulationsofgenes andenzymes.themicrowaveheatingtechniquemightalsobeusedtoset temperaturesrapidlyandcontrollablyinbiologicalandchemicalassays,suchasfor proteindenaturingstudies(arataetal.,2008)andenzymeoptimizationassays (Robertsonetal.,2004),whereobservationsofthermalresponsesaremadeonthe millisecondtimescale. 116

131 Chapter7.Conclusions 7.1Summary Thisthesisdescribesthedevelopmentofaversatileplatformforperformingbiology andchemistryexperimentsonachip,usingtheintegratedcircuit(ic)technologyof thecommercialelectronicsindustry.thisworkisanimportantsteptowards developingautomated,portable,andinexpensivedevicestoperformcomplex chemicalandbiologicaltasks.suchadevicewouldrevolutionizethewaythat biologicalandchemicalinformationiscollectedforapplicationssuchasmedical diagnostics,environmentaltesting,andscientificresearch.(ahnetal.,2004,chinet al.,2007,andmartinezetal.,2008) ThehybridIC/microfluidicchipsdevelopedinthisthesiscontrollivingcellsand smallvolumesoffluid.takinginspirationfromcellularbiology,phospholipid bilayervesiclesareusedtopackageplvolumesoffluidonthesechips.table7.1 summarizesthebasiclab on a chipfunctionsthatthehybridchipscanperformon thelivingcellsandvesicles.thechipscanbeprogrammedtotrapandposition, deform,setthetemperatureof,electroporate,andelectrofuselivingcellsand vesicles.thesebasicfunctionscanbestrungtogethertoperformcomplexchemical andbiologicaltasks.thefastelectronicsandcomplexcircuitryoficsenable thousandsoflivingcellsandvesiclestobesimultaneouslycontrolled,allowingmany parallel,well controlledbiologicalandchemicaloperationstobeperformedin parallel. 117

132 Table7.1Alistofthelab on a chipfunctionsperformedinthisthesisusing hybridic/microfluidicchips. 118

133 7.1FutureDirections ThisthesisdevelopshybridIC/microfluidicchipsanddemonstratesthattheycan performbasicfunctionsthatarenecessarybuildingblocksforbiologicaland chemicalexperimentsonachip.however,thegoalofaportabledevicethatcan performbiologicalandchemicaltasksremainsachallenge.forthisgoaltobefully realized,amoreself sufficientplatformmustbedeveloped.althoughahybrid IC/microfluidicchipcanbepackagedintoaportabledevice,thecurrentchip requiresalaboratory sizedfluorescencemicroscope,lightsource,andcomputerto fullyfunction.tocompletelyrealizethepotentialofhybridic/microfluidicchips, additionalfunctionsmustbeaddedtothechipssuchthatthelarge,external componentsarenotrequired. Thehybridchipsdescribedinthisthesiscouldbemademoreself sufficientby integratingsensorsontotheics.ahybridic/microfluidicchipthatincludedboth sensorsandthefunctionalitydemonstratedinthisthesiscouldperformcomplex biologicalandchemicalexperimentsandmeasuretheresults.theseexperiments couldincludemultiplestepssuchas:mixingalibraryofcellswithalibraryof chemicals,measuringtheoutcomeoftheexperimentswithsensorsbuiltintothe chip,analyzingtheresultsoftheexperiment,anddesigningthenextsetof experimentsbasedontheresultsofthefirst,andrepeat,untilfinallyreportingthe results.thissortofautomatedlab on a chipplatformcouldperformthecomplex, multi stepexperimentsthatarecurrentlyperformedinlaboratoriesbytrainedstaff usinglargeandexpensiveequipment. 119

