HybridIntegratedCircuit/MicrofluidicChips

fortheControlofLivingCellsandUltra­Small

BiomimeticContainers

ADissertationPresented

by

DavidAaronIssadore

to

TheSchoolofEngineeringandAppliedSciences

Inpartialfulfillmentoftherequirements

forthedegreeof

DoctorofPhilosophy

inthesubjectof

AppliedPhysics

HarvardUniversity

Cambridge,Massachusetts

May2009

©2009byDavidIssadore

Allrightsreserved.

iii

DavidAaronIssadoreAdviser:RobertWestervelt

HybridIntegratedCircuit/MicrofluidicChipsfortheControlof

LivingCellsandUltra­SmallBiomimeticContainers

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.

iv

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.

v

TableofContents

Abstract……………………………………………………………………………………………iiiAcknowledgements………………………………………………………………….............viiAbbreviationsandSymbols……………………………………………………………….ixListofTableandFigures………………………………………………………...…………xiChapter1.Introduction……………………………………………………….……………1

1.1Lab‐on‐a‐Chip–Motivation…………………………………...…………11.2HybridIC/MicrofluidicChips–Concept…………………………..41.3OverviewofThesis…………………………………………………………..7

Chapter2.Theory:DielectricandMagneticControlofMicroscopicObjects………………………………………………………………………….16

2.1TheDielectricPropertiesofWaterandSolutions………………182.2Dielectrophoresis…………………………………………………………….222.3DielectricModelsforCellsandVesicles…………………………….232.4TransmembranePotentialDifferenceanditsApplications...282.5DielectricHeating…………………………………………………………….322.6Magnetophoresis……………………………………………………………..34

Chapter3.HybridIntegratedCircuit/MicrofluidicChips…………………...36

3.1Overview…………………………………………………………………………363.2DielectrophoresisChip(Fabutron1.0)……………………………...37

3.2.1OperatingPrincipals……………………………………………383.2.2CharacteristicTimes………………………………………...…393.2.3IntegratedCircuitDesign…………………………………….413.2.4Capabilities………………………………………………………...46

3.3ExperimentalApparatus…………………………………………………..483.3.1Fluidics……………………………………………………………….483.3.2Electronics…………………………………………………………493.3.4Optics………………………………………………………………….503.3.5ThermalManagement…………………………………………513.3.6ComputerControl……………………………………………….54

Chapter4.AHybridIntegratedCircuit/MicrofluidicPlatformtoControlLivingCellsandpLBiomimeticContainers…………………………….56

4.1Overview…………………………………………………………………………564.2Methods…………………………………………………………………………..60

4.2.1TheHybridIntegratedCircuit/MicrofluidicChipPlatform………………………………………………………………604.2.2UnilamellarVesicles……………………………………………60

vi

4.2.3DielectrophoresisofVesiclesSuspendedinWater………………………………………………….…614.3.4ElectroporationandElectrofusion……………………….63

4.3Demonstrations……………………………………………………………….674.3.1TrappingandMovingCellsandVesicles……………....674.3.2TriggeredReleaseoftheContentsofVesicles………704.3.3ElectroporationofCells……………………………………....704.3.4TriggeredFusionofVesicles………………………………..714.3.5DeformingVesicleswithDielectrophoresis………….74

4.4Discussion…………………………………………………………………….…76Chapter5.HybridMagneticandDielectrophoreticIC/MicrofluidicChip………………………………………………………………………..78

5.1Overview………………………………………………………………………....785.2DescriptionoftheChip……………………………………………………..80

5.2.1FieldSimulations………………………………………………..805.2.2ChipArchitecture………………………………………………..83

5.3Demonstrations……………………………………………………………….865.3.1Dielectrophoresis:TrappingandPositioningVesicles……………………………………………....865.3.2Magnetophoresis:TrappingandPositioningMagneticBeads………………………………...…895.3.3DielectrophoresisandMagnetophoresis:DeformingVesicles……………………………………………………...91

5.4Discussion…………………………………………………………………….…93

Chapter6.MicrowaveDielectricHeatingofDropsinMicrofluidicDevices……………………………………………………...…….95

6.1Overview…………………………………………………………………………956.2ModelofDielectricHeatingofDrops…………………………………986.3TheMicrofluidicDevice……………………………………………………1006.4Demonstration………………………………………………………………...1086.5Discussion…………………………………………………………………….…115

Chapter7.Conclusions…………………………………………………………………..…117

7.1Summary………………………………………………………………………...1177.2FutureDirections………………………………………………………….…119

WorksCited…………………………………………………………………………..…………121AppendixA.DataSheetandUsersGuidefortheDEPChip(Fabutron1.0)……………………………………………………………...126 AppendixB.DataSheetandUsersGuidefortheHV‐DEPMagneticChip(Fabutron2.0)………………...………………………………..………...130AppendixC.FabutronControlSoftware………………………………....………...134

vii

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.

viii

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.

ix

AbbreviationsandSymbols

a Particleradius

€

B MagneticField

C Capacitance

Cmem Specificmembranecapacitance

CM ClausiusMosottiFactor

€

χ MagneticSusceptibility

CMOS ComplimentaryMetalOxideSilicon

D DiffusionConstant

DC Directcurrent

DEP Dielectrophoresis

ΔT ChangeinTemperature

€

E ElectricField

€

ε RelativePermittivity

€

εo VacuumPermittivity

€

ε' RealComponentofthePermittivity

€

ε' ' ImaginaryComponentofthePermittivity

f Frequency

€

F Force

gm SurfaceConductivity

GUI GraphicalUserInterface

HV HighVoltage

€

η Viscosity

I/O Input/Output

IC IntegratedCircuit

j

€

−1 kB Boltzmann’sConstant

L Thelengthofapixel

x

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 SteadyStateChangeinTemperature

τdiff CharacteristicTimeforanObjecttoDiffuseaHalfPixellengthL/2

τmem CharacteristicChargingTimeofaVesicleorCell’sMembrane

τmove CharacteristicTimetoMoveanObjectaPixelLengthL

τSS CharacteristicTimetoReachSteadyStateTemperature

τMW DielectricRelaxationTimeofavesicleorcell

τW DielectricRelaxationTimeofWater

€

µo VacuumPermeability

V Volume

V Voltage

VTM TransmembraneVoltage

ω AngularFrequency

xi

ListofFigures

Figure1.1aJackKilby’sIC………………….…………….……………………………….6

Figure1.1bAnIntelQuad‐CoreIC…..…....…….……………………………………..6

Figure1.1cASimpleMicrofluidicChip……………………………………..……….6

Figure1.1dAComplexMicrofluidicChip…………………….…………………….6

Figure1.1eAHybridIC/MicrofluidicChip……………………………………….6

Figure1.2TheDEPChipSpelling“LabonaChip”withYeastCells…..….9

Figure1.3aTheDEPHybridIC/MicrofluidicChip…..…………….………….15

Figure1.3bTheHighVoltageDEP/MagneticChip…….………………..…….15

Figure1.3cTheMicrowaveDielectricDropHeatingChip……………....…..15

Figure2.1FrequencyDomainPlotoftheEfficacyofDEP,Electroporation,andDielectricHeating………………...…………….….17

Figure2.2aPermittivityofWatervs.Frequency………………………………...21

Figure2.2bPermittivityofAqueousSolutionswithVaryingConductivityvs.Frequency…………………….…………...……….…………………….21

Figure2.3aLumpedCircuitModelforthePermittivityofaVesicle….…27

Figure2.3bClausius‐Mosottivs.InteriorConductivityforaVesicle... 27

Figure2.3cClausius‐Mosottivs.frequencyforaVesicle…………….……. 27

Figure2.4aTransmembraneVoltagevs.FrequencyforaVesicle.…….…31

Figure2.4bSchematicofElectrofusion……………………………………………..31

Figure2.5DielectricHeatingPowerDensityvs.Frequencyfora SolutionwithVaryingConduct...………………………………………..………………34

Figure3.1aPlotofSimulatedElectricFieldStrengthAboveTheDEPChip………………………………………………………………………….40

Figure3.1bPlotoftheForceExperiencedbyaVesicleonTheDEPChip…………………………………………………………..............40

Figure3.2aSchematicoftheArchitectureoftheDEPChip….……………...45

xii

Figure3.2bSchematicofanindividualDEPPixel………………………………45

Figure3.2cMicrographoftheDEPArray,ShiftRegister,andRowDecoder……………………………………………………………………………...45

Figure3.3aDemonstrationofTrappingandPositioningYeastontheDEPChip……………………………………………………………………….47

Figure3.3bDemonstrationofTrappingandPositioning DropsofWaterinOilontheDEPChip……………….............................................47

Figure3.3cTheDEPChipSpelling“Harvard”withYeastCells…...............47

Figure3.4aFlowChartoftheExperimentalApparatusSurroundingtheHybridIC/MicrofluidicChips……………………………………………………..53

Figure3.4bMicrographoftheDEPChip’sIC………………………………….….53

Figure3.4cPhotographoftheHybridIC/MicrofluidicChip initsChipCarrier………………………………………………………………….………..…53

Figure3.4dPhotographoftheExperimentalApparatus SurroundingtheHybridIC/MicrofluidicChips…………………….……………53

Figure3.5ScreenShotsoftheGUIthatControlsthe HybridIC/MicrofluidicChip…………………………………………………………….55

Figure4.1ASchematicofUnilamellarVesiclesandEmulsions……………59

Figure4.2aPlotoftheConcentrationGradientofaSubstance ReleasedfromaVesicle………………………………………………………………….....66

Figure4.2bPlotoftheConcentrationGradientofaSubstance ConsumedontheSurfaceofaCellorVesicle…………………………………...…66

Figure4.3aDemonstrationofIndependentlyTrappingand MovingSeveralVesiclesontheDEPChip…………………….………..........……..69

Figure4.3bTheDEPChipDrawing‘H’withVesicles…..………………………69

Figure4.3cSimultaneouslyTrappingandMovingYeastCellsandVesiclesontheDEPChip…………………………………………....69