134 SeveralsensorshavepreviouslybeendevelopedusinghybridIC/microfluidic chips.someexamplesofthesesensorsarenmrsensorsforchemicaldetection, (Lee etal.,2008)capacitivesensorsformeasuringeletrogeniccells, (DeBusschereetal., 2001)electricalsensorsforDNAdetection,(Thewesetal.,2002)andcharge coupleddevices(ccd)foropticalimaging.(cuietal.,2008)thesesensorsarebuilt usingstandardcommercialicprocesses,similartotheprocessesusedtocreatethe chipsdescribedinthisthesis.asingleiccouldbebuilt,usingacommercialfoundry, thatincludesthefunctionalitydemonstratedinthisthesisandthatofthesensors describedabove.sensorsthatrequirespecializedicprocessesorthataremade withe beamlithographycanalsobeincludedusingflip chipbonding.(leeetal., 2007) AhybridIC/microfluidicchipthatcontrols,performsexperimentson,and measuresmanylivingcellsandsmallvolumesoffluidwouldhaveabigimpact. Taskssuchasthedetectionandcountingofcells,sortingcells,geneticanalysis, proteindetection,andcombinatorialchemistrycouldbeperformedontheselowcost,automateddevices.(whitesidesetal.,2001,stoneetal.,2004,tabelingetal., 2005,Yageretal.,2006,Martinezetal.,2008,Leeetal.,2007,Huntetal.,2008, Maltezosetal.,2005)Thechipscouldbeprogrammedtoperformcomplex,multisteptasks,andreacttotheresultsofexperimentsinreal time.thedevicescouldbe usedforapplicationssuchaspoint of carediseasedetectionordetectingtoxic substancesorparasitesindrinkingwater.suchadevicewouldrealizetheinitial goalofmicrofluidics,tobringcomplexbiologicalandchemicaltestsfrom laboratoriesoutintotheclinicandthefield.(whitesides,2001) 120

135 WorksCited AhnC.H.,ChoiJ.W.,BeaucageG.,NevinJ.,LeeB.J.,PuntambekarA.,andLeeJ.Y. (2004)Proceedings IEEE,92,154. AlbertsB.,JohnsonA.,LewisJ.,RaffM.,WalterP.,andRobertsK.(2007)Molecular BiologyofTheCell,Taylor&FrancisGroup. AnnaS.L.,BontouxN.,andStoneH.A.(2003)Appl.PhysLett.,82,364. AngelovaM.I.andDimitrovD.S.(1986)FaradayDiscuss.Chem.Soc.,81,303. ArataH.F.,GillotF.,NojimaT.,FujiiT.,andFujitaH.(2008)LabChip,8,1436. BeanC.P.andLivingstonJ.D.(1959),J.Appl.Phys.,30,S120. BengtssonN.E.,OhlssonT.(1974)Proc.IEEE,62,44. BerryC.andCurtisA.S.G.(2003)J.Phys.D,2003,36,198. ChinC.D.,LinderV.,andSiaS.K.(2007)LabChip,7,41. ChiuD.T.,WilsonC.F.,RyttsénF.,StrömbergA.,FarreC.,KarlssonA.,NordholmS., GaggarA.,ModiB.P.,MoschoA.,Garza LópezR.A.,OrwarO.,andZareR.N. (1999)Science,283,5409. CuiX.,LeeL.M.,HengX.,ZhongW.,SternbergP.W.,PsaltisD.,YangC.(2008)Proc NatlAcadSci,105, CurtisA.S.G.andVardeM.(1964)J.NatlCancerRes.Int.,33,15. DeBusschereB.D.,andKovacsG.T.A.(2001)Biosensors&Bioelectronics,16,543. DillK.A.andBrombergS.(2003)MolecularDrivingForces,TaylorandFrancis. DittrichP.S.andManzA.(2006)Naturereviews.Drugdiscovery,5,210. DobsonJ.(2008)NatureNanotechnology,3,139. EltoukhyH.,SalamaK.,GamalA.E.(2006)Solid StateCircuits,IEEE,41,3. EricksonD.andLiD.(2004)AnalyticaChimicaActa,507,11. EversmannB.,JenknerM.,HofmannF.,PaulusC.,BrederlowR.,HolzapflB., FromherzP.,MerzM.,BrennerM.,SchreiterM.,GablR.,PlehnertK.,Steinhauser M.,EcksteinG.,Schmitt LandsiedelD.,andThewesR.(2003)J.Solid State Circuits,38,2306. FigeysD.andPintoD.(2000)Anal.Chem.,72,