Figure4.4aTriggeringtheReleaseoftheContentsofaVesicleontheDEPChip…………...………………………………………………….73

Figure4.4bElectroporatingaYeastCellontheDEPChip….………………..73

Figure4.4cElectrofusingTwoVesiclesontheDEPChip…..…………………73

xiii

Figure4.5DeformingVesiclesontheDEPChip…………………………..…..….75

Figure5.1aMicrographoftheHV‐DEP/MagneticIC…………………………83

Figure5.1bPlotoftheSimulatedElectricFieldStrengthAboveTheHV‐DEP/MagneticChip……………………………………………………………..83

Figure5.1cPlotoftheSimulatedMagneticFieldStrengthAboveTheHV‐DEP/MagneticChip………………………………………………………..……82

Figure5.2aMicrographoftheHV‐DEP/MagneticIC,showing thecircuitArchitecture……………………………………………………………………..85

Figure5.2bSchematicofaDEPPixelontheHV‐DEP/MagneticChip...85

Figure5.2cSchematicofaMagneticWireontheHV‐DEP/MagneticIC.………………………………………………………………........... 85

Figure5.3TrappingandMovingVesiclesontheHV‐DEP/MagneticChip…………………………………………………..……..88

Figure5.4TrappingandMovingMagneticBeadsontheHV‐DEP/MagneticChip…………...……………………………………….……90

Figure5.5DeformingVesiclesontheHV‐DEP/MagneticChip…...….…...92

Figure6.1aSchematicofMicrofluidicDielectricHeater……………………..102

Figure6.1bPlotofElectricFieldSimulationsfortheMicrofluidicDielectricHeater……………………………………………………………102

Figure6.1cCalibrationCurvefortheCdSeTemperatureSensor……..….102

Figure6.2aFlowDiagramoftheMicrowaveDielectricHeater…………...107

Figure6.2bMicrographoftheDropMaker………………………………………..107

Figure6.2cMicrographoftheDropSplitter………………………………………107

Figure6.2dMicrographoftheMicrowaveHeatingDevice………………....107

Figure6.2ePhotographoftheExperimentalSetupofthe MicrowaveDielectricDropHeater……………………………...………………..……107

Figure6.3aFluorescenceImageofDropsHeating……………………………..110

Figure6.3bLine‐averageofFluorescenceIntensityvs.Distance….……..110

Figure6.3cPlotofChangeinTemperaturevs.DistanceandTime……...110

Figure6.4aPlotofChangeinTemperaturevs.MicrowavePower….……114

xiv

Figure6.4bPlotofLog‐LinearplotofChangeinTemperaturevs.Time……………………………………………………………………….114

ListofTables

Table1.1ListofFunctionsthatarePerformedinthisThesis….............…13

Table7.1Lab‐on‐a‐ChipFunctionsDemonstratedinthisThesis………...118

1

Chapter1.Introduction

Lab­on­a­chip:ATechnologythatminiaturizesandintegratesthecomplexchemical

andbiologicaltasksusedfordiagnostics,research,andmanufacturingonto

automated,portable,andinexpensivechips.

1.1Lab­on­a­Chip–Motivation

Amajorchallengeofthe21stcenturyistobetterdiagnoseandtreatinfectious

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

tomeasurebiologicalandchemicalmarkersforinfectiousdiseaseininfrastructure‐

poorpartsoftheworld.(Ahnetal.,2004,Chinetal.,2007,andMartinezetal.,2008)

Thelaboratoriesthatperformthechemicalandbiologicalteststodiagnosedisease

2

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

3

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‐chipdevicesultra‐

lowcostbybeinglowtech,(Martinezetal.,2008)thechipsdescribedinthisthesis

4

usecutting‐edgetechnology.Thechipsremaininexpensive,however,byusingthe

integratedcircuit(IC)technologyofthecommercialelectronicsindustry.

1.2HybridIC/MicrofluidicChips­Concept

Integratedcircuitsarecentraltomanyofthetechnologicalwondersofthe21st

century.Builtonaslabofcrystallinesiliconnobiggerthanasquarecentimeterin

area(roughlythesizeofaquarter)anICcontains100sofmillionsoftransistorsand

amazeofwiresthatconnectthetransistorsintocomplexcircuitstoperformbillions

ofoperationspersecond.ThetransistorsandwiresofICsarefabricatedwithnano‐

lithography(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,

5

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.

6

Figure1.1(a)TheoriginalICbuiltbyJackKilbyatNationalInstrumentsin1958(Kilby,1976)(b)amicrographoftheIntelquad‐coreprocessor,whichhas820milliontransistos(IntelCorp.),(c)atwochannelglassmicrofluidicchip(MicronitCorp.),(d)anexampleofacomplexmicrofluidicdevicethatconsistsofhundredsofpneumaticallycontrolledgatesandvalvesandisusedtoperformgeneticanalysisonmicrobesinthehumanmouth,(Marcyetal.,2007)(e)aphotographofanIC/microfluidicchip,the‘DEPChip,’andamicrographofthearrayofpixelsthatareusedtotrapandmoveobjectswithdielectrophoresis(DEP).(Chapter3)

7

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

8

simultaneouslypositionthousandsofyeastcellswithDEPtospell“LabonaChip”.

ThechipshowninFig.1.2isdescribedinfulldetailinChapter3ofthisthesis.

9

Figure1.2ThecoverofLabonaChipfeaturingourhybridIC/microfluidicchipsimultaneouslypositioningthousandsofyeastcellswithDEPtospell“LabonaChip.”

10

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

11

complexcircuitryofICsallowthousandsoflivingcellsandvesiclestobe

simultaneouslytrappedandmovedonthechip,allowingmanyparallel,well‐

controlledbiologicalandchemicaloperationstobeperformedinparallel.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

12

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.

13

Table1.1AlistofthefunctionalitiesdemonstratedonthehybridIC/microfluidicchipsinthisthesis.

14

ThreedevicesaredescribedinthisthesisandareshowninFig.1.3.InChapter3a

customIC/microfluidicchipthatconsistsof128x256(32,768)11x11µm2pixels,

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)30x38µm2pixels,eachofwhichcanbedrivenwitha50Vpeak‐to‐

peakRFvoltagewithfrequenciesfromDCto10MHz.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.

15

Figure1.2(a)AmicrographoftheDEPChip(Fabutron1.0),(b)amicrographoftheHV‐DEP/MagneticChip(Fabutron2.0),(c)aphotographofthePDMSmicrofluidicdevice(MicrowaveHeater)usedtodemonstratemicrowavedielectricheatingofdrops.

16

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

17

negative,enablingvesiclesandcellstobepermeabilizedandfusedwithout

significantheatingorinadvertenttrapping.

Figure2.1.Asemi‐logplotoftheefficacyofheatingP/Pmax(red),dielectrophoresisCM(ω)(green),andelectroporation

€

VTM /VTMmax (blue)versusthefrequencyfofthe

appliedelectricfield.Thethreefrequencydomainsthatareusedforelectroporation,DEP,anddielectricheatingarelabeled.

18

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)

19

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)

€

εW = ε∞ +εs −ε∞1− jωτW

2.1

whereεs=78.4ε0isthelowfrequencydielectricconstantofwater,ε∞=1.78ε0isthe

opticaldielectricconstant,andτw=9.55psisthedielectricrelaxationtimeat

T=25°C.Thedielectricrelaxationtimeτwcanbeunderstoodasthecharacteristic

timethatittakesforwatermoleculestorealignthemselvestoaninstantaneous

appliedelectricfield.

Thedielectricresponseofwaterdependsontheconcentrationofionsinthe

solution.Dissolvedionschangetheconductivityofthesolutionσ byactingasfree

20

chargecarriers.Theeffectofionsinthesolutioncanbeincorporatedintothe

permittivityofthesolution:(A.Stogryn,1971)

€

εW = ε∞ +εs −ε∞1− jωτW

+ j σεoω

2.2

Figure2.2bshowsaplotoftheimaginarycomponentofthepermittivityε’’plotted

versusfrequencyonalog‐logscaleforsolutionswithseveraldifferent

conductivitiesσ.Forsolutionswithafiniteconductivity,theimaginarycomponent

ofthepermittivityε’’decreaseswithincreasingfrequencyuntilacharacteristic

frequency1/τI.Thecriticalfrequency1/τ1isdefinedbythedielectricrelaxation

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)

21

Figure2.2.(a)Therealε’andimaginarypartsε’’ofthepermittivityofwaterεWplottedversusfrequency,(b)theimaginarycomponentε’’ofthepermittivityofsolutionsofwaterplottedversusfrequencyforvariousconductivitiesσ.Theconductivityσ increasesgoingfromlefttoright(asisshownwiththeblackarrow)withvaluesof0S/m,0.03S/m,0.06S/m,0.14S/m,0.3S/m,0.62S/m.

22

2.2Dielectrophoresis

Dielectrophoresisistheinducedmotionofadielectricobjectinanon‐uniform

electricfield.Anyobjectwithapermittivitythatisdifferentthanthesurrounding

mediumcanbecontrolledwithDEP.TheDEPforceonasphericalobjectis:(Jones,

1995)

€

F DEP = 2πεma3CM(ω)∇

E RMS2 2.3

whereaistheradiusoftheparticle,εmisthedielectricconstantofthemedium,and

CM(ω)istheClausius‐Mosottifactor,arelationbetweenthefrequencydependent

complexpermittivityoftheparticleandthemedium.

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

€

Fdrag = −6πηa v whereηisthedynamicviscosity

!