136 GascoyneP.R.C.,VykoukalJ.V.,SchwartzJ.A.,AndersonT.J.,VykoukalD.M., CurrentK.W.,McConaghyC.,BeckerJ.A.,andAndrewsC.(2004)LabChip,4, 299. GeistJ.,ShahJ.J.,RaoM.V.,andGaitanM.(2007)J.Res.Natl.Inst.Stand.Technol., 112,177. GrantE.H.,ShephardR.J.,andSouthG.P.(1978)DielectricBehaviorofBiological MoleculesinSolution,OxfordUniv.Press,Oxford. GrosseC.andSchwanH.(1992)BiophysicalJ.,63,1632. HengX.,EricksonD.,BaughL.R.,YaqoobZ.,SternbergP.W.,PsaltisD.,andYangC. (2006)LabChip,6,1274. HoltzeC.,RowatA.C.,AgrestiJ.J.,HutchisonJ.B.,AngileF.E.,Schmitz,S.Koester, DuanH.,HumphryK.J.,ScangaR.A.,JohnsonJ.S.,PisiganoD.,andWeitzD.A. (2008)LabChip,8,1632. HuntT.(2007)IntegratedCircuit/MicrofluidicChipsforDielectricManipulation, PhDDissertation,HarvardUniversity. HuntT.,IssadoreD.,WesterveltR.M.(2008)LabChip,81,7. IsambertH.(1998)Phys.Rev.Lett.,80,15. IssadoreD.,HumphryK.J.,BrownK.A.,SandbergL.,WeitzD.A.,WesterveltR.M., LabChip,2009,DOI: /B822357B. JonesT.B.(1995)ElectromechanicsofParticles,CambridgeUniversityPress, Cambridge. Keehan,S.,SiskoA.,TrufferC.,SmithS.,CowanC.,PoisalJ.,ClemensM.K.andThe NationalHealthExpenditureAccountsProjectionsTeam(2008)HealthAffairs, doi: KhandurinaJ.,McKnightT.E.,JacobsonS.C.,WatersL.C.,FooteR.S.,and RamseyJ.M.(2000),Anal.Chem.,72,2995. KilbyJ.S.(1976),ElectronDevices,IEEETrans.,23,7. KosterS.,AngilèF.E.,DuanH.,AgrestiJ.J.,WintnerA.,SchmitzC.,RowatA.C., MertenC.A.,PisignanoD.,GriffithsA.D.,andWeitzD.A.(2008)LabChip,8, LagallyE.T.,SimpsonP.C.,MathiesR.A.(2000)Sens.Actuators,B63,138. LeeH.(2005)Microelectronic/MicrodluidicHybridSystemfortheManipulationof BiologicalCells,PhDdissertation,HarvardUniversity. 122

137 LeeH.,HamD.andWesterveltR.M.eds.(2007)CMOSBiotechnology,Springer,New York. LeeH.,LiuY.,AlsbergE.,IngberD.E.,WesterveltR.M.,HamD.(2005)ISSCC, LeeH.,LiuY.,WesterveltR.M.,andHamD.(2006)IEEEJ.Solid StateCircuits,41, LeeH.,PurdonA.M.,andWesterveltR.M.(2004)Appl.Phys.Lett.,85,1063. LeeH.,SunE.,HamD.,andWeisslederR.(2008)NatureMedicine,14,869. LeeJ.,MoonH.,FowlerJ.,SchoellhammerT.,andKimC.J.(2002),Sens.ActuatorsA, 95,259. LeeJ.andMudawarI.(2005)Int.J.HeatMassTransfer,48,928. LeeT.(1998)TheDesignofCMOSRadio FrequencyIntegratedCircuits,Second Edition,CambridgeUniversityPress,Cambridge. LeeW.G.,DemirciU.,andKhademhosseiniA.(2009),Integr.Biol.,1,242. LinkD.R.,AnnaS.L.,WeitzD.A.,andStoneH.A.(2004),Phys.Rev.Lett.,92, LiuJ.,EnzelbergerM.,andQuakeS.R.(2002)Electrophoresis,23,1531. LopezM.,CabreraA.,Agullo RuedaM.(1970)Electrooptics:Phenomena,Materials andapplications,academicpress. MaltezosG.,JohnstonM.,andSchererA.(2005)Appl.Phys.Lett.,87, MaltezosG.,RajagopalA.,andSchererA.(2006)Appl.Phys.Lett.,89, ManaresiN.,RomaniA.,MedoroG.,AltomareL.,LeonardiA.,TartagniM.,and GuerrieriR.(2003),IEEEJ.Solid StateCircuits,38,2297. MaoH.,YangT.,andCremerP.S.(2002)J.Am.Chem.Soc.,124,4432. MartinezA.W.,PhillipsS.T.,WileyB.J.,GuptaM.,andWhitesidesG.M.(2008)Lab Chip,8,2146. MurrellJ.N.andJenkinsA.D.(1994),PropertiesofLiquidsandSolutions,2ndEd. JohnWiley&Sons,Chichester,England. NakanoH.,MatsudaK.,YohdaM.,NagmuneT.,EndoI.,andYamaneT.(1994),Biosci. Biotechnol.Biochem.,58,349. NyholmL.(2005)Analyst,130,