CM(") = Re#p $#m#p + 2#m

%

& '

(

) *

23

ofthemedium,aistheradiusofthesphere,and

€

v isthevelocityofthesphere

relativetothemedium.ThecombinationoftheexpressionfortheDEPforceona

sphere,Eq.2.3,withtheStoke’sDragforcegivesthevelocityofaparticle

€

v DEPmoved

withDEP,

€

v DEP =εma2CM(ω)∇

E RMS2

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

24

assumedtobenegligible.(Jones,1995)Theinteriorofthevesicleisdefinedby

R=1/σcandC=εcinparallel.Bycombiningthelumpedcircuitelementsassuminga

sphericalvesicleandanelectricfieldinradialdirections,asisshowninFig.2.3a,an

effectivepermittivityfortheobjectisarrivedat

. 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:

2.7

Therearenowtwomorecharacteristictimesinthesystem,thedielectricrelaxation

timeofthesolutionoutsideofthevesicleτs=εs/σsandtherelaxationtimeofthe

chargesontheoutsideofthevesiclebuildinguponthemembraneτm’=cma/σs.

!

"eff = cmaj#$ c +1

j#($m + $ c ) +1

%

& '

(

) *

!

CM(") = Re" 2(# s#m $ # c#m

') + j"(#m

' $ # s $ #m ) $1

" 2(# s#m

'+ 2# s#m ) $ j"(#m

'+ 2# s + #m ) $ 2

%

& '

(

) *

25

AvesiclecanbetrappedandmovedwithpositiveDEPatRFfrequenciesifthe

internalconductivityσcislargerthantheexternalconductivityσs.InFig.2.3bthe

CMfactor,Eq.2.7,isplottedversustheinteriorconductivityσcofavesicleorcell

suspendedinasolutionwithσs=10‐3S/m,εs’=εc’,a=5µm,andf=1MHz.Ifthe

conductivityinsidethevesicleσc>10‐3S/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

26

vesicle τmw=(εp+2εm)/(σc+2σs),CM>0andtheDEPforceispositive.Atthese

intermediatefrequenciesthemembraneistransparenttotheelectricfieldandthe

solutioninsidethevesicleactslikeaconductor,makingthevesiclemorepolarizable

thanthemedium.Atfrequenciesabovetheinterfacialdielectricrelaxationtimeτmw

ofthevesicle,theDEPforceisnegative(CM<0).Atthesehighfrequenciesthe

vesicleistransparenttotheelectricfield,makingthevesiclelesspolarizablethan

themedium.

27

Figure2.3(a)Alumpedcircuitmodelforavesicleoralivingcellwitharadiusa,thatconsistsofaninternalsolutionwithaconductivityσcandadielectricconstantεc,wrappedinathinshellwithaconductivitygmemandacapacitanceperunitareacmem,(b)theClausiusMosottifactorCMplottedversustheinternalconductivityofavesiclesuspendedinasolutionwithanexternalconductivityσs=10‐2S/minafieldwithafrequencyω=1MHz,(c)CMofavesiclewithaninternalconductivityσc=0.1S/msuspendedinasolutionwithaconductivityσs=10‐2S/mfactorplottedversusthefrequencyωoftheappliedelectricfield.

28

2.4TransmembranePotentialDifferenceanditsApplications

InadditiontotheabilitytotrapandmoveobjectswithDEP,electricfieldscanbe

usedtoelectroporatevesiclestoreleasetheircontents,fusetwovesiclestogether,

orpermeabilizecellmembranes,asisdemonstratedinChapter4.Thesetasksare

accomplishedbyusinglowerfrequencyelectricfields(kHzfrequenciescomparedto

theMHzfrequenciesusedforDEP)toinducevoltagesacrossthemembranesof

vesiclesandcells.Alargetransmembranevoltagecancauseporestoformin

membranes(electroporation)orcausetwovesiclestofusetogether(electrofusion).

Theelectroporationofavesicleorcelloccurswhenthereisalargepotential

differenceacrossthemembraneofacellorvesicleVTM.Ingeneral,electroporation

andelectrofusionoccurwhenthemaximumtransmembranevoltage

€

VTMmax isgreater

than1V.(Sugaretal.,1987)Themaximumvoltage

€

VTMmax inducedacrossthe

membraneofasphericalvesicleinanexternalelectricfieldis:(Grosseetal.,1992)

€

VTMmax =

3 E a

2 1+ (ωτmem )2 2.8

whereτmem=acmem(1/σc+1/2σs)istheinterfacialchargingtimeofthemembrane,

asisdescribedintheprevioussection.Atypicalvesicleorcellhasacapacitanceper

unitareaCmem=10‐2F/m2.(Jones,1995)Foravesiclewitharadiusa=5μmwith

aninternalconductivityσc=0.1S/msuspendedinasolutionwithaconductivity

σs=10‐2S/m,thereisaninterfacialchargingtimeτmem=20μs.Thetransmembrane

voltageVTMisplottedversusfrequencyinFig.2.4foravesicleorcell,asisdescribed

insection2.3,inanelectricfield

€

E = 3 V /µm .Atthefrequenciesf≈1MHzusedfor

29

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

30

theinducedtransmembranevoltageismuchlessthanthenormalphysiological

transmembranevoltageVTM<<70mV,thenitwillhaveonlyasmalleffectoncell

physiology.(Olofssonetal.,2003)AcelltrappedinaDEPtrapwithanelectricfield

strength

€

E = 3V /µm andafrequencyf=1MHzwillhaveaninduced

transmembranevoltageVTM≈1mV.Therefore,acellinaDEPtrapshouldremain

healthyuntilthefrequencyoftheappliedelectricfieldispurposelyloweredto

induceelectroporationorelectrofusion.

31

Figure2.4(a)Thetransmembranepotentialofamodelofavesicleorcellwithaninternalconductivityσc=0.1S/m,suspendedinasolutionwithaconductivityσs=10‐2S/m,witharadiusa=5µmandacapacitanceofCmem=10‐2F/m2isplottedversusthefrequencyfofanappliedelectricfield

€

E = 3 V /µm ,(b)astep‐by‐step

illustrationofthefusionoftwovesicles.

32

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 =ωεoε' ' E

2

2.9

Thelossfactorofthematerialdependsonthefrequencyoftheelectricfieldandthe

characteristictimeτWofthedielectricrelaxationofthematerial,withthe

expression:

€

ε' '= (εs −ε∞)1+ (ωτ )2

+σωεo 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/m3]absorbedbywaterin

33

anelectricfieldwithamagnitude

€

E =105 V /m isplottedversusfrequencyfor

severalsolutionswithdifferentconductivitiesσ.Fordeionizedwaterthepower

densityincreasesmonotonicallyuntilitplateausatf=18GHz,thefrequency

associatedwiththecharacteristicrelaxationtimeofwaterτw.Forsolutionswitha

finiteconductivityσ,thepowerdensityhasaconstantvalue

€

P = εo

E

2σ 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).

34

Figure2.5.ThepowerdensityP(W/m3)plottedversusthefrequencyfoftheappliedelectricfield.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 . 2.11

35

Foraparamagneticparticlewithamagneticsusceptibilityχ,themoment

€

m = Vχ B /µoisproportionaltotheexternalmagneticfieldstrength

€

B ,thevolumeof

theparticleV,andthevacuumpermeabilityµ0.Theforceonaparamagneticparticle

is:(Leeetal.,2004)

€

F MAG =

−Vχµo

∇ B 2. 2.12

Intheworkpresentedinthisthesis,superparamagneticparticlesareused.

Superparamagnetismisaformofmagnetismwhereverysmallferromagnetic

particlesbehavelikeparamagnets.Theparticlesaresmallenoughthatthermal

fluctuationscanceltheirnetmomentwhenthereisnofieldapplied,butanapplied

fieldwillcausetheparticlestoalign.Assuch,superparamagneticparticlesmaybe

describedwithahighmagneticsusceptibilityχatfieldslowerthantheirsaturation

value.(Beanetal.,1959andLeeetal.,2005)

36

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.

37

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)

11x11μm2pixels,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)

38

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=10‐2S/m,inanelectricfieldwithafrequencyf=1MHzandanRMSmagnitude

showninFig.3.1a,isplottedinFig.3.1b.Thedielectricobjectistrappedatthe

interfaceoftwopixelsontheleftthatareturnedonandtwopixelsontherightthat

areturnedoff.ThemagnitudeofthetrappingforceisF~1nNandtheforceis

localizedwithintwopixellengthsofthetrap.Alineisfittotheforcecurveatthe

locationofthetrap,andtheeffectivespringconstantisfoundtobek=520pN/µm.

39

3.2.2CharacteristicTimes

Theratethatthedevicecanperformoperationsislimitedbythetimethatittakes

formicroscopicobjectssuspendedinsolutionabovethechip’ssurfacetoreactto

theelectricormagneticfields,notthespeedoftheelectronics.Therearetwo

importantcharacteristictimesthatdescribethemicroscopicobjectssuspendedin

solutiononourchips:thetimethatittakesforaparticletodiffuseawayifatrapis

turnedoffτdiffandthetimethatittakesforaparticletomovebetweentrapswhen

onepixelisturnedoffandthenextisturnedonτmove.Aparticlesuspendedina

solutiontakesatimeτdiff=L2/16Dtodiffusethedistanceofhalfofapixel

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.

40

Figure3.1.(a)Afiniteelementsimulationoftheelectricfieldstrength5μmabovethechip’ssurface,(b)aplotoftheforceonadielectricobject(asmodeledinchapter2.3)attheinterfaceoftwopixelsontheleftthatareturnedon(inyellow)andtwopixelsontherightthatareturnedoff(inwhite).

41

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

bitsofeachwordarereadinandoutofthechipwithasequentiallyloadedtwo‐

phaseclockedshiftregister.Eachwordisaddressedtoaspecificrowinthearray

usinga7bitrowdecoder.Therowdecodertakes7binaryinputsandusesthemto

choose1of27(128)rowsoftheSRAMarray.Amicrographofthechip,showinga

sectionofthearrayofDEPpixelssurroundedbythecontrolelectronics,isshownin

Fig.3.2c.

AschematicofthecircuitunderneatheachDEPpixelisshowninFig.3.2b.The

SRAMmemoryelementsunderneatheachpixelareaddressedwithaword‐lineand

itsvaluesetwithabit‐line.TheSRAMelementcontrolsamultiplexer(MUX)that

routesoneoftwosignals,producedoffofthechip,tothe11x11µm2pixelabove.