138 OdaR.P.,StrausbauchM.A.,HuhmerA.F.R.,BorsonN.,JurrensS.R.,CraigheadJ., WettsteinP.J.,EckloffB.,KlineB.,andLandersJ.P.(1998),Anal.Chem.,70, OlofssonJ.,NolkrantzK.,RyttsénF.,LambieB.,WeberS.G.,andOrwarO.(2003) Curr.Opin.Biotechnol.,14,29. PawleyJ.B.(2008)J.Biomed.Opt.,13, PollackM.G.,FairR.B.,andShenderovA.D.(2000),Appl.Phys.Lett.,77,11. PollackM.G.,ShendorovA.D.,andFairR.B.(2002),LabChip,2,96. PsaltisD.,QuakeS.R.,andYangC.(2006)Nature,442,381. RiskeK.A.andDimovaR.(2006),BiophysicalJ.,91,1778. RobertsonD.E.andSteerB.A.(2004),CurrentOpinioninChemicalBiology,8,141. Seifritz.W.(1924)Brit.J.Exper.Biol.,2,1. ShahJ.J.,SundaresanS.G.,GeistJ.,ReyesD.R.,BoothJ.C.,RaoM.V.,andGaitanM. (2007)J.Micromech.Microeng.,17,2224. SiegelA.C.,ShevkoplyasS.S.,WeibelD.B.,BruzewiczD.A.,MartinezA.W.,and WhitesidesG.M.(2006)Angew.Chem.,45,6877. SklavounosA.,MarchiarulloD.J.,BarkerS.L.R.,LandersJ.P.,andBarkerN.S. (2006)Proc.MicroTotalAnal.Syst.,2,1238. StoneH.A.,StroockA.D.,andAjdariA.(2004)AnnualReviewsofFluidMechanics, 36,381,andreferencestherein. SugarP.,ForsterW.,andNeumannE.(1987)BiophysicalChem.,26,321. SundaresanS.G.,PolkB.J.,ReyesD.R.,RaoM.V.,andGaitanM.(2005)Proc.Micro TotalAnal.Syst.,1,657. TabelingP.(2005)IntroductiontoMicrofluidics,OxfordUniversityPress. Thewes R., Hofmann F., Frey A., Holzapfl B., Schienle M., Paulus C., Schindler P., Eckstein G., Kassel C., Stanzel M., Hintsche R., Nebling E., Albers J., Hassman J., Schulein J., Goemann W., and Gumbrecht W. (2002) IEEE Int. Conf. Solid State Circuits:DigestofTechnicalPapers,1,350. TressetG.andIliescuC.(2007)Appl.Phys.Lett.,90, Tudos A.J.,G.A.J.Besselink,andR.B.M.Schasfoort(2001)LabChip,1,83. UngerM.A.,ChouH.P.,ThorsenT.,SchererA.,QuakeS.R.(2000)Science,288,

139 UnitedStatesDepartmentofHealthandHumanServices(2007)NationalHealth ExpenditureFactSheet.< UrdeaM.,PennyL.A.,OlmstedS.S.,GiovanniM.Y.,KasparP.,ShepherdA.,WilsonP., DahlC.A.,BuchsbaumS.,MoellerG.,andBurgessD.C.H(2006)Nature,444,73. VarmusH.,KlausnerR.,ZerhouniE.,AcharyaT.,DaarA.S.,andSingerP.A.(2003) Science,302,398. VykoukalJ.,SchwartzA.,F.F.Becker,andGascoyneP.R.C.(2001)MicroTotal AnalysisSystems,1,72. VyawahareS.,SitaulaS.,MartinS.,AdalianD.,andSchererA.(2008)LabChip,8, WangN.andIngberD.(1994)BiophysicalJournal,66,2181. WaughR.E.,SongJ.,SvetinaS.andZeksB.(1992)BiophysicalJournal,61,974. WheelerA.R.,ThrondsetW.R.,WhelanR.J.,LeachA.M.,ZareR.N,LiaoY.H.,Farrell K.,MangerI.D.,andDaridonA.(2003)AnalChem.,14,3581. WhitesidesG.M.,OstuniE.,TakayamaS.,JiangX.,andIngberD.(2001)Annual ReviewofBiomedicalEngineering,3,335. YagerP.,EdwardsT.,FuE.,HeltonK.,NelsonK.,TamM.R.,WeigB.H.(2006)Nature, 442,

140 AppendixA.DataSheetforthe DielectrophoresisChip(DEPChip) WedocumentthespecificationsfortheFabutron1.0,showthepinlayoutanddetail theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave alloftheinformationnecessarytomakethefabutron1.0workintheirexperiment. SummaryoftheFabutron1.0 TheFabutron1.0isacustomintegratedcircuitdesignedintheWesterveltlab.The chipconsistsofanarrayof128x256pixels,11x11μm 2 insize,controlledbybuilt in SRAMmemory;eachpixelcanbeindividuallyaddressedwitharadio frequency(rf)voltageupto5vpp,withfrequenciesfromdc 11MHz.TheICwas builtinacommercialfoundryandthemicrofluidicchamberwasfabricatedonits topsurfaceatharvard. TableA1SpecificationsfortheDEPchip Feature Description Bandwidth DC 11MHz Process 0.35µm,CMOSMOSISTSMC Pixels 128x256(32,768) 11x11µm 2 ChipSize 2.32x3.27mm 2 Addressing 8 bitwordlinedecoder 128bitshiftregister,twophase clocked DataLineBandwidth DC 20MHz PixelVoltage Vp p=3 5V PixelBandwidth DC 5MHz OperatingVoltage 5V OperatingCurrent 30mA 300mA 126

141 TableA2.AlistofthepinsfortheDEPChip Pin Description GND Ground VDD OperatingVoltage(5V) W(0:7)** WordAddressDecoderLines φ1 PixelVoltage1 φ2 PixelVoltage2 c2 (0)decouplesSRAMelementsfromthebitline,(1)connects SRAMelementsonactivatedrowtothebitline c1 (0)Prechargethebitlines,(1)disconnectprecharge WR ControlLine:(1)enablestheshiftregistertowriteitsvalueto thebitlines,(0)disablesshiftregisterfromwritingtobitlines PASS Controlline:(1)Shiftregisterelementslookstoprevious registerforinput,(0)registerslooktobitlineforinput DATA Datainputlineforshiftregisters CLK2 Clockforshiftregister CLK1 Clockforshiftregister OUT Dataoutputforshiftregister FigureA1PinlayoutfortheDEPchip 127

142 Protocols FigureA2.LoadDataIntoShiftRegisterandWritetoArray. 128

143 FigureA3.ReadDatafromSRAMArrayintoShiftRegisterandReadOutArray. **WordAddressDecoderLinesareincorrectlylabeledonthechip.Notethatgoing frommsbtolsbthecorrectorderingis:w3,w2,w1,w0,w7,w6,w5** ***Alsonotethattherewasanerrorinthedesignoftheshiftregisteranditactually functionsasa64bitshiftregister,withneighboringregisterstiedtogether.thefull 128bitsareaccessiblebyloadingin64bitsofdataatatime,anduseclock1toshift thedataintotheproperposition.*** 129