GenerallythetwosignalsareRFvoltagesthatare180°outofphase,butcanbeset

arbitrarily.TheoutputoftheMUXgoesthroughavoltage‐amplifierbeforedriving

thepixel.Thevoltage‐amplifierisatwotransistorinverter,appropriatelysizedto

drivethecapacitiveloadofthepixel.Thevoltage‐amplifiershaveanon‐resistance

ofRon≈10kΩ,drivingapixelcapacitanceoflessthanCload≈50fF,whichyieldsa

42

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.Foreachpixeltobefullyrandom‐

accesswouldrequiretwoextratransistorsunderneatheachpixel,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

43

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.Thedesignsoftwarechecksthatthecircuitsatthemask‐

levelmatchesthecircuitsatthemodule‐level.Theelectricaltestingbeginsatthe

bottomofthehierarchyatthemodule‐level,usingSPICEmodelsforeachelement,

totestthedigitalandanalog(bothtimedomainandfrequencydomain)responseof

thecircuits.Oncethelayoutforamoduleisdrawn,additionalunintended

capacitancesinthesystemareextractedfromthelayoutandincorporatedintothe

testing.Thenewlayoutcanbemodifiedbasedontheresultsofthetesting,andthis

cyclecanberepeatediniterationsuntilthedesignspecificationsofthemoduleare

met.Themodulesarecombinedandthesystemistestedworkingfromthebottom

44

ofthehierarchytothetop,untiltheentirechipislaidoutandfullytested.Forthe

chipsdesignedinthisthesis,thedesignprocesstakesabouttwomonthsfromstart

tofinishandresultsinachipwithN≈300,000transistors.

45

Figure3.2.(a)AschematicofthearchitectureoftheSRAMarrayandthelogicandmemorythatareusedtoreadandwritetoitontheDEPchip,(b)aschematicofthecircuitunderneatheachDEPpixel,showingtheSRAMmemoryelement,amultiplexer(MUX)thatdirectseithertheRFsignaloritslogicalinversetoavoltagedriverthatconnectstotheelectrode,(c)amicrographofa250x200µm2sectionshowingacornerofthechipwithaportionoftheDEParrayintheupperleftcorner,therowcontrolcircuitsonthebottom,andthebitcontrolcircuits,includingtheshiftregister,ontheright.

46

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).

47

Figure3.3(a)AtimesequenceofyeastandratalveolarmacrophagesbeingtrappedandmovedwithDEP.Theblackarrowsindicatethedirectionthatthecellsaremovedbetweenframes.Thecellsmoveatarateof~50µm/sec,(b)atimeseriesofdropsofcoloredwaterbetweenalayeroffluorocarbonoilandhydrocarbonoiltrapped,moved,split,andmergedwiththechip,(c)amicrographoftheentirechipbeingusedtopositionthousandsofyeastcellstospell“Harvard.”(Huntetal.,2008)

48

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.

3.3.1Fluidics

AfluidcellisbuiltdirectlyontopoftheICwithasiliconegasket(Invitrogen:p‐

24744)witha1.2mmholethatiscutwithaholepunch,asisshowninFig.3.4c.A

3x3mm2glasscoverslipisplacedontopofthefluidcelltosealit.Thefluidcell

canbefilledwithapipette.Directlypipettingfluidontothechip’ssurface,rather

thansettingupamicrofluidicnetwork,keepstheexperimentalapparatusflexible,

allowingmanyexperimentstobeattemptedinashorttime.

49

TheIC/microfluidicchipcanbeintegratedasacomponentinalargerPDMSor

glassbasedmicrofluidicsystem.Passivemicrofluidicscancoupletheoutsideworld,

anditsmacroscopicfluidsamples,tothechipinarolethatisanalogoustotherole

printedcircuitboardsplayforICsincomputers.TheIC/microfluidicchipcould

behaveasaprogrammablecomponenttoperformtaskssuchassortingor

combiningsamplesandreagents.Insuchasetup,thefluidoutputofthechipcould

befedtooutputchannelsofthepassivemicrofluidicdeviceforfurtherprocessingor

collection.

3.3.2Electronics

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.

50

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.

3.3.4Optics

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

51

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).

52

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.

53

Figure3.4.(a)AschematicoftheexperimentalapparatussurroundingtheIC/microfluidicchip.Thegreenboxesrepresentcomponentsthatareelectrical,bluerepresentfluidic,orangerepresentoptic,andyellowrepresentswherethecomponentscometogether,(b)anopticalmicrographoftheIC(b)84pinchipcarrierholdingtheIC,ontopoftheICisaredfluidcell,(c)aphotographoftheexperimentalsetupofthehybridIC/microfluidicsystem.

54

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

55

thehumanfromthecontrolloop,andallowcomplexautomatedtaskssuchas

sortingtobedoneinaclosed‐loop.

Figure3.5.ScreenshotsoftheGUI,(a)ThemainscreenfortheGUIwhereobjectscanbedrawnonabitmapthatrepresentsthestateofthechip.Theblackandwhitestripesarethearbitrarypatternthatisbeingwrittentothechipinthescreen‐shot,(b)the“advancedmode”oftheGUIwhereobjectsthatarecreatedinthemainscreencanbemovedwithacursor.

56

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

57

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.

58

Thefrequencydependenceofthedielectricpropertiesofvesiclesandcellsenable

thedielectricphenomenonusedinthischaptertobeindependentlyaccessedwith

electricfieldsatdifferentfrequencies.VoltagesatMHzfrequenciesareusedfor

DEP,totrap,position,anddeformcellsandvesiclesasisdescribedinSection4.2.3.

Voltagepulseswithmspulse‐widthsareusedforelectroporationandelectrofusion,

totriggerthereleaseofthecontentsofvesicles,fusetwovesiclestogether,and

permeablizethemembranesofcellsasisdescribedinSection4.2.4.

TheDEPChipisusedinthischapterdemonstratebasicfunctionsonlivingcellsand

unilamellarvesicles,whichcanbestrungtogethertoperformcomplexbiological

andchemicallaboratorytasks.InSection4.3.1theDEPChipisusedto

simultaneouslyandindependentlytrapandmovemanyindividualvesiclesand

livingcellswithdielectrophoresis(DEP).InSection4.3.2voltagespulsescreatedby

theDEPChipareusedtoselectivelyelectroporatevesiclestoreleasetheircontents,

fusetwovesiclestogether,andpermeabilizelivingcells.InSection4.3.3vesiclesare

deformedbychangingtheshapeoftheDEPtraps.Theplatformdemonstratedin

thischaptercanbeusedforawiderangeofchemicalandbiologicalapplicationsand

isastepforwardforthefieldofhybridIC/microfluidics.

59

Figure4.1.Aschematicrepresentationofawaterdropinoilemulsionstabilizedwithsurfactantsisshownontheleft.Aschematicrepresentationofaunilamellarvesicleisshownontheright.Thephospholipidmoleculesthatformthesurfactantandthevesiclemembraneareshownwithredcirclesforthehydrophilicheadsandblacklinesforthehydrophobictails.

60

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)11x11µm2pixels,eachofwhichcanbedriven

withanRFvoltagewithabandwidthfromDC–11MHz.Theentirearrayofpixels

canbeupdatedhundredsoftimesinasecond.AmicrographoftheIC/microfluidic

chipisshowninFig.1.1b.

4.2.2UnilamellarVesicles

Lipidvesiclesaredropsofwaterencapsulatedbyathinphospholipidbilayer

membrane.Aschematicofthecross‐sectionofaphosphlipidunilamellarvesicleis

showninFig.4.1.Thephospholipidmembraneconsistsofamphipathic

phospholipidsthatself‐assembleintobilayerssuchthattheirhydrophilicheadsface

theinsideandoutsideofthevesicle,providingarobustcontainerforaqueous

61

solutions.(Albertsetal.,2007)Aunilamellarvesicleconsistsofasinglebilayer

membrane,asopposedtoseveralassomevesiclesdo.

ThevesiclesaremadethroughcollaborationwithThomasFrankeatUniversityof

AugsburginGermany.Thevesiclesarepreparedwithliposomeelectroformation

usingamodificationoftheAngelovaMethod.(Angelovaetal.,1986)Therecipe

usedtocreatethevesiclesisasfollows.Briefly,asmallamountoflipidin

chlorophorm(ca.20microliterofa5mg/mlsolution)isdepositedontotwoindium‐

tin‐oxidecoatedglassslides.Afterevaporationoftheorganicsolventforatleast

6hourstheslidesareassembledinparallelwithaseparating2mmteflonspacerto

formthepreparationcellandincubatedforonehourinasaturatedwatervaporat

roomtemperatureforprehydration.Subsequently,theelectroformationcellisfilled

with200mOsmaqueoussolution(50mMsodiumchlorideandsucrose)andalow

frequencyelectricfieldof500Hzandamplitude1V/mmisappliedtotheITO‐

slides.Afterapproximately8hourslipidvesiclesofdiameterslargerthe20µmare

harvested.Theexternalsaltysolventisremovedbyrepeatedlywashingwithaniso‐

osmoticaqueousglucosesolutionandgentlecentrifugationatlowrotation

frequencies.ThisprocedureprovidesthedielectriccontrastnecessaryforDEP

actuationandstillensurestheintegrityofthevesiclecontainers.

4.2.3Dielectrophoresis(DEP)ofVesiclesSuspendedinWater

ThehybridchipusespositiveDEPtotrapandmovecellsandvesiclessuspendedin

water.Anyobjectthathasacomplexpermittivitythatisdifferentthanthe

surroundingmediumcanbecontrolledusingDEP,(Jones,1995)asisdescribedin

62

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=10‐3S/m.Iftheconductivityinsidethevesicleisgreaterthanσint>10‐2S/m

thentheCMfactorispositive,andthevesiclecanbetrappedandpositionedwith

positiveDEP.