144 AppendixB.DataSheetfortheHighVoltage Dielectrophoresis/MagneticChip(HV DEP/ MagneticChip) WedocumentthespecificationsfortheFabutron2.0,showthepinlayout,anddetail theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave alloftheinformationnecessarytomakethefabutron2.0workintheirexperiment. SummaryoftheFabutron2.0 TheFabutron2.0isacustomintegratedcircuitdesignedintheWesterveltlab.The chipconsistsofanarrayof60x61pixels,30x38μm 2 insize,controlledbybuilt in SRAMmemory;eachpixelcanbeindividuallyaddressedwitharadio frequency(rf)voltageupto50vpp,withfrequenciesfromdc 10MHz.Interlaced withthedeppixelsareamatrixof60x61wiresthatmaybeaddressablysourced with100matoapplyforcesonmagneticallypolarizableobjects.theicwasbuilt inacommercialfoundryandthemicrofluidicchamberwasfabricatedonitstop surfaceatharvard.thechipsuffersfromlocalheatingproblemsduetothe magneticwires.animprovedversionwouldswitchtopulsedcurrentstominimize heating.thechipalsohasissueswiththemagneticpowerlinesandthedigital powerlinesbeingcoupled,whichleadstopowerlineissuesonthechip.anupdated versionwouldtakeextracaretomakesurethatpowerlinesaredecoupled. TableB1.SpecificationsfortheDEPchip Feature Bandwidth Process Pixels Description DC 10MHz 0.6µm,CMOSXFABXC06 60x61(3,660) 130

145 30x38µm 2 ChipSize 3.4x3.7mm 2 Addressing 5 bitwordlinedecoder 128bitshiftregister,twophase clocked DataLineBandwidth DC 20MHz PixelVoltage Vp p=20 50V PixelBandwidth DC 1MHz OperatingVoltage 5V (Logic) OperatingCurrent 20mA (logic) OperatingMagnetic 5V WireVoltage OperatingMagnetic 0 800mA* WireCurrent OperatingHigh 50V Voltage OperatingHigh 20 60mA** VoltageCurrent TableB2.AlistofthepinsfortheDEPChip Pin Description GND Ground VDD OperatingVoltage(5V) HV HighVoltage(50V) HVg HighVoltageGate(45 50V),controlstheactiveimpedanceintheRF voltagedrivingcircuitunderneatheachcircuit Vmag PowerSupplyfortheMagneticLines(5V) W(0:5) WordAddressDecoderLines THERM AnalogOutputfortheThermometer(0 5V) φ1 PixelVoltage1 φ2 PixelVoltage2 c2 (0)decouplesSRAMelementsfromthebitline,(1)connectsSRAM elementsonactivatedrowtothebitline c1 (0)Prechargethebitlines,(1)disconnectprecharge WR ControlLine:(1)enablestheshiftregistertowriteitsvaluetothe bitlines,(0)disablesshiftregisterfromwritingtobitlines PASS Controlline:(1)Shiftregisterelementslookstopreviousregisterfor input,(0)registerslooktobitlineforinput DATA Datainputlineforshiftregisters CLK2 Clockforshiftregister CLK1 Clockforshiftregister OUT Dataoutputforshiftregister 131

146 FigureB1PinlayoutfortheDEPchip Protocols TheprotocoltoupdatetheSRAMarrayisidenticalasfortheFabutron1.0 TheSRAMaddressesoftheDEPpixels,magneticwires,andthermometersareas follows: TableB3.Amapofthefeaturesofthe50V/magneticchipontheSRAMarray Array DEPArray(1:60,1:161) MagneticMatrixXdirection(1:2,1:122) MagneticMatrixYdirection(1:2,1:122) Thermometers(1:16) SRAMAddress(word,bit) SRAM(3:32,5:126)*** SRAM(1:2,5:126)*** SRAM(1:30,1:4)*** SRAM(1:8,127:128)*** *Notethatspecialcaremustbetakenwhenthechipisfirstturnedon.TheSRAM willbesettoarandomstateandmorethanafewmagneticlineswillbeturnedon. ItisadvantageoustoleavetheVmagturnedoffuntilthestatesofthewirescanbeset suchthatnoneorfewofthewiresareturnedon. 132

147 **Notethataspecialfuseandvoltageconditioning/currentlimitingcircuitrymust bebuiltintothepcbthathousestheic.spikesinthehvlinecandestroythechip. ***FormoredetailonhowtheSRAMarraymapsontotheDEPandMagneticarray pleaseseelinetheattachedmatlabcodewherethedetailedmappingislaidout. 133

148 AppendixC.FabutronControlSoftware LabviewProgramsusedtoCommunicatewiththeChips FigureC1.Labviewcodeusedtoreaddatacomingfromthechips. 134

149 FigureC2.Labviewcodeusedtowritedatatothechips FigureC3.BlockdiagramofLabviewcodeusedtoreaddatafromthechips 135

150 FigureC4.BlockdiagramofLabviewcodeusedtowritetothechip 136