Thedielectrophoreticresponseofcellsandvesiclesisafunctionofthefrequencyof

theappliedelectricfield.InFig.2.3caplotofCMversusfrequencyisshownfora

vesiclewithinteriorconductivityσint=50mS/m.Belowacut‐offfrequencyof

ωd=50MHzandaboveacut‐offfrequencyofωmem=2kHz,theCMfactorispositive

andinvariabletochangesinfrequency.Itisinthisfrequencybandthatpositive

DEPisusedinthischapter.Thehighfrequencycut‐offissetbythedielectric

63

relaxationtimeτd=εp/σintofthesolutioninsidethevesicle.Thelowfrequencycut‐

offissetbythechargingtimeofthemembraneofthevesicle

τmem=aCmem(1/σint+1/σsol).

4.2.4ElectroporationandElectrofusion

Voltagepulsescreatedbythechipcanbeusedtoelectroporateandelectrofuse

vesiclesandcells,asisexplainedinfulldetailinChapter2.Electroporation

generallyoccurswhenthereisatransmembranevoltageVTM>1V.(Sugaretal.,

1987)ThemaximumvoltageinducedacrossamembraneVTMofasphericalvesicle

orcellinanexternalelectricfieldisgivenbyEq.2.8.Thecharacteristiccharging

timeofthemembraneisgivenbytheexpression:(Jones,1995)

€

τmem = aCmem (1σ int

+1

2σ sol

) 4.1

whereCmem=10‐2F/m2isthespecificmembranecapacitanceofaunilamellar

vesicle.(Jones,1995)Foravesiclewitharadiusa=5µmandinternalconductivity

σint=0.1S/minasolutionwithconductivityσsol=10‐2S/m,thereisamembrane

chargingtimeτmem=20ms.Electricfieldswithfrequencies1/ω<τmemareusedfor

DEP.Atthef=1MHzfrequenciesusedforDEPthetransmembranevoltage

VTM≈10mVandnodamageiscausedtothemembrane.(Albertsetal.,2007and

Grosseetal.,1992)Electricfieldscreatedwithvoltagepulseswithapulsewidth

τ>τmemcreatetransmembranevoltagesVTM~1Vandareusedtoelectroporateand

electrofusevesiclesandcells.

64

Theabilitytoselectivelyelectroporatevesiclesenablesthechiptolocallyrelease

substancesintothesolution.Thislocalreleaseofsubstancescreatesagradientin

theconcentrationasafunctionofthedistancerfromthecenterofthevesicle.

Consideravesiclewitharadiusafilledwithaconcentrationn=nsofasubstance

suspendedinasolutionthathasnoneofthesubstancen=0.Thegradientinthe

concentrationiscalculatedbysolvingthediffusionequationinspherical

coordinatesaroundthevesiclewiththeboundaryconditionsthattheconcentration

n=nsadistancer=afromthecenterofthevesicleandn=0atadistancer=∞.

(DillandBromberg,2003)Thesolutiontothediffusionequationisplottedfora

vesiclewithradiusa=5µminFig.4.2aandisgivenbytheexpression:

€

n(r) =nSar . 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−ar) 4.3

65

Theconcentrationgradientsdependonlyontheconcentrationofthesubstances,

theradiusofthevesicle,andthedistancefromthevesicle.Theabilitytocreate

well‐controlledconcentrationgradientsinthesolutionisatoolthatcanbeusedfor

deliveringreagentsandsamplestocellsortootherreagentsforchemistryand

biologyexperimentsonthechip.

Thevoltagepulsesusedforelectroporationcanbetime‐multiplexedwiththe

voltagesusedforDEP.Thisispossiblebecauseduringthedurationofthems

voltagepulses,duringwhichtheDEPforceisturnedoff,cellsorvesiclesdonot

diffuseasubstantialamount.Aparticlesuspendedinasolutiontakesτdiff=L2/16D

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.

66

Figure4.2(a)Aplotoftheconcentrationgradientcreatedaroundavesicle,shownastheorangecircle,thatisreleasingasubstanceatitssurface,(b)aplotoftheconcentrationgradientcreatedaroundavesicleorcell,shownasthepurplecircle,thatisreactingwithasubstanceinthesolutionatitssurface.

67

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

68

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.

69

Figure4.3(a)TimesequenceofvesiclespositionedwithDEP.Pixelsareturnedoninsequencetotrapandmovethevesicles.Thegreenlineshowsthedirectionthatthechipmovesthevesicle.Themaximumspeedofavesicleis70μm/sec.Eachframeisseparatedby~1sec,(b)amicrographofhundredsofvesiclessimultaneouslypositionedtospellan‘H,’(c)timesequenceofvesiclesandcellssimultaneouslytrappedandmovedonthechip.Thefluorescenceimageofthevesiclesissuperimposedontothebrightfieldimageofthecellsandiscoloredred.Thegreenlineshowsthedirectionthatthechipismovingthevesiclesorcells.Eachframeisseparatedby~1sec.

70

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.

4.3.3ElectroporationofCells

InthissectionitisdemonstratedhowthehybridIC/microfluidicchipscan

permeabilizeacell’smembrane,allowingsubstancesinthesolutiontoenterthecell.

71

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.

4.3.4TriggeredFusionofVesicles

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.

72

ThefusionoftwovesiclesallowspLvolumesofsamplestobecontrollablymixed.

Thevoltagepulsesusedtofusethevesiclesdonotleadtoanappreciablemixingof

thecontentsofthevesiclewiththesolution.Thevesiclesareobservedtofuseafter

0.5secofthepulsesequence.IntheexperimentshowninFig.4.4a,ittakes~4sec

forasimilarvesicletohaveappreciablemixingwiththeoutsidesolutionusingthe

samepulsesequence.Thereforeinthetimethatittakestofusethevesiclesthere

shouldnotbeappreciablemixingofthevesicleswiththesolution.Theabilityto

selectivelyfusetwovesiclestogetherenablessmallisolatedvolumesofsamplesto

bemixedonthechip,animportantfunctionforperformingchemistryandbiology

experiments.

73

Figure.4.4(a)TimesequenceofavesicleheldinplacewithDEPfieldswhileitscontentsarereleasedintothesolutionwithelectroporation.Thevesicleissuspendedinaconcentratedfluorosceinsolution.Theelectroporationcausethevesicletomixitscontentswiththesolution,makingthevesicletofluoresce.Inthefirstthreeframes,eachseparatedby4seconds,thevesicleiselectroporated.ThevesicleisheldinaDEPtrapfor2minutes.Inthenextthreeframes,separatedby4seconds,thevesicleiselectroporateduntilthecontentsofthevesiclefullymixeswiththesolutionandthevesiclestopsfluorescing,(b)acellisheldinplacewithDEPandelectroplatedinasolutioncontainingTrypanBluestain.Theleftandrightframeshowthecellbeforeelectroporationandafter40sec.ofelectroporation,respectively,(c)twovesiclesarebroughtintocontactwithDEPandthenfusedtogetherwithelectrofusion.Theschematiconthebottomshowwhichpixelsareturnedon.AgreenpixelindicatesapixelthatisturnedonforDEPandaredpixelindicatesapixelthatisturnedonwithvoltagepulsesmultiplexedwiththeMHzfrequencyvoltage.

74

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)

75

Figure4.5VesiclesaredeformedintoavarietyofshapesbychangingtheshapeoftheDEPtrap.Theschematicsontheleftshowwhichpixelsareturnedon.Redandwhitepixelsindicatepixelsthatareturnedonoroff,respectively.Thecentergraphicsareplotsofthesimulatedelectricfieldstrength

€

E 5µmabovethechip’s

surface.Ontherightaremicrographsofthedeformedvesicles.

76

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

77

optics(Cuietal.,2008)wouldopenupmoreapplicationsandleadtoamore

versatileandself‐sufficientplatform.(Leeetal.,2007)

78

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.

79

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)

80

5.2DescriptionoftheChip5.2.1FieldSimulations

Thechipcreateselectricfieldsaboveitssurfacewitha60x61pixelarrayof

30x38µm2electrodesthatcanbeindividuallyaddressedwith50Vpeak‐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

81

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)

82

Figure5.1.(a)Amicrographoftheintegratedcircuit,(b)themagnitudeoftheelectricfield|

€

E |,fromaquasi‐staticelectricfieldsimulation,plotted5µmabove

thechipsurface.Thepixelsareshownasbluetilesthatcoverthesurfaceofthechip.Twopixelsareheldat50Vrelativetothesurroundingpixels,(c)themagnitudeofthemagneticfield|

€

B |,fromsimulation,plotted5µmabovethechip’ssurface.The

wiresareshownasbluestripesrunningacrossthesurfaceofthechip.Twowiresaresourcedwith120mAandallsurroundingwiressetto0mA.

83

5.2.2ChipArchitectureThechip’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.Theshiftregisterusesatwo‐

phaseclockingscheme.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

84

lineshavedrivingcircuitryonbothends.Bysettingthememoryelementsoneither

sideofthewireacurrentof±120mAor0mAflowsthroughthewire.Interspersed

throughoutthearrayare16distributedtemperaturesensorsthatcanbe

individuallyaddressedtoberoutedtoananalogvoltageoutput.

ThehybridchipisconstructedbybuildingasimplefluidcelldirectlyontopoftheIC

withasiliconegasket.Thefluidcellispreparedbycuttinga1.2mmholeoutof

Press‐to‐SealTM0.5mmthicksiliconesheetsfromInvitrogen(Invitrogen:p‐24744)

withahole‐punch.ThesiliconegasketisplacedontotheICunderastereoscope

withtweezers.A3x3mm2glasscoverslipsealsthefluidcell.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.

85

Figure5.2.(a)AmicrographofapartoftheintegratedcircuitshowingtheDEParray,themagneticcurrentdrivers,andthelogicandmemoryusedtoupdatetheSRAMarray,(b)aschematicofthecircuitryunderneatheachDEPpixel,showingtheSRAMmemoryelement,amultiplexer(MUX)thatdirectseithertheRFsignaloritslogicalinversetoavoltagedriverthatconnectstotheelectrode,(c)aschematicofthecircuitryforeachmagneticwireinthematrix,showingtheSRAMmemoryelements,andtheMUXsthatconnecttothecurrentdriverstodrivethewireswitheither±120mAor0mA.

86

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.

5.3.1Dielectrophoresis: Trapping and Positioning Vesicles

ThissectiondemonstrateshowtheHV‐DEP/MagneticChipsimultaneouslyand

independentlycontrolsthepositionofobjectswithDEP.Figure5.3shows

87

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.

88

Figure5.3AtimesequenceoftheDEPpositioningofvesicles.Theframes(a‐d)arefluorescenceimagesoftherhodaminestainedmembranesofthevesicles.TheDEPpixelsareturnedoninsequencetopositionthevesicles.Thedashedgreenlineshowsthedirectionthatthechipismovingthevesicles,theyellowsquareshowstheDEPpixelthatisactivated.Themaximumspeedofavesicleis80μm/sec.Eachframeisseparatedbyroughly1sec.

89

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.

90

Figure5.4Timesequenceofmagnetictrappingandpositioningofamagneticbead.Magneticwiresinthematrixareturnedoninsequencetopositionthebead.Thepositionoftheparticleismarkedwithagreencircle.Thedirectionthatthebeadisbeingpulledismarkedwithagreenarrow.Theredlinesshowswhichmagneticwiresinthematrixhavebeenturnedon,theactualwiresareburiedundertheDEPpixelsandarenotvisible.Eachframe(a‐d)isseparatedbyroughly2sec.

91

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)

92

Figure5.5AvesicleisheldinplacewithaDEPpixelwhileatetherispulledusingthemagneticmatrix.TheyellowsquareshowstheDEPpixelthatisactivated,andtheredlinesshowthemagneticwiresthatareturnedon.Thevesicleisimplantedwithironoxidenanoparticles.Afterthefirstframe(a)themagneticfieldisturnedoffandthetetherispulledbackintothevesicle.Eachframe(a‐f)isseparatedby0.2seconds.

93

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‘laboratory‐

on‐a‐chip’functionsdemonstratedinChapter4,tocreateageneral‐purpose

platformforbiologyandchemistryexperimentsonindividuallivingcells.The

techniquesdemonstratedinChapter4canbeusedtomixsmallvolumesofreagents

94

andsamplestogetherandtocontrolthechemicalenvironmentofindividualcells.

Themagneticactuationofnanoparticles,demonstratedinthischapter,canbeused

tocontrolcellsembeddedwithmagneticnanoparticlestoactivateordeactivate

specificsurfacereceptorsorionchannels.Thehybridchipscansimultaneously

performmanyoftheseexperimentsinparallel,allowingastatisticalnumberof

independentlycontrolledsingle‐cellexperimentstobecarriedout.

95

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

96

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

97

microfluidicdevicescontrolthelocalizationofthetemperaturechangeandtendsto

beontheorderofcentimeters.(Nakanoetal.,1994andKhandurinaetal.,2000)

Temporalcontrolislimitedbytheheatcapacityandthermalcouplingofthe

microfluidicdevicetotheenvironmentandthermalrelaxationtimestendtobeon

theorderofseconds.(Nakanoetal.,1994andKhandurinaetal.,2000)Alternative

techniquestoimprovethelocalizationandresponsetimehavebeendeveloped,

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’

98

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

99

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:

€

ΔTSS =VALDkoil

P. 6.1

Thesystemhasacharacteristictimescaleτs=RCthatdescribesthethermal

responsetimeofthesystem,

€

τ S =VALDkoil

CW. 6.2

Thecharacteristictimescaleτsdescribesthethermalrelaxationtime,thetimeit

takesforthedroptoreachanequilibriumtemperaturewhenthemicrowavepower

isturnedonandthetimethatittakesforthedroptoreturntothebasetemperature

ofthedevicewhenthemicrowavesareturnedoff.

100

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‐glassdrop‐

basedmicrofluidics.Microwavepowerislocallydeliveredviametalelectrodesthat

aredirectlyintegratedintothemicrofluidicdeviceandthatrunparalleltothefluid

channel,asisshowninFig.6.1a.Thedropsarethermallyinsulatedfromthebulkby

beingsuspendedinlowthermalconductivityoil.Aschematicofthedeviceisshown

inFig.6.2a.Syringepumpsprovidetheoilandwateratconstantflowratestothe

microfluidicdevice.Adropmakerandtwodropsplittersinseriescreatedropsthat

areproperlysizedforthemicrowaveheater.Themicrowavepoweriscreatedoff

chipusinginexpensivecommercialcomponents.

101

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.

102

Figure6.1(a)Aschematicofthemicrowaveheater.Theblacklinesrepresentthemetallineswhichareconnectedtothemicrowavesource,thecenterfluidchannelcarriesdropsofwaterimmersedinfluorocarbon(FC)oil,(b)acrosssectionofthemicrowaveheaterwithaquasi‐staticelectricfieldsimulationsuperimposedisshown,theelectricfieldisplottedinlogscale,(c)thecalibrationcurveoftheCdSenanoparticleswhichareusedastemperaturesensors,wherethecirclesaredatapointsandtheredlineisthefit.

103

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.

104

Theelectronicsthatcreatethemicrowavepowerareassembledusinginexpensive

modules.Themicrowavesaregeneratedwithavoltagecontrolledoscillator(ZX95‐

3146‐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

dimensions50x50μm2.Paralleltoand20μmawayfromeachsideofthefluid

channelaremetallinesthatare100μmwideand50μmhigh.Superimposedonthe

schematicinFig.6.1bisaquasi‐staticelectricfieldsimulationoftheelectricfield

(Maxwell,Ansoft).Fora12Vpeak‐to‐peaksignalacrossthemetallines,theRMS

electricfieldwithinadropwitha15μmradiussuspendedinfluorocarbonoilis

|E|~8×103V/m.Theelectricfieldlinearlyscaleswiththevoltageacrossthemetal

lineswhichallowsustocalculatethefieldwithinthedropforanyvoltage.The

105

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

106

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.

107

Figure6.2(a)Aschematicofthemicrowaveheatingdevice,showingtheoilinorange,thewaterinblue,andtheelectronicconnectionsinred,(b)amicrographoftheflow‐focusingdropmaker,(c)thetwosetsofdropsplittersinseries,(d)amicrographofthemicrowaveheater,thedarkregionsthatrunparalleltothefluidchannelarethemetallines,(e)aphotographofthemicrofluidicdevice,connectedtothemicrowaveamplifierwithacablewithanSMAconnector,ontopofahotplate,underneaththefluorescencemicroscope.

108

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,thetemperaturerisestoasteady‐

statevalueof26°Cabovethebasetemperatureof21°Cin15ms.Thecurvein

Fig.6.3cisarrivedatbyusingthecalibrationcurveoftheCdSenanocrystals,

Fig.6.1c,toconvertthefluorescenceintensityinFig.6.3bintoachangein

temperatureΔT.Thesumoftheflowratesoftheoilandwaterareusedtocalculate

thespeedofthedropsthroughthechannel,whichmaybeusedtotransformthe

109

lengthinFig.6.3aintothetimethatthedropshavespentintheheater.Eachdata

pointconsistsoftheaverageof20independentlytaken2secexposures.

110

Figure6.3(a)A2secondexposureofthefluorescencesignalnormalizedtoanimagetakenwiththemicrowavesourceturnedoffsuperimposedontoabrightfieldimageofthedevice,(b)thelineaverageofthenormalizedintensityplottedversusdistancedownthechannel,(c)thetemperaturechangeΔTplottedversustimeandversusthedistancethedropshavetraveleddownthechannel.

111

Thecombinedstatisticalerrorofthemeasurementoftheheatingisalsoplottedin

Fig.6.3c.Theaverageerroris~1.4°C.Thestatisticalerrorinthesteady‐state

temperatureisfoundtocomeprimarilyfromvariationsinthesizeofthedrops.The

steady‐statetemperaturechangeofeachdropislinearlyrelatedtothevolume‐to‐

arearatioofthedropandtothedistanceofthedroptothechannelwalls,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

112

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

113

predictionsofLDcanbeatleastpartiallyexplainedbynotingthattheheatcapacity

inEq.6.2islargerthaniscalculated,duetothefactthatthechannelwallsarenota

perfectheatsinkasthemodelassumes.Theagreementbetweenthetwo

independentmeasurementssupportsthesimplemodelformicrowaveheatingof

dropsoutlinedinthebeginningofthischapter.

114

Figure6.4(a)Thesteady‐statetemperaturechangeofthedropsiscontrolledbyvaryingtheamplitudeofthemicrowavevoltage.Intheinsetthegreencurveshowsthedropheatingversustimeformicrowaveswithanamplitudeof11.7V,thered11.0V,theyellow10.3V,thelightblue9.3V,thepurple8.5V,thegrey7.5V,andtheblue4.5V.Thesteady‐statetemperaturechangeisplottedversusthepowerdensity,ascalculatedbyEq.2.1,inthemainplot,(b)themagnitudeofthechangeintemperatureΔTsubtractedfromthemaximumchangeintemperatureΔTSSversustimeisplottedonalog‐linearscale.Thetemperaturerisesasasingleexponentialwithacharacteristictime,τs=14.7±0.56ms.Theinsetshowsthetemperatureversustimeplotfromwhichthelog‐linearplotistaken.

115

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

116

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.

117

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.

118

Table7.1Alistofthelab‐on‐a‐chipfunctionsperformedinthisthesisusinghybridIC/microfluidicchips.

119

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.

120

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,andcombinatorialchemistrycouldbeperformedontheselow‐

cost,automateddevices.(Whitesidesetal.,2001,Stoneetal.,2004,Tabelingetal.,

2005,Yageretal.,2006,Martinezetal.,2008,Leeetal.,2007,Huntetal.,2008,

Maltezosetal.,2005)Thechipscouldbeprogrammedtoperformcomplex,multi‐

steptasks,andreacttotheresultsofexperimentsinreal‐time.Thedevicescouldbe

usedforapplicationssuchaspoint‐of‐carediseasedetectionordetectingtoxic

substancesorparasitesindrinkingwater.Suchadevicewouldrealizetheinitial

goalofmicrofluidics,tobringcomplexbiologicalandchemicaltestsfrom

laboratoriesoutintotheclinicandthefield.(Whitesides,2001)

121

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AppendixA.DataSheetfortheDielectrophoresisChip(DEPChip)

WedocumentthespecificationsfortheFabutron1.0,showthepinlayoutanddetail

theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave

alloftheinformationnecessarytomaketheFabutron1.0workintheirexperiment.

SummaryoftheFabutron1.0

TheFabutron1.0isacustomintegratedcircuitdesignedintheWesterveltlab.The

chipconsistsofanarrayof128x256pixels,11x11μm2insize,controlledbybuilt‐in

SRAMmemory;eachpixelcanbeindividuallyaddressedwitharadio

frequency(RF)voltageupto5Vpp,withfrequenciesfromDC‐11MHz.TheICwas

builtinacommercialfoundryandthemicrofluidicchamberwasfabricatedonits

topsurfaceatHarvard.

TableA1SpecificationsfortheDEPchip

Feature DescriptionBandwidth DC‐11MHzProcess 0.35µm,CMOSMOSISTSMCPixels 128x256(32,768)

11x11µm2ChipSize 2.32x3.27mm2Addressing 8‐bitwordlinedecoder

128bitshiftregister,twophaseclocked

DataLineBandwidth DC‐20MHzPixelVoltage Vp‐p=3‐5VPixelBandwidth DC–5MHzOperatingVoltage 5VOperatingCurrent 30mA–300mA

127

TableA2.AlistofthepinsfortheDEPChip

Pin DescriptionGND GroundVDD OperatingVoltage(5V)W(0:7)** WordAddressDecoderLinesφ1 PixelVoltage1φ2 PixelVoltage2c2 (0)decouplesSRAMelementsfromthebitline,(1)connects

SRAMelementsonactivatedrowtothebitlinec1 (0)Prechargethebitlines,(1)disconnectprechargeWR ControlLine:(1)enablestheshiftregistertowriteitsvalueto

thebitlines,(0)disablesshiftregisterfromwritingtobitlinesPASS Controlline:(1)Shiftregisterelementslookstoprevious

registerforinput,(0)registerslooktobitlineforinputDATA DatainputlineforshiftregistersCLK2 ClockforshiftregisterCLK1 ClockforshiftregisterOUT Dataoutputforshiftregister

FigureA1PinlayoutfortheDEPchip

128

Protocols

FigureA2.LoadDataIntoShiftRegisterandWritetoArray.

129

FigureA3.ReadDatafromSRAMArrayintoShiftRegisterandReadOutArray.

**WordAddressDecoderLinesareincorrectlylabeledonthechip.Notethatgoing

fromMSBtoLSBthecorrectorderingis:w3,w2,w1,w0,w7,w6,w5**

***Alsonotethattherewasanerrorinthedesignoftheshiftregisteranditactually

functionsasa64bitshiftregister,withneighboringregisterstiedtogether.Thefull

128bitsareaccessiblebyloadingin64bitsofdataatatime,anduseclock1toshift

thedataintotheproperposition.***

130

AppendixB.DataSheetfortheHighVoltageDielectrophoresis/MagneticChip(HV­DEP/MagneticChip)

WedocumentthespecificationsfortheFabutron2.0,showthepinlayout,anddetail

theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave

alloftheinformationnecessarytomaketheFabutron2.0workintheirexperiment.

SummaryoftheFabutron2.0

TheFabutron2.0isacustomintegratedcircuitdesignedintheWesterveltlab.The

chipconsistsofanarrayof60x61pixels,30x38μm2insize,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 DescriptionBandwidth DC‐10MHzProcess 0.6µm,CMOSXFABXC06Pixels 60x61(3,660)

131

30x38µm2ChipSize 3.4x3.7mm2Addressing 5‐bitwordlinedecoder

128bitshiftregister,twophaseclocked

DataLineBandwidth DC‐20MHzPixelVoltage Vp‐p=20‐50VPixelBandwidth DC–1MHzOperatingVoltage(Logic)

5V

OperatingCurrent(logic)

20mA

OperatingMagneticWireVoltage

5V

OperatingMagneticWireCurrent

0–800mA*

OperatingHighVoltage

50V

OperatingHighVoltageCurrent

20‐60mA**

TableB2.AlistofthepinsfortheDEPChip

Pin DescriptionGND GroundVDD OperatingVoltage(5V)HV HighVoltage(50V)HVg HighVoltageGate(45‐50V),controlstheactiveimpedanceintheRF

voltagedrivingcircuitunderneatheachcircuitVmag PowerSupplyfortheMagneticLines(5V)W(0:5) WordAddressDecoderLinesTHERM AnalogOutputfortheThermometer(0‐5V)φ1 PixelVoltage1φ2 PixelVoltage2c2 (0)decouplesSRAMelementsfromthebitline,(1)connectsSRAM

elementsonactivatedrowtothebitlinec1 (0)Prechargethebitlines,(1)disconnectprechargeWR ControlLine:(1)enablestheshiftregistertowriteitsvaluetothe

bitlines,(0)disablesshiftregisterfromwritingtobitlinesPASS Controlline:(1)Shiftregisterelementslookstopreviousregisterfor

input,(0)registerslooktobitlineforinputDATA DatainputlineforshiftregistersCLK2 ClockforshiftregisterCLK1 ClockforshiftregisterOUT Dataoutputforshiftregister

132

FigureB1PinlayoutfortheDEPchip

Protocols

TheprotocoltoupdatetheSRAMarrayisidenticalasfortheFabutron1.0

TheSRAMaddressesoftheDEPpixels,magneticwires,andthermometersareas

follows:

TableB3.Amapofthefeaturesofthe50V/magneticchipontheSRAMarray

Array SRAMAddress(word,bit)DEPArray(1:60,1:161) SRAM(3:32,5:126)***MagneticMatrixXdirection(1:2,1:122) SRAM(1:2,5:126)***MagneticMatrixYdirection(1:2,1:122) SRAM(1:30,1:4)***Thermometers(1:16) SRAM(1:8,127:128)***

*Notethatspecialcaremustbetakenwhenthechipisfirstturnedon.TheSRAM

willbesettoarandomstateandmorethanafewmagneticlineswillbeturnedon.

ItisadvantageoustoleavetheVmagturnedoffuntilthestatesofthewirescanbeset

suchthatnoneorfewofthewiresareturnedon.

133

**Notethataspecialfuseandvoltageconditioning/currentlimitingcircuitrymust

bebuiltintothePCBthathousestheIC.SpikesintheHVlinecandestroythechip.

***FormoredetailonhowtheSRAMarraymapsontotheDEPandMagneticarray

pleaseseelinetheattachedMATLABcodewherethedetailedmappingislaidout.

134

AppendixC.FabutronControlSoftware

LabviewProgramsusedtoCommunicatewiththeChips

FigureC1.Labviewcodeusedtoreaddatacomingfromthechips.

135

FigureC2.Labviewcodeusedtowritedatatothechips

FigureC3.BlockdiagramofLabviewcodeusedtoreaddatafromthechips

136

FigureC4.BlockdiagramofLabviewcodeusedtowritetothechip

137

MatlabCodeusedtoGenerateandReadPatternstotheChip

ProgramtowritedatafromamatrixcreatedintheGUIandpackageitintoalineofcommandssenttothechipwiththeLabviewprogram

functionfabwrite(image,protocol)

%functionfabwrite(image,protocol)

%

%1.30.2008

%

%CreateaTextfilethatLabViewcanreadwhichdirectsittowriteafile

%totheFabutronandreadfromitatthesametime.theoutputfilez.txt

%WrittenbyDavidIssadoreandKeithBrown

%

%Changedon2.4.2008toalsoworkwith5and50voltfabutrons

%Changedon2.24.2008toaccomidatemultiplelayerimages

%changedon8­15­08toswitchclock1andclock2online90

%

%%%

%%addedbydavetorearrangethedataforthe50Vchip.fornowwewill

%%justconcernourselveswiththeDEParrayandnotmagneticlinesor

%%thermometers

ifprotocol==50

%image=arrange50V(image);

end

%%SetupVariables

frames=size(image,3);

bits_per_row=size(image,2);

rows=size(image,1);

138

ecorr=10;

bcorr=10;

icorr=10;

command_lines=13+bcorr+ecorr+icorr;

cycles_per_bit=4;

commands_per_row=command_lines+bits_per_row*cycles_per_bit;

%%DefineRowWriteCommand

%Loadsdatafromdataportormemoryintoshiftregister

clock1=zeros(1,commands_per_row);

clock1(linspace(2,commands_per_row­command_lines­2,bits_per_row))=ones(1,bits_per_row);

%clock1(bits_per_row*cycles_per_bit+12)=1;

%Advancestheshiftregister

clock2=zeros(1,commands_per_row);

clock2(linspace(4,commands_per_row­command_lines,bits_per_row))=ones(1,bits_per_row);

clock2(bits_per_row*cycles_per_bit+10+bcorr+icorr)=1;

%Modulatesthedataimagesothatithastherightform

data=zeros(commands_per_row,bits_per_row);

fori=0:bits_per_row­1

data(i*cycles_per_bit+1:(i+1)*cycles_per_bit,i+1)=[1;1;1;1];

139

end

%Writeenablesdatatobewrittenintomemoryfromtheshiftregister

write=zeros(1,commands_per_row);

write(bits_per_row*cycles_per_bit+2+bcorr:bits_per_row*cycles_per_bit+5+bcorr)=ones(1,4);

%Passtellsitwhentolookatthepreviosregister/data(1)andwhento

%lookatmemory(0)

pass=ones(1,commands_per_row);

pass(bits_per_row*cycles_per_bit+8+bcorr+icorr:bits_per_row*cycles_per_bit+11+bcorr+icorr)=zeros(1,4);

%Tellswhenthewordlinesshouldbeenegaged

%words=zeros(1,commands_per_row);

%words(bits_per_row*cycles_per_bit­10:commands_per_row­1)=ones(1,command_lines+10);

words=ones(1,commands_per_row);

%Clock_C1Loadsdatato/frommemoryto/fromthebitline

clock_c1=zeros(1,commands_per_row);

clock_c1(bits_per_row*cycles_per_bit+3+bcorr:bits_per_row*cycles_per_bit+4+bcorr)=ones(1,2);

clock_c1(bits_per_row*cycles_per_bit+7+bcorr+icorr:bits_per_row*cycles_per_bit+11+bcorr+icorr)=ones(1,5);

%clock_c1(bits_per_row*cycles_per_bit+3:bits_per_row*cycles_per_bit+11)=ones(1,9);

%Clock_C2Turnsoffpre­chargingofpull­upcircuitry

clock_c2=zeros(1,commands_per_row);

clock_c2(bits_per_row*cycles_per_bit+bcorr:bits_per_row*cycles_per_bit+6+bcorr)=ones(1,7);

clock_c2(bits_per_row*cycles_per_bit+6+bcorr+icorr:bits_per_row*cycles_per_bit+12+bcorr+icorr)=ones(1,7);

140

%outputtriggertellsthecodewhentostartrecording

output_trigger=zeros(1,commands_per_row);

output_trigger(bits_per_row*cycles_per_bit­10)=1;

%output_trigger(1)=1;

%%

temp=clock1;

clock1=clock2;

clock2=temp;

%%

%plotthecontrolsingalstomakesuretheylookok

%Testcodetoberemoved

%

%figure

%plot(clock_c1­1.5,'b­')

%holdon

%plot(clock_c2+0,'r­')

%plot(words+1.5,'g­')

%plot(write+3,'k')

%plot(pass+4.5,'y')

%plot(clock1+6,'b­o','MarkerSize',3)

%plot(clock2+7.5,'r­o','MarkerSize',3)

%plot(data*ones(bits_per_row,1)+9,'k­o','MarkerSize',3);

%plot(output_trigger+10.5,'g­o','MarkerSize',3);

%legend('clockc1','clockc2','wordlines','write','pass','clock1','clock2','data','outputtrigger');

%%BuildCommand

141

temp=[];

forcf=1:frames

ifprotocol==5

k=linspace(0,255,256);

k=dec2bin(k,8);

%Thisistheorderthatweoriginallysuspected

%changed6­27­08,foundinTom'sbinder.

w0=str2num(k(:,4));

w1=str2num(k(:,3));

w2=str2num(k(:,2));

w3=str2num(k(:,1));

w4=str2num(k(:,8));

w5=str2num(k(:,7));

w6=str2num(k(:,6));

w7=str2num(k(:,5));

%Thisistheorderthatistheoppositeoftheonethatwe

%origniallysuspected.pshaw.

%w0=str2num(k(:,1));

%w1=str2num(k(:,2));

%w2=str2num(k(:,3));

%w3=str2num(k(:,4));

%w4=str2num(k(:,5));

%w5=str2num(k(:,6));

%w6=str2num(k(:,7));

%w7=str2num(k(:,8));

%%buildbitmap

bit_map=zeros(32,(rows+1).*commands_per_row);

142

fori=0:rows­1

%0.1­0.8notset

%0.9<­>CLK1

bit_map(9,i*commands_per_row+1:(i+1)*commands_per_row)=clock1;

%0.10­0.13notset

%0.14<­>CLK2

bit_map(14,i*commands_per_row+1:(i+1)*commands_per_row)=clock2;

%0.15<­>OUT_TRIGGER­notsetinthisloop

%0.16<­>DATA

%bit_map(16,i*commands_per_row+1:(i+1)*commands_per_row)=0;

bit_map(16,i*commands_per_row+1:(i+1)*commands_per_row)=(data*image(i+1,:,cf)')';

%0.17<­>PASS

bit_map(17,i*commands_per_row+1:(i+1)*commands_per_row)=pass;

%0.18notset

%0.19<­>W0

bit_map(19,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w0(i+1);

%0.20<­>W1

bit_map(20,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w1(i+1);

%0.21<­>WR

bit_map(21,i*commands_per_row+1:(i+1)*commands_per_row)=write;

%0.22<­>C1

bit_map(22,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c1;

%0.23<­>W4

bit_map(23,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w4(i+1);

%0.24<­>W5

bit_map(24,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w5(i+1);

%0.25<­>C2

bit_map(25,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c2;

143

%0.26­0.27notset

%0.28<­>W6

bit_map(28,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w6(i+1);

%0.29<­>W2

bit_map(29,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w2(i+1);

%0.30<­>W7

bit_map(30,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w7(i+1);

%0.31<­>W3

bit_map(31,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w3(i+1);

%0.32notset

end

%Settheoutputtriggertogooffonlyonce

bit_map(15,1:commands_per_row)=output_trigger;

%Setclock1,clock2andpassforthefinaldataout

bit_map(9,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock1;

bit_map(14,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock2;

bit_map(17,rows*commands_per_row+1:(rows+1)*commands_per_row)=pass;

elseifprotocol==50

%%preparewordlines

%preparewordlines

k=1:32;

k=dec2bin(k,5);

w0=str2num(k(:,5));

w1=str2num(k(:,4));

w2=str2num(k(:,3));

144

w3=str2num(k(:,2));

w4=str2num(k(:,1));

%%buildbitmap

bit_map=zeros(32,(rows+1).*commands_per_row);

fori=0:rows­1

%0.1­0.16notset

%0.17<­>PASS

bit_map(17,i*commands_per_row+1:(i+1)*commands_per_row)=pass;

%0.18<­>C1

bit_map(18,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c1;

%0.19<­>C2

bit_map(19,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c2;

%0.20<­>W4

bit_map(20,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w4(i+1);

%0.21<­>DATA

bit_map(21,i*commands_per_row+1:(i+1)*commands_per_row)=(data*image(i+1,:)')';

%0.22<­>OUT

%0.23<­>W1

bit_map(23,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w1(i+1);

%0.24<­>W0

bit_map(24,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w0(i+1);

%0.25<­>WR

bit_map(25,i*commands_per_row+1:(i+1)*commands_per_row)=write;

%0.26and0.27notset

%0.28<­>CLK1

bit_map(28,i*commands_per_row+1:(i+1)*commands_per_row)=clock1;

%0.29<­>W3

145

bit_map(29,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w3(i+1);

%0.30<­>CLK2

bit_map(30,i*commands_per_row+1:(i+1)*commands_per_row)=clock2;

%0.31<­>W2

bit_map(31,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w2(i+1);

end

%Settheoutputtriggertogooffonlyonce

bit_map(15,1:commands_per_row)=output_trigger;

%Setclock1,clock2andpassforthefinaldataout

bit_map(28,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock1;

bit_map(30,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock2;

bit_map(17,rows*commands_per_row+1:(rows+1)*commands_per_row)=pass;

end

%convertto32­bitnumber

exponent=(1:32)'*ones(1,size(bit_map,2));

temp=[temp,sum(bit_map.*2.^exponent,1)];

end

globalcommand;

command=temp;

%tellittowrite

globalstatus;

status=1;

Programtoreaddatafromthechipandrepackageitasamatrix

Function[pixel_array]=fabload(sizes,protocol)

%function[pixel_array]=fabload(sizes)

%CreatedbyKeithBrown1.30.2008toreadfilescreatedbytheLabView

%scriptthatreadsthefabutron

%2.12.2008Workswith5Vchip

146

%commentedoutbydavidissadoretoletlabviewpassthedatawithout

%savingtotheharddrive

%[headerdata]=hdrload('out.txt');

%%

globalouts

data=outs;

size(data)

%%

ifprotocol==5

%fabwrite

icorr=10;

bcorr=10;

ecorr=10;

overhead_per_row=13+icorr+bcorr+ecorr;

offset=26+icorr+bcorr+ecorr;

%%fabverify

%icorr=0;

%bcorr=0;

%ecorr=0;

%overhead_per_row=13+icorr+bcorr+ecorr;

%offset=21+icorr+bcorr+ecorr;

pixel_array=zeros(sizes(1),sizes(2));

bits_per_row=4.*sizes(2)+overhead_per_row;

temp=zeros(sizes(1),sizes(2)*4);

forj=1:sizes(1);

temp(j,:)=data(offset+bits_per_row*(j­1):offset+bits_per_row*(j­1)+sizes(2)*4­1);

end

pixel_array=temp(:,4*(1:sizes(2))­1);elseifprotocol==50

147

icorr=20;

bcorr=20;

ecorr=20;

overhead_per_row=(15+icorr+bcorr+ecorr).*1;

pixel_array=zeros(sizes(1),sizes(2));

bits_per_row=8.*sizes(2)+overhead_per_row;

%bits_per_row=4.*sizes(2)+overhead_per_row;

%offset=45

offset=­11+icorr+bcorr+ecorr;

temp=zeros(sizes(1)./2,sizes(2)*8)';

%temp=zeros(sizes(1),sizes(2)*4);

size(temp)

forj=1:sizes(1)./2;

%forj=1:sizes(1);

temp(:,j)=data(offset+bits_per_row*(j­1):offset+bits_per_row*(j­1)+sizes(2)*8­1);

%temp(j,:)=data(offset+bits_per_row*(j­1):offset+bits_per_row*(j­1)+sizes(2)*4­1);

end

%pixels(1:60,1:30)=DEP_array(1:60,1:30)

%pixels(61:120,1:30)=DEP_array(1:60,31:60);

pixel_array(1:60,1:30)=temp(4*(1:60)­3,1:30);

pixel_array(1:60,31:60)=temp(4*(61:120)­3,1:30);

%pixel_array=temp(:,4*(1:sizes(2))­1);

size(pixel_array)

end